HPLC Columns Guide Br7614en MK

HPLC and
UHPLC Column
Selection Guide
The comprehensive
Supelco® portfolio of analytical
solutions to meet your
U/HPLC and LC-MS needs
The Life Science business

of Merck operates as
MilliporeSigma in the
U.S. and Canada.
Table of contents
Our HPLC and LC-MS Workflow 4
Supelco® HPLC and UHPLC Column selection 5

Stationary phases at a glance 6
Selection of stationary phase carrier by their benefits 8
Selection of phase bonding by compound class 9
Selection by Chemical Structure of the Analyte using the log P Value 10
Selection of Chromatographic mode and Stationary Phase by log P Value 12
Selection by Specific Chromatographic Need (C18) 13
Selection by USP classification 14
Selection by Column Dimension 20
HPLC Columns hardware 22
Column Accessories and Pre-column Holder 24
Fused-Core® (Superficially Porous Silica Particles, SPP) HPLC and UHPLC columns 26
Ascentis® Express HPLC and UHPLC Columns 28
BIOshell™ HPLC and UHPLC Columns for Biomolecule Separation 38
Monolithic Silica HPLC Columns 46

Chromolith® and Chromolith® HR HPLC Columns 50
Chromolith WP 300 HPLC Columns for Biomolecule Separation
®
54
Fully Porous Particulate Silica (FPP) Columns 60

Purospher™ STAR HPLC and UHPLC Columns 62
Discovery HPLC Columns
®
70
Discovery® BIO 74
Ascentis HPLC Columns
®
76
Titan™ UHPLC Columns 80
SeQuant® HILIC HPLC and Capillary Columns 82
LiChrospher HPLC Columns
®
86
Superspher HPLC Columns
®
88
LiChrosorb® HPLC Columns 89
SUPELCOSIL™ HPLC Columns 90
Chiral HPLC Columns 94
Biomacromolecule Characterization: A Multipronged Separation Challenge 100

Sepax Technologies: HPLC Columns for Bioanalysis 102
Ion Exchange Chromatography 104
Hydrophobic Interaction Chromatography (HIC) 106
Affinity Chromatography 112
Synthetic Carbons for Chromatographic Applications 114
Customized Packings 120
2
The Supelco® portfolio of analytical
solutions is developed by analytical
chemists for analytical chemists to
ensure your results are accurate, precise
and reproducible. Every product is
meticulously quality controlled to maintain
the integrity of your testing protocols
and, with our dedicated scientists, the
expertise you need is always on hand.
We provide a premier selection of proven

analytical tools and consumables that meet
the requirements of scientists who primarily
use HPLC and LC-MS for separation and
analysis of drugs and biomolecules, or for
other analytical assays. Our selection of
columns, solvents, standards and sample
preparation products are exclusively
designed for HPLC and LC-MS, meeting the
critical need for purity.
3
Our HPLC and LC-MS Workflow
Sample Collection Sample Preparation Standardization & Chromatographic

Callbration Separation & Analysis
• Full Labware Portfolio • SPE Cartridges and • Certified • U/HPLC-MS Columns
• Pipettes, Pipettors 96-well Plates Reference Materials • U/HPLC-MS Solvents
and Tips • SPME (BioSPME) 96-well • Analytical Standards • LC-MS Reagents
• Multiwell Plates Tips and Needle Probes
• TLC-MS
• Microcentrifuge Tubes • QuEChERS
• Reference Materials
(dispersive SPE)
• Vials • Accessories
• Dispersive Pipette
Extraction (DPX) Tips
• SLE Cartridges
• Online SPE Cartridges
• Millex® Filters
• Samplicity®
Filtration System
4
Supelco® HPLC and UHPLC Columns
Our HPLC & UHPLC column portfolio meets today’s challenging needs of Fast
HPLC including UHPLC, LC-MS, biopolymer separation, high pH conditions, as
well as traditional pharmacopeia and agency methods within pharmaceutical,
environmental, clinical, and food industries etc. – from nano LC to
semi-preparative applications
Fused-Core® technology, monolithic silica, ultra-pure backpressure and provide a very high matrix-tolerance
and monodisperse silica, and polymeric particles making this material perfectly suitable for the
are some of the particle platforms that make up the separation of matrix-rich samples.
Supelco® HPLC product line so you can find the right
Our extensive range of chiral HPLC & LC-MS columns,
column for your specific application.
derivatization reagents and mobile phase additives
We have a tradition of providing innovative make us one of the leaders in chiral chromatography
HPLC Columns; while our trusted SUPELCOSIL™, technology. Our comprehensive range of well-respected
LiChrospher®, Discovery®, Ascentis®, SeQuant® and brands includes Astec® CHIROBIOTIC®, CYCLOBOND™,
Purospher™ STAR columns have a proven track record, ChiraDex®, and CLC columns.
our Purospher™ STAR and Titan™ columns deliver
We can help you find a solution for your chiral
leading edge UHPLC performance at an affordable cost,
separation application, including clinical, food,
and Ascentis® Express and BIOshell™ (based on Fused-
environmental, or drug discovery. Depending on your
Core® technology) have the capability to turn any
chromatography needs, our chiral columns can be used
HPLC system into a Fast HPLC workhorse. Chromolith®
with several of the most common mobile phase modes,
HPLC and UHPLC columns, based on monolithic silica,
and many are suitable for LC-MS.
enable rapid separations at extremely low column
NEW
Supel™ Carbon HPLC columns
enable the use of extreme pH and
temperature without a compromise
in efficiency. This column is an
excellent choice for the retention and
separation of polar compounds using
reversed phase conditions
Supel™ Carbon HPLC Columns
5
Stationary Phase Carrier
Stationary phase base material Methods / Use
Silica based stationary phases
Type B Superficially porous silica particles First choice for new methods Silica SPP
(SPP) / Fused-Core® under development or
Si(OC2H5)4 during method transfer in
SPP - Superficially porous silica HPLC
and UHPLC columns provide very high Pharma.
[-Si(OC2H5)2 - O - efficiencies, which are typically 40% higher Excellent for UHPLC
Si(OC2H5)2 - O -]n in comparison to fully Porous Particles of applications (2 µm particles)
the same particle size.
-Si - O - Si - O -
O O Monolithic silica Best choice for matrix-rich Monolithic
-Si - O - Si - O - samples and applications silica
n
Monolithic silica HPLC, UHPLC and
semi-preparative columns enable rapid were lifetime and robustness
SiO2 separations at very low column back- is a concern. As well for
pressure with very high matrix-tolerance rapid separations at low
• NO Metal content and extended column lifetime. column backpressure.
• Provides optimal peak
shape for basic and Fully porous silica particles (FPP) For established HPLC Silica FPP
chelating compounds Type B (high purity silica) methods, for special
Fully porous silica particles provide the full selectivities such as HILIC
loadability of the stationary phase due to and Chiral as well as
its fully porous physical characteristics. columns for biomolecule
This ensures high sensitivities because separation
the peak broadening effect of overloading They are in use in thousands
the stationary phase is minimized. Type of methods and ensure
B silica particles are produced from reliable results over the
tetraalkoxysilane in a sol-gel process. This complete range of use,
metal free stationary phase base material particle sizes and column Silica FPP
can be used for the analysis of acidic, dimensions in
basic, and chelating compounds providing Nano-LC (Capillary columns)
excellent peak symmetries with less need Silica FPP
for strong buffer concentrations. UHPLC
Analytical HPLC
Semi-preparative LC
Preparative LC
Type A Fully porous silica particles (FPP) Silica FPP

M2O - nSiO2 M=Na, Type A (conventional silica)
K, Fe Spherical
[-Si(OH)2 - O - Traditional silica made from sodium
Si(OH)2 - O -]n waterglass established in many applications
and methods.
-Si - O - Si - O -
O O
-Si - O - Si - O - n
SiO2 (Na, K, Fe)

Irregular Silica FPP
Reliable stationary phase providing same irregular
• Contains metals
properties for HPLC and large scale
• Peak-Tailing for
preparative LC (bulk sorbent) as well as TLC
basic and chelating
compounds
Other stationary phase materials

Carbon particles Excellent choice for the Porous
Fully porous graphitic carbon (PGC) retention of separation of Graphitic
particles manufactured using a patented polar compounds using Carbon
synthetic process, enable extreme pH and reversed phase conditions.
temperature stability without a compromise
in efficiency.
Fully porous polymeric particles Polymeric

Enable the use of the full pH-rage for the FPP
mobile phase (0-14)
Zirconia and Alumina Particles

Outstanding thermal and pH stability.
6
Separation mode Solution for small molecule separation Page Solution for Biomolecule separation Page
Reversed Phase Ascentis® Express 28 BIOshell™ MS

38
HILIC HPLC Columns MS HPLC Column
UHPLC columns UHPLC columns
Capillary columns Capillary columns
Reversed Phase Chromolith® 50 Chromolith® WP 300 54

MS HPLC columns MS
HILIC HPLC columns
Affinity Capillary columns
Semi-preparative Columns
Reversed Phase Purospher™ STAR MS

62 Discovery® BIO 74
HILIC HPLC and UHPLC columns HPLC columns
Normal phase Discovery® HPLC columns MS 70 Capillary columns
MS 76
Ascentis® HPLC columns
Titan™ UHPLC columns MS 80
SeQuant® HILIC HPLC columns and 82

MS
capillary columns
Chiral Astec® Chiral HPLC and UHPLC columns
Size Exclusion Sepax 102

Ion Exchange U/HPLC columns
HIC TSKgel® 107
U/HPLC columns
Reversed Phase LiChrospher® 86

Normal Phase HPLC columns
HILIC Semi-preparative Columns
Ion Exchange Superspher® 88
HPLC columns
SUPELCOSIL™ 90
HPLC columns
Reversed Phase LiChrosorb® 89
Normal Phase HPLC columns
Bulk sorbent
Reversed Phase Supel™ Carbon 114 Supel™ Carbon MS

MS
HILIC apHera™ HPLC columns For SeQuant® 118 Cytiva FPLC and HPLC columns 107
Reversed Phase SUPELCOGEL™ HPLC columns ZIC®-pHILIC TSKgel® HPLC and UHPLC columns
Size Exclusion Hamilton® HPLC columns
MS
Ion Exchange SeQuant® ZIC®-pHILIC 82
HIC
Discovery® Zr HPLC Columns 119
Aluspher® HPLC columns
Note: As chromatographic separation depends on many physical and

chemical parameters, we cannot guarantee the success of a separation 1st choice for method development MS Recommended for LC-MS use
based on the recommended column modification.
7
Selection of stationary phase carrier
by their benefits
The performance of HPLC columns has improved dramatically in recent years, particularly in terms of separation
power as measured by the number of theoretical plates per meter. The improvement in performance has been
achieved primarily by a reduction in particle size.
Superficially Porous Particles (SPP) show higher separation efficiencies than Fully Porous Particles (FPP) with the
same particle size. To achieve this very high efficiency it is essential to avoid any column overloading that could
cause peak broadening effects.
HPLC columns filled with small and very small particles can be plugged or blocked more easily. Matrix-rich samples
require extensive sample preparation when analyzed with HPLC and UHPLC columns using small particles. This
added sample preparation step is a substantial time and cost factor. When analyzing challenging, matrix-rich
samples, the benefit of monolithic silica columns is significant.
Loadability
10 µm
FPP +++
SPP +
Monolith ++
Matrix tolerance
5 µm 5 µm
3 µm
2.7 µm
2 µm
<2 µm
100 000 N/m 150 000 N/m 200 000 N/m 300 000 N/m
Separation efficiency
Need for Speed

Plates per pressure (N/bar)
SPP (Core-shell) It goes without question that the development
a 2.7 µm of faster separation processes is one of the most
SPP (Core-shell) important issues in HPLC. Particularly in industry,
b 2.6 µm
chromatographers wish to speed up separations, and
Porous particles 3 µm analyze more samples with the limited financial and
human resources available. One of the main issues
Porous particles 5 µm preventing speed is congestion. With conventional
particle-packed HPLC columns, higher efficiency always
SPP (Core-shell) 5 µm comes at the expense of higher back pressure. Even
Chromolith® core-shell particle (equal to SPP) columns, which are
HighResolution designed for lower resistance, still exhibit rather high
Chromolith® back pressure. Hence, the task is to minimize back
Performance pressure in order to maximize speed.
0 100 200 300 400 500 600 700
All columns are C18 modified, 100-4.6 mm. Sample: anthracene, eluted
isocratically using acetonitrile/water (60/40) at 2 mL/min flow rate.
Injection volume: 5 μL, detection at 254 nm UV. All analyses performed
at room temperature.
8 HPLC and UHPLC Column Selection Guide

Selection of stationary phase bonding
by compound class
Selecting the most suitable column is highly dependent upon the sample undergoing analysis. Compound structure,
solubility, and log P values of analytes all need to be taken into consideration when selecting column phase
chemistry and mode of separation. While compounds can often be separated using various column chemistries,
some column selectivities are better suited than others for certain compound classes. The table below shows a
selection of classes of compounds typically analyzed by HPLC methods.
Compound ZIC®- ZIC®- ZIC®- RP- Propyl Phenyl RP-

Class HILIC cHILIC pHILIC NH2 Si OH5 DIOL CN F5 Amide Phenyl Hexyl Biphenyl RP-4 RP-8 8e RP-18 RP-18e PAH C30
Aflatoxins 2 2 2 1 1
Alcohols 1 1 2 1 2 2 2 2 2
Aldehydes 2 2 2 2 1 1
Alkaloids 2 2 1 2 2 2 2 1 1
Aliphatic amines 1 2 1 1 2 2
Amino Acids 1 1 1 1 2 1 2 2
Antibiotics 2 2 1 2 2 1 2 2 2 1 1
Aromatic amines 1 2 1 1 1 1 2 2
Carboxylic acids 2 1 2 1 2 1 1
Carotenoids 1 1 2 2 2 2
Catecholamines 2 1 1
Explosives 2 1 2
Oils 1 2 2 2 2
Oligonucleotides 1 1
Esters 2 2 2 2 1 1 1 1
Fat soluble
1 1 2 2 2 1 1
vitamins
Lipids 2 2 1 1 1
Fatty acids 1 2 2 1 1
Flavonoids 2 2 1 1 1 1 1 1
Glycans 1 1 1 2 2 1 1
Glycols 1 1 2 1 2 2
Inorganic ions 1 1 1 2
Ketones 2 2 2 2 2 1 1
Nitrosamines 1 1 1 2 2 1 1
Nucleosides 2 2 1 1
Nucleotides 1 1 1 1 2 2 2 2 2
PAH 1 1 1 1 1
PCB 2 2 2 2 1 1
Peptides 1 1 2 1 1 1 2 2 2 1 1
Pesticides 2 2 2 1 1
Phenols 2 1 1 1 2 2 1 1
Phospholipids 1 1 2 2 2 2 2
Phthalates 1 1 1
Preservatives 1 1 2 1 1
Proteins 2 1 1 1 2 2
Organic
1 1 2 2
phosphates
Steroids 1 2 1 2 2 2 2 2 1 1
Metabolized
2 2 2 2 2 2 2 1 1
steroids
Sugars 2 2 1 1 2 1 1
Sugar Alcohols 1 1 2 2 1
Sulfonamides 2 2 2 2 1 1
Sweeteners 1 1 1 1 2 2 2 1 1
Water soluble
1 1 1 1 1 1 1
vitamins
1 Most suitable column 2 Possible alternative column
9
Selection by Chemical Structure of the Analyte
using the log P Value
The selection of the most appropriate stationary phase depends on the chemical structure of the compound to be
separated. One important parameter that describes the chemical structure of a compound is the log P value (water
octanol logorithmic partition coefficient). This table shows the log P value of representative compounds of important
analyte groups.
Analyte group Example Structure log P value
A Aflatoxins Aflatoxin G1 1.8
Alcohols Ethyl alcohol -0.1
Aldehydes Benzaldehyde 1.5
Alkaloids Quinine 2.9
Amino Acids Aspartic acid 2
Antibiotics Amoxicillin -2
Ranitidine 0.3
Aromatic amines Aniline 0.9
C Carboxylic acids Glucuronic acid -2.3
Carotenoids Canthaxanthin 11.4
D Dyes Rhodamine 4.4
E Enatiomers Thalidomide 0.3
Essential oils Safrole 3
Esters Atropine 1.8
F Fat soluble vitamins Retinol 5.7
Fatty acids Stearidonic acid 5.9
Flavonoids Quercetin 1.5

Analyte group Example Structure log P value
G Glycols Ethylene glycol -1.4
I Inorganic ions Chloride Cl- 0.8

K Ketones Cyclohexanone 0.8
N Nitrosamines N-Nitrosodimethylamine -0.6
P PAH Anthracene 4.4
PCB Pentachlorobiphenyl Cl Cl
7.3
Cl
Cl Cl
Peptides Neurokinin B -1.6
Pesticides Glyphosate -4.6
Phenols Bisphenol A 2.2
Phospholipids Phosphatidylserine -3.5
S Steroids Progesterone 3.9
Sugars Lactose -4.7
Sugar Alcohols Maltitol -5.2
Sulfonamides Furosemide 2
Sweeteners Aspartame -2.7
W Water soluble vitamins Folic Acid -1.1
11
Selection of Chromatographic Mode and
Stationary Phase by log P Value
C18 is usually the first choice for starting a new method. However, when a C18 doesn’t give the desired
separation, or your sample contains compounds that are known to be difficult to retain or resolve on a C18, then
you should consider changing the stationary phase, modification or both.
If a compound is predominantly hydrophobic with a positive log P value (water octanol logarithmic partition
coefficient), then the use of a reversed phase column is recommended. For low/medium polarity analytes, normal
phase HPLC or HILIC are viable techniques.
Hydrophobic Hydrophilic
Too much Poor Peak

Aromatic Retention too short or
Isomers retention Shape (Basic
compounds inadequate Separation on RP-18
on C18 Compounds)
Closely Polar
related Pi-Pi Interactions compounds with HILIC
high aqueous
compounds mobile phases
RP- F5 Phenyl ES- Si

C30 C18 C8 Biphenyl AQ C18 OH5
Amide (PFP) Hexyl Cyano HILIC
C18 C8 Diol Cyano Amino Si
RP- F5 Phenyl
C18 C8 Diol Cyano Amino Si ZIC
Amide (PFP) Hexyl
(SPP) Ascentis® Express (monolithic) Chromolith® (FPP) Purospher™ STAR, Discovery®, Ascentis®, SeQuant®, Titan™
Choose the right HPLC column

[RS]
Chromatographic resolution is most influenced by the 3

selectivity (α) (when k>2 and N> 3000). Changing the
mobile phase composition or the stationary phase, is the
most powerful way of optimizing selectivity whereas the 2.5
particle size, pore size, length of the column, temperature,
mobile phase strength have much less effect. Therefore,
if satisfactory results are not met, or no retention is 2
achieved, it is better to change to another selectivity using
a different column type and/or a different mobile phase.
1.5
Resolution is mainly controlled by selectivity
Resolution (Rs or R) is impacted by three parameters 1
(k, α, and N) which are directly related to experimental
conditions. k is the average retention factor for the two
bands, N is the number of theoretical plates and α is the
0.5
separation factor (or selectivity factor).
The parameters k and α are determined by the
experimental conditions (composition of the mobile phase; 0
stationary phase chemistry and temperature), and N is 1 1.05 1.1 1.15 1.2 1.25
affected by column length, particle size and pore size.
0 5000 10000 15000 20000 25000
0 5 10 15 20 25
Selection by Specific
Chromatographic Need (C18)
Prioritizing specific needs helps to select the best suitable column material for different chromatographic demands.
Depending on the sample, analyte or matrix, the lab environment (e.g. instrumentation) and the separation
goal, the best column choice can be very different from task to task. While the selection of the column chemistry
is the first step in this process, the next step is to determine the most suitable column material. Many different
selectivities are available based on different column materials. Nevertheless, C18 is still the most widely used
column modification. The table below lists a selection of specific needs in HPLC on column materials with
C18 modifications. The ranking is from 1 (lowest ranking) to 5 (highest ranking).
Fused® Core Silica (SPP) Monolithic Silica Fully porous silica particles (FPP) Type B FPP Type A
Ascentis® Ascentis® Purospher™
Need Chromolith® Discovery® Ascentis® Titan™ Superspher® LiChrospher® SUPELCOSIL™
Express Express STAR
C18 AQ C18 RP-18e HR RP-18e RP-18e C18 HS-C18 C18 C18 RP-18 (e) RP-18 (e) LC-18 LC-18-DB
Particle size (µm) 2 2.7 5 2 2.7 5 2 3 5 5 3 5 10 3 5 10 1.9 4 5 10 3 5 12 3 5
Separation efficiency 5 5 4 5 5 4 3 4 5 4 3 2 3 2 1 4 3 2 5 3 2 1 3 2 1 3 2
Peak symmetry 5 5 5 5 5 5 3 4 5 5 5 3 3 3 3 4 4 4 5 3 3 3 3 3 3 3 3
Need for sample preparation 1 2 3 1 2 3 5 4 1 2 3 3 2 3 4 2 3 4 1 2 3 4 1 2 3 1 2
Lifetime (for Matrix-rich samples) 1 2 3 1 2 3 5 4 1 2 3 3 2 3 4 2 3 4 1 2 3 4 1 2 3 1 2
Lifetime (based on particle mechanical stability) 3 3 3 3 3 3 5 4 4 4 2 5 5 5 5 4 4 4 4 5 5 5 5 5 5 5 5
100% Aqueous mobile phase compatibility 1 1 1 5 5 5 1 1 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
pH stability (range) 3 3 3 3 3 3 1 2 4 4 4 2 2 2 2 2 2 2 2 1 1 1 2 2 2 2 2
Bleeding (for MS) 5 5 5 5 5 5 5 5 4 4 4 3 3 3 3 5 5 5 5 2 2 2 3 3 3 3 3
Reproducibility (Column-to-Column) 4 4 4 4 4 4 3 4 4 4 5 4 4 4 4 4 4 4 3 3 5 3 3 3 3 3 3
Column Back Pressure 1 2 3 1 2 3 5 4 1 2 3 3 2 3 4 2 3 4 1 2 3 4 2 3 4 2 3
Useable flow rate ranges 5 4 2 5 4 2 5 5 5 3 2 2 3 2 1 3 2 1 5 2 2 1 3 2 1 3 2
Loadability 3 3 3 3 3 3 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
Quantitation - linear response range 4 4 4 4 4 4 4 5 5 5 5 3 4 3 3 4 3 3 5 3 3 3 3 2 2 3 2
Sample throughput 5 5 4 5 5 4 5 5 5 4 3 3 4 3 2 4 3 2 5 3 3 2 4 3 2 4 3
UHPLC use (Stability at high pressure) 5 4 4 5 4 4 1 1 5 5 2 2 2 2 2 2 2 2 5 2 2 2 2 2 2 2 2
Temperature stability 4 4 4 4 4 4 3 3 5 5 5 5 5 5 5 5 5 5 4 4 4 4 5 5 5 5 5
Up-Scalability (above 4.6 mm id) 1 1 1 1 1 1 5 1 1 1 4 1 1 3 4 1 4 5 1 1 5 5 1 4 5 1 4
Down-Scalability (to below 1 mm id) 1 5 1 1 5 1 5 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Criteria - Ranking:
Separation efficiency (N/m) Temperature (Max °Celcius) pH stability (range) UHPLC (Stability at high pressure)
1 = < 50,000 1 = 30 1 = 2-7.5 1 = 200 bar
2 = 50,000 - 75,000 2 = 40 2 = 2-8 2 = 400 bar
3 = 75,001 - 100,000 3 = 50 3 = 2-9 3 = 500 bar
4 = 100,001 - 150,000 4 = 60 4 = 1.5-10.5 4 = 600 bar
5 = > 150,000 5 = 70 5 = 1.5-12 5 = 1000 bar
Validation kits
The success of an HPLC method depends strongly on the
consistent quality of the stationary phase. Long-term
reproducibility is a key factor in achieving reliable results.
Supelco® validation kits consists of three HPLC columns,
packed with three different sorbent lots to confirm the
reliability of HPLC methods and their robustness.
13
Selection by USP Classification
HPLC Packings for USP Compendial Methods

The following list describes the main USP
classes and the corresponding Supelco®
stationary phases. The official pharmaceutical
analysis monographs in the United States
Pharmacopeia (USP) detail the methods used
by pharmaceutical manufacturers for quality
control of bulk drug substances and dosage
form preparations. Each method specifies a
particular high pressure liquid chromatography
(HPLC) or gas chromatography (GC) column
or column type and the conditions under
which the analysis is performed. This table
lists the USP Codes for the HPLC phases used
in these methods, descriptions of the columns,
and information about our products that
conform to these descriptions.
Available Columns (2) (3)

Particles Monolithic
Fused-Core® Silica Fully Porous Silica Non-Silica Silica Based
USP
Particles Particles Particles
Code
(1)
Description (1) Type B Silica (4) Type B Silica (4) Type A Silica (5) Type B Silica (4)
L1 Octadecyl silane chemically Ascentis®
Ascentis C18
®
LiChrosorb RP-18
®
Chromolith®
bonded to porous or non- Express C18 Discovery C18
®
LiChrospher ® CapRod®
porous silica or ceramic BIOshell™ RP-18e HighResolution
micro-particles, 1.5 to Discovery® HS C18 RP-18e
Peptide C18 LiChrospher® RP-18
10 µm in diameter, or a Discovery® BIO Chromolith®
monolithic rod. Ascentis® Wide Pore C18 LiChrospher® PAH
Express PAH CapRod® RP-18e
Purospher™ RP-18e SUPELCOSIL™ Chromolith®
Ascentis® LC-18
Express PFAS Purospher™ RP-18 HighResolution
Purospher™ SUPELCOSIL™ RP-18e
BIOshell™ IgG C18 LC-18-DB
STAR RP-18e Chromolith®
Ascentis® SUPELCOSIL™ Performance RP-18e
Express Titan™ C18
LC-318 Chromolith®
AQ C18
SUPELCOSIL™ Prep RP-18e
LC-18-S Chromolith®
SUPELCOSIL™ SemiPrep RP-18e
LC-18-T Chromolith® WP
Superspher® RP-18e 300 RP-18
Superspher® RP-18
L3 Porous silica particles, 1.5 to Ascentis® Ascentis® Si LiChrosorb® Si 60 Chromolith®
10 µm in diameter, or a Express Purospher™ LiChrospher® Si 60 Performance Si
monolithic silica rod. HILIC STAR Si Chromolith® Prep Si
SUPELCOSIL™ LC-Si
SUPELCOSIL™ Chromolith®
LC-3Si SemiPrep Si
Superspher® Si 60
1st choice for method development

Fused-Core Silica
®
Fully Porous Silica Non-Silica Silica Based
USP
Code
(1)
L7 Octylsilane chemically Ascentis® Ascentis® C8 LiChrosorb® RP-8 Chromolith®
bonded to totally or Express C8 Discovery® C8 LiChrospher® CapRod® RP-8e
superficially porous silica RP-8e 100 Chromolith®
particles, 1.5 to 10 µm in Discovery® BIO
Wide Pore C8 LiChrospher ® HighResolution
diameter, or a monolithic RP-8e
silica rod. Purospher™ RP-8 100
STAR RP-8e LiChrospher® Chromolith®
RP-Select B 60 Performance RP-8e
SUPELCOSIL™ LC-8 Chromolith®

WP 300 RP-8
SUPELCOSIL™
LC-8-DB
SUPELCOSIL™
LC-308
Superspher®
RP-8e 60
Superspher®
RP-8 60
Superspher®
RP-Select B 60
L8 An essentially Purospher™ LiChrospher® Chromolith® NH2
monomolecular layer of STAR NH2 NH2 100
aminopropylsilane SUPELCOSIL™
chemically bonded to totally LC-NH2
porous silica gel support, 1.5
to 10 µm in diameter, or a SUPELCOSIL™
monolithic silica rod. LC-NH2-NP
L9 Irregular or spherical, totally *TSKgel® SUPELCOSIL™

porous silica gel having a SP-2SW LC-SCX
chemically bonded, strongly
acidic cation-exchange
coating, 3 to 10 µm in
diameter.
L10 Nitrile groups chemically Ascentis® Ascentis® ES Cyano LiChrospher® CN Chromolith® CN
bonded to porous silica Express Discovery Cyano
®
SUPELCOSIL™
particles, 1.5 to 10 µm in ES-Cyano LC-CN
diameter, or a monolithic BIOshell™
silica rod. Peptide CN
L11 Phenyl groups chemically Ascentis® Ascentis® SUPELCOSIL™
bonded to porous silica Express Phenyl LC-DP
particles, 1.5 to 10 µm in Phenyl-Hexyl Purospher™ SUPELCOSIL™
diameter, or a monolithic BIOshell™® IgG STAR Phenyl LC-3DP
silica rod. Diphenyl
L13 Trimethylsilane chemically *TSKgel® SUPELCOSIL™ LC-1
bonded to porous silica TMS-250
particles, 3 to 10 µm in
diameter.
L14 Silica gel having a *TSKgel® SUPELCOSIL™
chemicallly bonded strongly QAE-2SW SAX1
basic quaternary ammonium
anion-exchange coating, 5
to 10 µm in diameter.
L17 Strong cation-exchange Proteomix®
resin consisting of sulfonated WCX-NP10
cross-linked styrene- SUPELCOGEL™
divinylbenzene copolymer in C-610H
the hydrogen form, 6 to
12 µm in diameter. SUPELCOGEL™ H
*Tosoh Bioscience columns are available in select countries.

For a list, please go to the page 107 of this brochure 1st choice for method development
15
Fused-Core Silica
®
USP
Code
(1)
L19 Strong cation-exchange Proteomix®
resin consisting of sulfonated SCX-NP5
cross-linked styrene- Proteomix®
divinylbenzene copolymer in SCX-NP10
the calcium form, 5 to SUPELCOGEL™
15 µm in diameter. Ca
L20 Dihydroxypropane groups TSKgel®* LiChrosorb® Diol
chemically bonded to porous QC-PAK GFC LiChrospher® Diol
silica or hybrid particles, TSKgel®* SuperSW
1.5 to 10 µm in diameter, or SUPELCOSIL™
a monolithic silica rod. TSKgel®* SW LC-Diol
TSKgel®* SWXL
L21 A rigid, spherical styrene- Hamilton®
divinylbenzene copolymer, 3 PRP-1
to 30 µm in diameter. Hamilton® PRP-3
TSKgel®* SuperH
TSKgel®* SuperHZ
TSKgel®* HHR
TSKgel®* HXL
L22 A cation-exchange resin Hamilton®
made of porous polystyrene PRP-X200
gel with sulfonic acid groups, Hamilton®
5 to 15 µm in diameter. PRP-X300
SUPELCOGEL™
C-610H
SUPELCOGEL™ H
L25 Packing having the capacity TSKgel®*
to separate compounds with G2500PWXL
a molecular weight range TSKgel®* G2500PW
from
100 – 5000 (as determined TSKgel®* G2000PW
by polyethylene oxide), TSKgel®* G1000PW
applied to neutral, anionic,
and cationic water-soluble
polymers.
L26 Butyl silane chemically BIOshell™ SUPELCOSIL™ Chromolith®
bonded to totally porous or Protein C4 LC-304 WP 300 RP-4
superficially porous silica BIOshell™
particles, IgG C4
1.5 to 10 µm in diameter.
L27 Porous silica particles, 30 to Pelliguard™ LC-Si
50 µm in diameter. Supelclean™ LC-Si
L29 Gamma alumina, reverse- Aluspher® RP-
phase, low carbon Select B 100
percentage by weight,
alumina-based
polybutadiene spherical
particles, 5 µm in diameter
with a pore volume of 80 Å
units.
L32 A chiral ligand-exchange Astec® CLC-D
resin packing-L-proline Astec® CLC-L
copper complex covalently
bonded to irregularly shaped
silica particles, 5 to 10 µm in
diameter.

1st choice for method development For a list, please go to the page 107 of this brochure

HPLC Packings for USP Compendial Methods (continued)
Fused-Core Silica
®
USP
Code
(1)
to separate dextrans by G2000SWXL
molecular size over a range TSKgel®*
of 4,000 to 500,000 Da. It is G4000SWXL
spherical, silica-based, and
processed to provide pH
stability.
L34 Strong cation-exchange SUPELCOGEL™
resin consisting of sulfonated Pb
cross-linked styrene-
divinylbenzene copolymer in
the lead form, 7 to 9 µm in
diameter.
to separate proteins by G3000PWXL
molecular size over a range TSKgel®* G3000PW
of
2,000 to 40,000 Da. It is a
polymethacrylate gel.
L38 A methacrylate-based size- TSKgel®*
exclusion packing for water- SuperAW
soluble samples. TSKgel®* PW
TSKgel®* PWXL
TSKgel®* PWXL-CL
L39 A hydrophilic TSKgel®*
polyhydroxymethacrylate gel SuperAW
of totally porous spherical TSKgel®* PW
resin.
TSKgel®* PWXL
TSKgel®* PWXL-CL
L40 Cellulose tris-3,5- Astec® Cellulose
dimethylphenylcarbamate DMP
coated porous silica
particles,
3 to 20 µm in diameter.
L41 Immobilized α1-acid CHIRALPAK®
glycoprotein on spherical AGP
silica particles, 5 µm in
diameter.
L43 Pentafluorophenyl groups Ascentis® Discovery® HS F5
chemically bonded to silica Express F5
particles by a propyl spacer,
1.5 to 10 µm in diameter.
L45 Beta cyclodextrin, R,S- Astec® ChiraDex®
hydroxypropyl ether CYCLOBOND™ I ChiraDex® HR
derivative, bonded to porous 2000 Series
silica particles,
3 to 10 µm in diameter.
L49 A reversed-phase packing Discovery®
made by coating a thin layer Zr-PBD
of polybutadiene on to
spherical
porous zirconia particles, 3

17

Fused-Core® Silica Fully Porous Silica Non-Silica Silica Based
USP
Code
(1)
L52 A strong cation exchange SUPELCOSIL™
resin made of porous silica LC-SCX
with sulfopropyl or sulfoethyl
groups, 1 to 10 µm in
diameter.
L59 Packing for the size- SRT® SEC Series
exclusion separations of TSKgel®*
proteins (separation by SuperSW
molecular weight) over the
TSKgel®* SW
range of 5 to 7,000 kDa. The
packing is spherical 1.5 to TSKgel®* SWXL
10 µm, silica or hybrid
packing with a hydrophilic
coating.
L60 Spherical, porous silica gel, Ascentis® Ascentis® RP-Amide SUPELCOSIL™
10 µm or less in diameter, Express Discovery® ABZ+Plus
the surface of which has RP-Amide RP-AmideC16 SUPELCOSIL™
been covalently modified LC-ABZ
with alkyl amide groups and
endcapped.
L62 C30 silane bonded phase on Ascentis®
a fully porous spherical Express C30
silica 3 to 15 µm in
diameter.
L63 Glycopeptide teicoplanin Astec®
linked through multiple CHIROBIOTIC® T
covalent bonds to a 100 Å Astec®
units spherical silica. CHIROBIOTIC®
T2
Astec®
CHIROBIOTIC®
TAG
L67 Porous vinyl alcohol apHera™ C18
copolymer with a C18 alkyl
group attached to the
hydroxyl group of the
polymer, 2 to 10 µm in
diameter.
L68 Spherical, porous silica, SUPLEX™
10 µm or less in diameter, pKb-100
the surface of which has
been covalently modified
with alkyl amide groups and
not endcapped.
L82 Polyamine chemically apHera™
bonded to cross-linked NH2
polyvinyl alcohol polymer, 4
L86 A 5 µm fused core particle Ascentis®
with a highly polar ligand Express OH5
possessing 5 hydroxyl BIOshell™
groups tethered Glycan
to the silica gel outer layer.

1st choice for method development For a list, please go to the page 107 of this brochure

HPLC Packings for USP Compendial Methods (continued)
Fused-Core Silica
®
USP
Code
(1)
L88 Glycopeptide vancomycin Astec®
linked through multiple CHIROBIOTIC® V
covalent bonds to 100 Astec®
Ånsgtroms spherical silica. CHIROBIOTIC®
V2
L95 Highly polar alkyl ligand Ascentis® Express
comprising five hydroxyl OH5
groups that are chemically
BIOshell™ Glycan
bonded to totally porous or
superficially porous silica or
a monolithic silica rod.
L114 Sulfobetaine graft- SeQuant®
polymerized to totally or ZIC®-HILIC
superficially porous silica,
1.5 to 10 µm in diameter, or
a monolithic rod. Packing
having densely bonded
zwitterionic groups with 1:1
charge balance.
L122 ZIC®-pHILIC ZIC®-pHILIC
Footnotes:
1
United States Pharmacopeia. Request from United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD USA 20852
(tel. 800-227-8772).
2
Indicates availability of material(s) matching the description. We are not necessarily the manufacturer of the material.
3
Purple text indicates our recommendation(s).
4
Type B silica is obtained from a synthetic source, and is virtually free of metal contant.
5
Type A silica is obtained from a natural source, so may contain varying degrees of metal content.

19
Selection by Column Dimension
Depending on the scale and/or efficiency of the separation required, the table below can help you to choose a
column by the most appropriate inner diameter (I.D.) and column length for your needs.
Which column length is best for my needs?

• If you want to maximize the speed of your application. . . . . 20 mm to 75 mm length
• If you want a balance of resolution and speed. . . . . . . . . . . 100 mm
• If you want the best resolution possible . . . . . . . . . . . . . . . 150 mm
• Also available in both analytical and semi-prep dimensions
Column dimension
[length × I.D. in mm] Application Reason
4×4 Guard-column Protection from mechanical contamination
5 × 2 / 3 / 4.6 Sample contaminated to low extent
10 × 4.6 / 10 / 25
25 × 4 Precolumn High capacity precolumn
30 × 2 / 2.1 / 3 / 4 Method development Short retention time
55 × 2 / 2.1 / 3 / 4 Rapid HPLC and UHPLC Rapid equilibration
75 × 4 (if pressure stable) Low solvent consumption (small I.D.)
Low pressure drop
100 × 2.1 High detection sensitivity (mass selectivity) Semi-micro column for low injection volumes and low peak
125 × 2 / 3 dispersion
150 × 2.1 / 3 Low solvent consumption
100 × 4.6 Standard column Adequate performance for most applications
125 × 4 / 4.6 (average performance 8000 – 10000 N/column)
150 × 4.6
250 × 2 / 2.1 / 3 High detection sensitivity Semi-micro column for low injection volumes and low peak
High performance separation dispersion
Low solvent consumption
For complex samples
250 × 4.6 High robustness/higher tolerance to injection volume For very complex samples
250 × 10 Semi-preparative For mg quantities of pure substance on lab scale
250 × 25 Preparative For g quantities of pure substance
Guidelines for typical flow rates and orientation values for the loading capacities of analytical
and semi-preparative columns
Column dimensions
[length x I.D. in mm] Typical flow rates Sample amount Sample volume
150 × 1 0.06 mL/min ≈ 0.05 mg 0.05 – 1 µL
250 × 2 0.25 mL/min ≈ 0.2 mg 0.2 – 5 µL
250 × 3 0.6 mL/min ≈ 1 mg 1 – 20 µL
250 × 4 1 mL/min ≈ 5 mg 5 – 80 µL
250 × 10 6 mL/min ≈ 30 mg 30 – 500 µL
250 × 25 39 mL/min ≈ 200 mg 200 – 3000 µL
Guidelines for Typical flow rates and values for the loading capacities of Nano and capillary columns
packed with particles
Capillary column
Inner dimension (mm) Typical flow rate (µL/min) Sample amount (g) Sample volume (µL)
1 20 - 200 10 – 10
-6 -10
1
0.5 5 - 50 10-7 – 10-11 0.25
0.3 2 - 20 10-8 – 10-12 0.128
0.2 1 - 10 10 – 10
-9 -13
0.057
0.1 0.25 – 2.5 10-10 – 10-14 0.014
0.075 0.05 – 0.5 <10-13 0.002

Increase sensitivity and save solvents with small I.D. HPLC Columns
The use of smaller inner diameter (I.D.) columns results in decreased solvent usage. Using small I.D. columns less
mobile phase is required to achieve the same linear velocity, analysis time can be reduced by increasing flow rate.
This allows significant cost and time savings.
In addition, the peak response is increased with small I.D. columns – the peak height increases as the column
diameter decreases. The peak response for 2 or 2.1 mm I.D. columns is 3 times higher in comparison to 4.6 mm
I.D. columns. This is beneficial when analyzing mass limited samples, typically used in LC-MS applications.
Loadability Dead Volume

Column I.D. Typical Particle Size Technique
>20 mm 10 – 25 µm Prep
10 mm 5 - 10 µm Semi-prep
3 / 4 / 4.6 mm 3 - 5 µm Conventional HPLC
2 / 2.1 mm ≤ 2 - 3 µm UHPLC
1.0 mm ≤ 2 - 5 µm Micro LC
200 µm ≤ 2 - 5 µm Capillary LC
<100 µm ≤ 2 - 5 µm Nano LC
Sensitivity Solvent Saving
Column Length 250 mm 100 - 150 mm 30 - 50 mm 4 – 25 mm

Use Very complex adequate rapid separations Guard columns
samples performance for most
applications
Resolution
Speed
Increase sensitivity and save solvents with 2 mm I.D. Chromolith® RP-18 endcapped columns
Chromolith® Performance RP-18e 100-4.6 mm Chromolith® Performance RP-18e 100-2 mm
Column: Chromolith® Performance RP-18 endcapped Column: Chromolith® Performance RP-18 endcapped
100-4.6 mm 100-2 mm
Mobile phase: A: 100 % Acetonitrile Mobile phase: A: 100 % Acetonitrile
B: 100 % Water + 0.1 % TFA (v/v) B: 100 % Water + 0.05 % TFA (v/v)
C: 100 % Methanol C: 100 % Methanol
Isocratic: A/B/C 30/60/10 (v/v/v) Isocratic: A/B/C 30/60/10 (v/v/v)
Flow rate: 2 mL/min Flow rate: 0.38 mL/min
Pressure: 45 bar (4.5 MPa, 65.3 psi) Pressure: 48 bar (4.8 MPa, 70 psi)
Detection: Dionex Ultimate 3000 VWD-3400, 2.5 Hz, Detection: Dionex Ultimate 3000 VWD-3400, 2.5 Hz,
Response time 0.1 s, UV = 210 nm Response time 0.1 s, UV = 210 nm
Vol. detector cell: 11 µL Vol. detector cell: 1.4 µL
Temperature: ambient Temperature: ambient
Injection volume: 1 µL Injection volume: 1 µL
Sample: Bimatoprost Sample: Bimatoprost
Bimatoprost free acid Bimatoprost free acid
150 150
120 120
[mAU]
[mAU]
90 90
60 60
30 30
0 0
0 1 2 3 4 5 0 1 2 3 4
Retention time (min) Retention time (min)
The same separation on a Chromolith® 2 mm I.D. column demonstrates improved sensitivity and solvent savings of 81 %.
21
HPLC Column Hardware
Supelco® HPLC Columns perfectly fit to every HPLC and U/HPLC instrument. All Supelco® columns have a
Parker style endfitting. A 1/16 inch outer diameter capillary connection of stainless steel or PEEK is typically
used to connect the HPLC column to the HPLC system. 0.5 mm outer dimension flexible stainless steel capillary
connections are suitable as well using a 1/16 inch connection part.
Trademark Pressure
Hardware Trademark Sorbent Column Use Precolumn Material stability
Purospher™ STAR
LiChrospher® Requires
HPLC direct integration of precolumn—
LiChoCART® Superspher® manu-CART® Stainless Steel 250 bar
Cartridge no separate holder needed
LiChrosorb ® to use
Aluspher®
Purospher™ STAR
LiChrospher®
Hibar® RT HPLC Column ready to use column separate precolumn holder required Stainless Steel 400 bar
Superspher®
LiChrosorb ®
Hibar® HR Purospher™ STAR UHPLC Column ready to use column No precolumns available Stainless Steel 1000 bar
SeQuant® U/HPLC Column ready to use column separate precolumn holder required PEEK lined Stainless Steel 550 bar*
Chromolith® U/HPLC Column ready to use column separate precolumn holder required PEEK 200 bar
Discovery®
Ascentis®
SUPELCOSIL™ HPLC Column ready to use column separate precolumn holder required Stainless Steel 400 bar
[LiChrospher®]
[LiChorsorb®]
Ascentis® Express
HPLC Column ready to use column separate precolumn holder required Stainless Steel 600 bar
BIOshell™
Ascentis® Express (2 µm)
BIOshell™ (2 µm) UHPLC Column ready to use column separate precolumn holder required Stainless Steel 1000 bar
Titan™
*SeQuant® ZIC®-pHILIC: 200 bar
Supelco® HPLC, UHPLC and Capillary

columns are available in different column
hardware designs:
Ascentis® Express and BIOshell™ as well as
SUPELCOSIL™, Discovery®, Ascentis®, Titan™ and
Astec® columns are designd for direct connection to any
HPLC system. Supelguard™ pre-columns protect the
analytical HPLC column.
SeQuant® PEEK lined stainless steel ready to

use U/HPLC and capillary columns
SeQuant® columns are PEEK lined stainless steel
columns making them best suitable for HILIC
separations including phosphorylated compounds and
meeting the need for bioinertness

Chromolith® PEEK columns LiChroCART® HPLC cartridge
Chromolith® monolithic silica HPLC columns are With LiChroCART® cartridges, the user works with
cladded in inert PEEK re-usable manu-CART®
(polyetheretherketone) endittings which fit different
polymeric material and can cartridge lengths and inner
be connected directly to dimensions. Since these
HPLC or UHPLC system as a cartridge holders may remain in the system, and the
“ready to use” column. capillary connections do not need to be detached,
the cartridges may be changed within the shortest
Hibar® HR ready to use UHPLC columns possible time.
Purospher™ STAR Hibar® HR UHPLC

manu-CART® holder for LiChroCART®
columns are fully compatible with
any UHPLC instrument and provide a
HPLC cartridges
pressure stability of 1000 bar. The “one-turn” cartridge system for simple, rapid hand
tight fitting of cartridges
Hibar® RT ready to use HPLC columns and pre-columns. manu-
CART® cartridge holder
Hibar® RT HPLC columns are made of stainless steel for the LiChroCART®
providing a pressure stability of 400 bar. For the cartridge system are
use of a pre-column, a separate pre-column holder reusable and it allows for
is required. every cartridge length with different internal diameter
a simple turn permits an easy and problem-free
integration of a guard cartridge. For coupling of two
LiChroCART® cartridges, a coupling unit can be used
and for connecting a LiChroCART® HPLC cartridge with
a LiChroCART® 25-4 pre-cartridge the coupling kit. The
manu-CART® cartridge holder is for 2, 3, 4 and 4.6 mm
I.D. LiChroCART® cartridges and 75, 100, 125, 150 and
250 mm length.
manu-CART® NT cartridge
holder [1.51486.0001]
for LiChroCART® cartridge
of 75, 100, 125, 150 and
250 mm length and 2, 3, 4
and 4.6 mm I.D.
LiChroCART® cartridge
Use of LiChroCART® 4-4

or 10-2 guard cartridges
with manu-CART® NT
23
Column Accessories and Pre-column Holder
Supelco® HPLC Columns perfectly fit to every HPLC and U/HPLC instrument. All Supelco® columns
have a Parker style endfitting. A 1/16 inch outer diameter capillary connection of stainless steel or
PEEK is typically used to connect the HPLC column to the HPLC system. 0.5 mm outer dimension
flexible stainless steel capillary connections are suitable as well using a 1/16 inch connection part.
For protection of the analytical HPLC column from contamination it is recommended to use guard- or
pre-columns. These short columns are placed typically into a guard column holder. For the different
column hardwares described on the previous pages, corresponding pre-column holders are available.
Supelguard™
59660-U Stand-Alone, for use with Supelguard™ pkg of 1 ea For 2.1, 3.0, 4.0 and 4.6 mm I.D.
cartridges (2 cm L x 2.1 to 4.6 mm I.D.) fittings included Supelco® columns
21150AST Stand-Alone (Swivel-type), pkg of 1 ea Holder fits 2cm x 2, 3, and 4 mm I.D.
for use with Supelguard™ cartridges Astec® CYCLOBOND™, CHIROBIOTIC®,
(2 cm L x 2 to 4.6 mm I.D.) Cellulose DMP, guard cartridges. Cartridges,
tubing, nuts and ferrules are not included 21150AST
55205 Direct-Connect (Swivel-type), pkg of 1 ea Connects guard column directly to a

for use with Supelguard™ cartridges 3.0, 4.0 and 4.6 mm I.D. Supelco®
(2 cm L x 3 to 4.6 mm I.D.) analytical column 504254
504254 Direct-Connect (Swivel-type), pkg of 1 ea Directly connects a 2 cm Supelguard™

for use with Supelguard™ cartridges cartridge to a Supelco® analytical cartridge
(2 cm L x 3 to 4.0 mm I.D.) after the column endfitting is removed
504262 Direct-Connect (Swivel-type), pkg of 1 ea For direct connection to 2.1 mm I.D.

for use with Supelguard™ cartridges Supelco® columns 581392-U
(2 cm L x 2.1 mm I.D.)
581392-U Stand-Alone, for use with Supelguard™ pkg of 1 ea For use with Supelguard™ catridges
cartridges (1 cmL x 21.2 mm I.D.) (1 cm L x 21.2 mm I.D.)
For coupling the

precolumn holder
remove endfitting
Hibar® RT column
2, 3, 4, and 4.6 mm I.D.
Ordering information
Guard column holder for Hibar® RT columns

Contents of one package
Product Ordering No. [see figure on next 2 pages]
Precolumn holder for 4-4 LiChroCART® cartridges 1.16217.0001 1 piece
for capillary connection to Hibar® RT column
Precolumn holder for 4-4 LiChroCART® cartridges 1.51487.0001 1 piece

for direct coupling to Hibar® RT column

manu-CART® cartridge holder, manu-CART® endfittings for stainless steel cartridges LiChroCART®
Product Ordering No. Contents of one package
manu-CART NT cartridge holder for 2, 3, 4 and 4.6 mm I.D.
®
1.51486.0001 2 complete stainless steel units for mounting one
LiChroCART® cartridges LiChroCART® cartridge
manu-CART® “10” II cartridge holder for 10 mm I.D. 1.51419.0001 2 complete stainless steel units for mounting one
LiChroCART® cartridges LiChroCART® cartridge
manu-CART® coupling kit for coupling with LiChroCART® 25-4 1.50082.0001 1 coupling unit 1 endfitting for LiChroCART® 25-4
pre-cartridge
manu-CART® coupling unit to connect two LiChroCART® 1.50083.0001 1 piece

cartridges
manu-CART® holder 25-4 and 25-2 1.50017.0001 1 piece
manu-CART® holder 30 mm for 30-2, 30-3 and 30-4 1.50227.0001 1 piece

LiChroCART® cartridges
manu-CART® holder 55 mm for 55-2, 55-3 and 55-4 1.50226.0001 1 piece

LiChroCART® cartridges
Pressure cone for manu-CART® endfitting 1.51258.0001 2 pieces
Split collets for manu-CART® endfitting 1.51257.0001 4 pieces
manu-CART® holder 30 mm [1.50227]

for LiChroCART® 30-4, 30-3 and 30-2
manu-CART® holder 55 mm [1.50226]

for LiChroCART® 55-4, 55-3 and 55-2
manu-CART® cartridge holder
Mounting with guard cartridge 4-4 or 10-10 Mounting without guard cartridge
1. Slide sleeve 1. Slide sleeve with external
over cartridge. thread over cartridge.
2. Fix split-collets in the 2. Using your finger, hold
groove in direction of the one split-collet at the
guard cartridge. Apply grove. Apply the second
guard cartridge with its split-collet, slide sleeve
cone in direction of the over it and fasten with
main cartridge, slide cap nut.
sleeve on top and fasten
with cap nut.
25
Fused-Core® (Superficially Porous Silica
Particles, SPP) HPLC and UHPLC columns
Maximum Resolution for Small and Large Molecule Separation
Ascentis® Express and BIOshell™ HPLC and UHPLC Superior for small molecule separation:
columns are based on Fused-Core® particle technology
enabling fast results with highest resolution. Ascentis® Express HPLC and UHPLC columns
Fused-Core® columns feature narrower particle Ascentis® Express HPLC and UHPLC columns provide
size distribution as well as a shorter diffusion path about 40% more efficiency in comparison to columns
compared to fully Porous Particles. The result with fully Porous Particles of the same size. This
is increased resolution, added sensitivity and performance enhancement is applicable to all HPLC
higher throughput. instruments (in addition to UHPLC systems).
Fused-Core® particles consist of a The very broad range of column chemistries makes
solid silica core and a porous silica it easy to select the best suitable column for any
shell allowing a shorter diffusion HPLC and UHPLC application – from Capillary column
path compared to conventional fully dimensions to analytical 4.6 mm I.D. dimensions.
Porous Particles.
Features of Fused-Core® particles over Fully

As well as for biomolecule separation:
Porous Particles:
BIOshell™ UHPLC and HPLC Columns
• Narrower particle size distribution
BIOshell™ UHPLC and HPLC columns deliver maximum
• More consistently packed bed
speed and efficiency for the separation of biomolecules
• Shorter diffusion path on both UHPLC and HPLC systems. The Fused-Core®
superficially porous silica particles (SPP) with pore sizes
from 90 Å up to 1000 Å allow superior separation of
glycans as well as very large proteins. In particular,
a pore size of 1000 Å shows very clear advantages
over common 300 Å pores for the separation of very
large proteins in biotherapeutic drug development
such as monoclonal antibodies (mAbs) or proteins with
molecular weights greater than 100 kDa.
Fused-Core® Particles (SPP)

Fused-Core® Fully Porous Particles
(Superficially Porous Particles, SPP) (FPP)
Size Distribution
The innovative manufacturing process

for Fused-Core® particles produces a
very narrow particle size distribution.
Particle
Narrow
This allows for the use of larger porosity

frits that clogg less, resulting in a more
rugged column. Traditional fully porous
particles provide a larger particle size
distribution, requiring smaller pore frits
that clogg more easy.
The “A” term in the van Deemter

More Consistent
equation accounts for the effects of

heterogeneities in the packed bed of
an HPLC column. Narrow particle size
distributions form a more consistently
Bed
packed bed and more consistent path

lengths, minimizing analyte dispersion
(peak broadening) through the column.
This eddy diffusion is effectively
independent of mobile phase velocity.
The short diffusion path of the

Fused-Core® particle yields sharper
peaks than on traditional fully porous
Diffusion
particle columns. The minimized

Shorter
Path
resistance to mass transfer, the “C”

term in the van Deemter equation,
of the Fused-Core® particle provides
sharper peaks than traditional fully
porous particles. The short diffusion
path also permits the use of higher
flow rates without significant peak
broadening / loss in efficiency.
The factors that affect chromatographic efficiency are eddy diffusion, longitudinal diffusion, and resistance to mass
transfer, the A, B and C terms respectively from the van Deemter equation.
35.00
dp=10 µm
30.00
25.00
dp=5 µm
HETP (µm)
20.00 B
_ + Cu
H = A +u H Height equivalent to theoretical plate
(column length/efficiency)
15.00 dp=3 µm A Eddy diffusion
B Longitudinal diffusion
10.00 C Resistance to Mass Transfer
dp=1.7 µm
u Mobile phase linear velocity
5.00
d p=2.7 µm Fused-Core®
0
0 1 2 3 4 5 u
Mobile Phase Velocity (mm/sec)
27
Ascentis® Express HPLC and UHPLC columns
Maximum Resolution on any System
Based on Fused-Core® particle technology, Ascentis® Twice the Speed at Equivalent Pressure vs.
Express columns provide an exceptional advancement sub-2 µm Fully Porous Particles
in HPLC column performance and the benefits of high
sample throughput at maximum resolution. Compared to fully porous sub-2 µm particles typically
used in UHPLC, Ascentis® Express Fused-Core® 2.7 µm
Feature and benefits: particles generate approximately half the backpressure
while providing the same high resolution. This trait
• Fused-Core® technology (Superficially Porous permits both longer columns, for more resolving
Particles; SPP) power, and faster flow rates, for higher throughput.
• Maximum speed and efficiency on both UHPLC and Demonstrating this point, the separation below shows a
HPLC systems (particle sizes: 2 µm, 2.7 µm and 5 µm) steroid mixture on Ascentis® Express (top) and a
sub-2 µm UHPLC column (bottom) of the same
• 40% more efficiency in comparison to Fully Porous
dimensions. Due to the lower backpressure of the
Particles (FPP) of same particle size
Ascentis® Express, an increased flow rate (double in
• UHPLC columns with 2 μm particles (pressure this case) can be applied, providing the same back
stable 1000 bar) pressure, separation efficiency and resolution as on
• Column dimensions from 0.075 mm I.D. (capillary a sub-2 μm UHPLC column, just with a 50% shorter
columns) to 4.6 mm I.D. (analytical HPLC columns) runtime, increasing sample throughput.
• Broadest range of phases/selectivities for optimal
method development
Chromatographic conditions:
Ascentis Express C18

® Columns: Ascentis® Express C18, 10 cm x 2.1 mm I.D.,
0.4 mL/min flow rate
2.7 µm particles (53823-U) and sub-2 µm
particle column (same dimensions)
2
1 Mobile phase: water/acetonitrile 49:51 (for Ascentis® Express);
4 water/acetonitrile 55:45 (for sub-2 µm)
3000 psi Column temp: ambient
Detector: UV, 200 nm

Injection: 1 μL
TWICE
5 THE SPEED
3 AT EQUAL
PRESSURES
1.0 2.0 3.0

Min
Sub-2 µm competitor
0.2 mL/min flow rate
1 3000 psi
2
4
1. Estrad io l
2. β -Estradiol
5
3 3. Imp urit y
4. Estro ne
5. Estro ne d eg rad ant
0 2 4 6
Min

Best fit for HPLC and UHPLC
Best Fused-Core® UHPLC column Fast HPLC on any System The Lab Work-horse Column
Fused-Core ® Fused-Core ®
Fused-Core ®
1.2 µm 1.2 µm 1.7 µm 1.7 µm 3.3 µm 3.3 µm

1.2 µm 1.7 µm 3.3 µm
0.4 µm 0.4 µm 0.5 µm 0.5 µm 0.6 µm 0.6 µm

0.4 µm 0.5 µm 0.6 µm
2.0 µm 2.0 µm 2.7 µm 2.7 µm 5 µm 5 µm
2.0 µm 2.7 µm 5 µm
An optimized solution for high throughput A practical solution that delivers UHPLC True plug and play solution for improving
small molecule analysis performance from any HPLC separations on existing 3 µm or 5 µm fully
porous particle HPLC columns
Pressure stability: 2 µm: 1000 bar 2.7 µm: 600 bar 5 µm: 600 bar
More separation power per unit pressure Higher Sample Throughput

Designed to deliver speed and resolution on all UHPLC Without Compromises
and HPLC systems, Ascentis® Express columns with The outstanding separating power of Ascentis® Express
Fused-Core® technology exceed the benefits of HPLC columns allows the use of shorter column
sub-2, 3 and 5 µm particles. Ascentis® Express 2.7 µm dimensions while maintaining good resolution. This
particles deliver more resolving power per unit pressure trait results in higher sample throughput and reduction
than even sub-2 µm particles on any HPLC system in costs.
(including UHPLC). Ascentis® Express 5 µm columns are
able to achieve greater speed and efficiency than any Ascentis® Express C18 column
other 5 µm particle-based column. This fact means that 10 cm × 4.6 mm I.D., 2.7 µm particles
Ascentis® Express 5 µm columns can be the standard
column for all fully porous 5 µm-based methods.
With the addition of 2.0 µm Ascentis® Express UHPLC
columns, we now offer three U/HPLC Fused-Core®
particle sizes, making the Ascentis® Express column 0 10 20 30
0 Min 10 20 30 40
Min
line truly scalable from HPLC to UHPLC. Fully porous particle C18
25 cm × 4.6 mm I.D., 5 µm particles
16,000
1.7 µm
14,000
0 10 20 30 40
12,000 0 10 Min 20 30
Min
Pressure (psi)
10,000
8,000 2.7 µm
Fused-Core®
6,000
3.0 µm
4,000
2,000
0
0 2 4 6 8 10 12
Mobile Phase Velocity (mm/sec)
Ascentis® Express HPLC and UHPLC columns
29
A Broad Range of Column Selectivities for all Compound Classes
Column selectivity has the highest influence on resolution in chromatography. Selection of the best column
chemistry for your target analytes is therefore an important selection parameter. C18 column chemistries are
typically the first choice. Nevertheless, when a C18 does not give the desired separation or the sample contains
compounds that are known to be difficult to retain or resolve on a C18, consider changing stationary phase
chemistry early in method development for more optimal applications. The range of selectivity provided by
Ascentis® Express makes this easy.
Hydrophobic Hydrophobic Closely Related Polar Very Polar Peptides/Proteins
Compounds Compounds Compounds Compounds
Too Much Retention too short or inadequate Poor Peak Shape

separation on RP-Amide (Basic Compounds) Not Enough RP Retention
Retention on C18
Basic Use with H-Bonding, π-Electron Nitroaromatics Strong

Phenolic, and HILIC Mode
Compounds 100% aqueous Acceptors Dipole
mobile phase Acidic, or Heteroaromatics
Hydroxylated
Compounds
Phenyl HILIC BIOshell

C30 C18 C8 F5 (PFP) AQ-C18 RP-Amide ES-Cyano Biphenyl F5 (PFP) RP-Amide OH5 ES-Cyano
Hexyl (Si) U/HPLC
USP
Ascentis® Express Phase Bonding Designation Bonding Chemistry Chromatographic Properties / Use
C18 L1 Dimethyloctadecyl Outstanding performance for a broad range of analytes. Excellent peak
shape for acids, bases and neutral compounds.
R - Si - R
AQ-C18 L1 Polar modified Octadecyl Resistant to dewetting; compatible with 100% aqueous mobile phase.
Suitable for the separation of polar compounds in RP-mode.
O O O
Polar
Si
Si
Peptide ES-C18 L1 Diisobutyloctadecyl Fast separation of peptides and polypeptides with high peak capacity. Ideal
for pharmaceutical/therapeutic peptide separation, peptide mapping, natural
R - Si - R
and synthetic peptide analysis and oligonucleotide analysis.
C30 L62 Triacontyldimethyl Excellent selectivity for hydrophobic, long chain and structurally
Si
related isomers.
C8 L7 Dimethyloctyl Enhanced retention for less hydrophobic compounds or faster separation if

retention on C18 is too high
Si
Phenyl-Hexyl L11 Dimethylphenyl-hexyl Enhanced selectivity for aromatic compounds; strong pi-pi donor. Ideal for
O Si
the separation of ketones, nitriles and alkenes.
Phenyl-Hexyl
Biphenyl L11 Dimethylbiphenyl High selectivity for aromatic compounds with enhanced pi-pi and mild
Si
steric interactions due to the two sequential phenyl groups. Ideal for rapid,
efficient drug and metabolite analysis using conditions that are compatible
with MS detection.
F5 (PFP) L43 Pentafluorophenylpropyl Outstanding selectivity for stereoisomers, strong pi-pi acceptor. Enhanced
F F
O
Si F F selectivity for aromatic and electron-rich compounds. Can be used in
Reversed-phase and HILIC mode. In comparison with the C18 phases, the
F5 phase shows longer retention time of basic analytes and less retention of
hydrophobic analytes.
ES-Cyano L10 Diisopropylcyanopropyl Enhanced retention for polar compounds and much less retention for
Si
hydrophobic compounds. Ideal for the separation of non-polar bases in
O
HILIC mode (Ion-exchange mechanism). Compatible to 100% aqueous

mobile phase and stable at low pH and high temperature.
O
RP-Amide L60 C16-Amide Complementary selectivity to alkyl phases with improved peak shape
N
H
Si for basic compounds compared to older EPG amide phases. Ideal for
the separation of organic acids, phenols, catechins, alcohols, and bases.
Compatible in 100% aqueous mobile phase.
HILIC L3 Bare silica Enhanced separation of polar compounds. Can be used in HILIC and
normal-phase mode. Offers both ion-exchange and partition mechanisms of
separation in HILIC mode.
OH5 L95 Penta-hydroxy Ideal for the HILIC separation of very polar compounds with a LogP
HO OH
OH
O
Si
HO OH
value close to 0 or less than 0. Exhibits predominantly HILIC partitioning
retention, limited silanol anionic character, and is relatively insensitive to
ionic strength.

Excellent Lot-to-Lot Reproducibility Ascentis® Express Reproducibility Data (2010-2020)
(QA Retention Factor)
4.0
The consistency of chromatographic results depends 3.9
on many factors. One major contributor to consistent 3.8
3.7
and reliable results is the HPLC column. Therefore, 3.6
the lot-to-lot and column–to-column reproducibility is 3.5
Retention Factor
3.4
a major concern. Ascentis® Express HPLC and UHPLC 3.3
columns show excellent reproducibility. Over the last 3.2
3.1
10 years, the relative standard deviation (RSD) of the 3.0
RSD = 1.2%
QA retention factor was 1.2%. 2.9

2.8
2.7
Visit: SigmaAldrich.com/express 2.6
2.5
2.4
2.3
2.2
2.1
2.0
0 10 20 30 40 50 60 70 80 90 100
Chronological Lot Data
Pore Surface Area Carbon Load Surface Coverage Low pH Limit/ High pH Limit/
Particle Size (µm) Size (Å) (m2/g) (%) (µmol/m2) Max T Max T Endcapped
2 90 120 7.2 3.6 2/60 °C 9/40 °C Yes
2.7 135 7.7 3.4
5 90 6.4 4
2 90 120 6.5 3.1 2/60 °C 9/40 °C Yes
2.7 135 6.7 3.2
5 90 5.6 3.6
2.7 160 90 4.6 2.1 1/90 °C 8/40 °C No
2.7 160 90 4.5 1.4 2/60 °C 9/40 °C Yes
2 90 120 4.8 3.6 2/60 °C 9/40 °C Yes

2.7 135 5.4 3.6
5 90 3.7 3.6
2 90 120 6.3 3.4 2/60 °C 9/40 °C Yes

2.7 135 7.1 3.5
5 90 5.2 3.7
2 90 120 6.7 3.6 2/60 °C 9/40 °C Yes
2.7 135 7.0 3.4
5 90 5.5 3.9
2 90 120 5.3 3.8 2/60 °C 8/40 °C Yes
2.7 135 5.5 3.5
5 90 3.9 3.6
2 90 120 3.4 2.5 1/80 °C 8/40 °C Yes
2.7 135 3.5 2.3

5 90 2.5 2.4
2 90 120 7.3 3 2/60 °C 9/40 °C Yes

2.7 135 8.2 3
5 90 5.1 3
2 90 120 Unbonded Unbonded 1/60 °C 8/40 °C N.A.
2.7 135
5 90
2 90 120 2.8 3 2/60 °C 9/40 °C No
2.7 135 3.2 3
5 90 2.1 3
31
Ascentis® Express UHPLC Columns
Ascentis® Express columns with 2 µm particles are dedicated UHPLC columns which deliver
reliable high speed and high resolution separations at pressures lower than fully porous
sub-2 µm columns. Fused-Core ®
• Highest UHPLC performance possible without the ultra-high pressure of sub-2 µm fully
porous columns
• Highest efficiency and best performance obtained with UHPLC instruments
1.2 µm
• Excellent for fast method development due to full range of column chemistries
0.4 µm
2.0 µm
• 1 µm inlet frit enable high ruggedness
• Pressure stability: 1000 bar/14,500 psi
Ascentis® Express 2 µm C18 FPP 1.7 µm C18

N = 15500 N = 9600
180 P = 190 bar 180 P = 280 bar
Plates/bar = 82 Plates/bar = 34
130 130
Aborbance, mAU
Aborbance, mAU
80 80
30 30
-20 -20
0 0.5 1.0 1.5 2.0 2.5 0 0.5 1.0 1.5 2.0 2.5
Time, min Time, min
Chromatographic Conditions:
Peak Identities:
Dimensions of both 2.1 x 50 mm Flow rate: 0.5 mL/min
Columns: 1. Uracil
Injection Volume: 0.2 µL
Mobile Phase A: A: water 2. Pyrene
B: acetonitrile Detection: 254 nm
3. Decanophenone
Isocratic: A/B: 15/85 Temperature: 25 °C 4. Dodecanophenone
Ascentis® Express 2 µm UHPLC Columns enable full suitability in UHPLC applications providing very high
efficiencies at lower column backpressure in comparison to 1.7 µm fully Porous Particles (FPP)
UHPLC Analysis of Methotrexate and Related Compounds on Ascentis® Express C18, 2.0 μm
1. Aminopterin
1
2. Folic acid Column: Ascentis® Express C18, 10 cm x 2.1 mm
3. Methotrexate I.D., 2.0 µm particles (50813-U)
Column temp.: 35 °C
Mobile phase: [A] 10 mM ammonium formate, pH 3.0 with
2 formic acid; [B] acetonitrile; (90:10, A:B)
3
Flow rate: 0.7 mL/min
Pressure: 1153 psi (795 bar)
Sample: 50 µg/mL in 85:15, water:methanol
Injection: 1 µL
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Min

High resolution UHPLC Analysis of Cannabinoids on Ascentis® Express C18, 2.0 μm
With increasing cannabis and hemp legislation, there has been increased demand for development and validation
of accurate and precise testing methods for potency quantitation. Ascentis® Express 2 µm UHPLC columns enable
the high resolution separation of 17 Cannabinoids in 6 minutes.
300- Column: Ascentis® Express C18, 2.7 µm,

150 x 3 mm (53816-U)
250- Mobile Phase A: 5 mM Ammonium Formate

+ 0.1% Formic acid in water
CBDVA
Mobile Phase B: 0.1% Formic acid in acetonitrile

200- Flow: 0.4 mL/min
CBDA
CBGA
CBN
mAU
Gradient: 75% B to 90% B in 2 min, hold at

THCVA
150- 90% B 5 min
CBDV
CBC
Injection: 3 µL, 25 µg/mL
CBNA
∆-9-THC
THCA
THCV
CBG
∆-8-THC
CBD
100- Column Temp.: 25 °C
CBLA
CBL
Detection: UV, 228 nm
CBCA
50- Max Pressure: 530 bar (7690 psi)
0-
1 2 3 4 5 6 7 Learn more
Minutes
UHPLC Separation of Explosives on Ascentis® Express C18 2 µm UHPLC Column compared to FPP C18 UHPLC Column
300
Ascentis® Express C18, 2.0 µm Chromatographic Conditions:
4
250 Dimensions of 100 mm x 2.1 mm ID
3 both Columns:
200 550 bar Mobile Phase: A: Water
1 Pw = 0.071 Pw = 0.103 B: Methanol
mAU
150 2 5 Isocratic: A/B: 72/28

7 10
100 6 8 Flow rate: 0.4 mL/min
9 11 Detection: PDA @ 254 nm
Pw = 0.168
50 12
13 14 Temperature: 42 °C
0
Peak Identities:
-50
0 2.5 5.0 7.5 10.0 12.5 15.0 1. Peak Identities:
Time (min)
2. HMX
300 3. RDX
4. 1,3,5-Trinitrobenzene
250 FPP C18, 1.7 µm, 2.1 x 100 mm
5. 1,3-Dinitrobenzene
6. Nitrobenzene
200
640 bar 7. Tetryl
150 3 4 8. 2,4,6-Trinitrotoluene
Pw = 0.140
mAU
1 9. 2-Amino-4,6-Dinitrotoluene
100 2 Pw = 0.094 9 10 10. 4-amino-2,6-dinitrotoluene
5 7 11. 2,4-Dinitrotoluene
50 6 8 Pw = 0.213
11 12. 2,6-Dinitrotoluene
12 13 14
13. 2-Nitrotoluene
0
14. 4-Nitrotoluene
-50 15. 3-Nitrotoluene
0 2.5 5.0 7.5 10.0 12.5 15.0
Time (min)
Ascentis® Express UHPLC columns
33
Ascentis® Express columns for environmental testing
Ascentis® Express PAH HPLC columns delivers a fast and high efficiency separation of 16 standard PAH
compounds with a resolution value of at least 1.5 in under five minutes for EPA 8310 and EPA 610.
• Application-assured through method qualified lot analysis
• 2.7 µm Fused-Core® particle for UHPLC-like separation with maximum resolution at lower
back-pressure in comparison to sub-2 µm particles
• Well suited for UV, fluorescence and MS detection
Ascentis® Express PAH, 2.7 µm

7
FPP 95 Å PAH, 1.8 µm
1 5 6
3
Absorbance @ 280 nm
11
4 16
2
9 13 15
12 17 18
8 10 14
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Time, min
Column: Ascentis® Express PAH, Flow Rate: 1.8 mL/min

Peak Identities
2.7µm, 5cm x 4.6mm, 90A
Ascentis Express
®
256 bar 1. Naphthalene 11. Benzo[a]anthracene
Part Number: 53539-U Back Pressure:
2. Acenaphthylene 12. Chrysene
Competitor FPP 95 Å PAH, FPP Back 344 bar 3. 1-Methylnaphthalene 13. Benzo[b]fluoranthene
Column: 1.8 µm, 4.6 x 50mm Pressure:
4. 2-Methylnaphthalene 14. Benzo[k]fluoranthene
Mobile Phase: A: Water Temperature: 30 °C 5. Acenaphthene 15. Benzo[a]pyrene
B: Acetonitrile
Detection: 280 nm 6. Fluorene 16. Dibenzo[a,h]
Gradient: Time %B anthracene
Injection Volume: 2 µL 7. Phenanthrene
0.0 50 8. Anthracene 17. Benzo[g,h,i]perylene
Sample Solvent: Methanol
4.0 100 9. Fluoranthene 18. Indeno[1,2,3-c,d]
Data Rate: 100 Hz pyrene
5.0 100 10. Pyrene
Response Time: 0.025 sec.
5.01 50
Flow Cell: 1 µL
Ascentis® Express PFAS HPLC Columns and Delay Columns

The new Ascentis® Express PFAS HPLC column is designed for the separation of novel and legacy short chain
and long chain PFAS compounds containing branched and linear isomers, whilst adhering to EPA methodology
requirements. Furthermore, a specific PFAS delay column prevents background PFAS contamination from
interfering with the sample results in quantitative LC-MS methods. The Ascentis® Express PFAS HPLC column,
with its Fused-Core® technology and a particle size of 2.7 µm, delivers fast and high-resolution separations with
excellent selectivity, peak shape, and necessary retention to perform in EPA methods 537.1, 533 and 8327. These
advantages are demonstrated, in one particular example, by the separation of all PFAS analytes from EPA methods
537.1, 533, and 8327 in under five minutes.
• Application-assured through method qualified lot analysis
• 2.7 µm Fused-Core® particle for UHPLC-like separation with maximum resolution at lower
back-pressure in comparison to sub-2 µm particles
• Endcapped alkyl phases for high sensitivity (no bleed) LC-MS analysis
PFAS Testing Ascentis® Express PFAS HPLC Columns and Delay Columns

Ascentis® Express PFAS column Delay is a high-performance liquid chromatography column based on Fused-Core®
particle design. The highly retentive, endcapped silane of the Ascentis® Express PFAS Delay column provides high
retention of PFAS compounds across various mobile phase conditions and is used to delay background instrument
PFAS contamination from interference with analyzed samples. For this reason, the Ascentis® Express PFAS Delay
column is placed upstream of the sample injector and after the mixer. See diagram below.
Sample
Injector
Pump PFAS Delay
PFAS Analytical Data
Solvent Column
Column Processing/
Reservoir
Mixer System
Detector/Tandem
Mass Spectrometer
Peak No. Compound

1 PFBA
16 2 4:2FTS
3 PFPeA
40000 4 PFBS
23
19 24 5 PFHpS
6 PFPeS
7 PFMPA
8 PFHxA
30000 14 21
9 9 PFEESA
10 HFPO-DA
11 PFHpA
15
12 PFHxS
20000 13 ADONA
25
12 14 PFOA
1 5 13 15 PFMBA
6 10 22 16 PFNA
11 17
17 PFOS
10000 4 18 9Cl-PF3ONS
3 19 PFDA
7 20 8:2FTS
2 8 18
20 21 6:2FTS
22 NFDHA
0
2.5 23 PFUnA
5.0 7.5 10.0 12.5
24 11Cl-PF3OUdS
min 25 PFDoA
Access full application data:

LC-MS Analysis of PFAS Compounds in EPA Methods 537.1, 533 and 8327 | LC-MS Analysis of 33 PFAS Compounds in 5 minutes
Ordering Information Ascentis® Express PAH and PFAS HPLC Columns

Column dimension
Length (mm) I.D. (mm) PAH PFAS Length (mm) I.D. (mm) PAH PFAS
50 x 2.1 53513-U 53557-U 100 x 4.6 53540-U
100 x 2.1 53532-U 53559-U 150 x 4.6 53541-U
150 x 2.1 53533-U 53560-U 250 x 4.6 53550-U
250 x 2.1 53562-U Guard Columns Delay column
50 x 3 53534-U 53563-U 50 x 2.1 53551-U
100 x 3 53535-U 53564-U 50 x 3 53555-U 53572-U
150 x 3 53538-U 53565-U 50 x 4.6 53556-U 53573-U
250 x 3 53570-U
50 x 4.6 53539-U
35
Length I.D. Phenyl-
(mm) (mm) C18 C8 RP-Amide Hexyl Biphenyl F5 (PFP) ES-Cyano OH5 HILIC (Si)
20 x 2.1 50507-U 50362-U 50732-U 50442-U on request 50603-U 50557-U 50313-U 50255-U
50 x 2.1 50509-U 50364-U 50734-U 50446-U 584585-U 50605-U 50559-U 50317-U 50257-U
100 x 2.1 50517-U 50368-U 50737-U 50454-U 584586-U 50612-U 50563-U 50322-U 50260-U
150 x 2.1 50518-U 50372-U 50739-U 50455-U 584587-U 50613-U 50564-U 50327-U 50261-U
250 x 2.1 50521-U 50373-U 50747-U 50456-U 584588-U 50614-U 50566-U 50328-U 50262-U
30 x 3.0 50522-U 50376-U 50749-U 50459-U 584589-U 50615-U 50567-U 50329-U 50264-U
50 x 3.0 50523-U 50377-U 50751-U 50464-U 584590-U 50616-U 50568-U 50335-U 50265-U
100 x 3.0 50526-U 50381-U 50753-U 50469-U 584591-U 50622-U 50570-U 50338-U 50269-U
150 x 3.0 50527-U 50382-U 50758-U 50470-U 584592-U 50623-U 50574-U 50339-U 50270-U
30 x 4.6 50529-U 50386-U 50767-U 50474-U 584593-U 50625-U 50577-U 50343-U 50278-U
50 x 4.6 50530-U 50389-U 50768-U 50477-U 584594-U 50626-U 50581-U 50344-U 50284-U
75 x 4.6 50533-U 50390-U on request 50479-U on request 50627-U 50583-U 50345-U 50286-U
100 x 4.6 50536-U 50391-U 50773-U 50482-U 584595-U 50628-U 50585-U 50346-U 50288-U
150 x 4.6 50537-U 50392-U 50774-U 50483-U 584596-U 50631-U 50588-U 50347-U 50289-U
Guard 5 mm x 2.1 mm 50539-U 50395-U 50776-U 50496-U 584597-U 50633-U 50592-U 50349-U 50295-U
Guard 5 mm x 3 mm 50541-U 50396-U 50777-U 50497-U 584598-U 50634-U 50593-U 50350-U 50297-U
Guard 5 mm x 4.6 mm 50542-U 50399-U 50779-U 50498-U 584599-U 50635-U 50597-U 50355-U 50298-U
Ascentis® Express (2.7 µm)

Length I.D. Peptide C18*, Phenyl- HILIC
(mm) (mm) C30 C18 AQ-C18 ES-C18 PCP C8 RP-Amide Hexyl Biphenyl F5 (PFP) ES-Cyano OH5 (Si)
20 x 2.1 on request 53799-U 577320-U on request on request 53795-U on request on request 64043-U 53592-U 53494-U 53779-U
30 x 2.1 on request 53802-U 577321-U 53299-U on request 53839-U 53910-U 53332-U 64054-U 53566-U 53468-U 53748-U 53933-U
50 x 2.1 577100-U 53822-U 577322-U 53301-U on request 53831-U 53911-U 53334-U 64057-U 53567-U 53470-U 53749-U 53934-U
250 x 2.1 577103-U on request on request on request on request on request on request on request on request on request on request on request on request
20 x 3.0 on request on request 577326-U on request on request on request on request on request 64047-U on request on request on request on request
20 x 4.6 on request on request 577332-U on request on request on request on request on request 64051-U on request on request on request on request
100 x 4.6 577135-U 53827-U 577336-U 53324-U 50461-U 53837-U 53929-U 53352-U 64067-U 53590-U 53491-U 53776-U 53979-U
150 x 4.6 577136-U 53829-U 577337-U 53328-U 50462-U 53838-U 53931-U 53353-U 64071-U 53591-U 53492-U 53778-U 53981-U
Guard 5 x 2.1 577137-U 53501-U 577338-U 53536-U on request 53509-U 53514-U 53524-U 64074-U 53594-U 53495-U 53780-U 53520-U
Guard 5 x 3 577138-U 53504-U 577339-U 53537-U on request 53511-U 53516-U 53526-U 64076-U 53597-U 53496-U 53781-U 53521-U
Guard 5 x 4.6 577139-U 53508-U 577340-U 53542-U on request 53512-U 53519-U 53531-U 64078-U 53599-U 53497-U on request 53523-U
*Ascentis® Express C18 PCP: pre-conditioned with phosphoric acid.
HPLC Columns "on request" are available as Custom Product. Please see page 120/121

Ascentis® Express UHPLC Columns
Length I.D. Phenyl-
(mm) (mm) C18 C8 RP-Amide Hexyl Biphenyl F5 (PFP) ES-Cyano OH5 HILIC (Si)
50 x 2.1 50811-U 51656-U 51569-U 51603-U 584600-U 50859-U 51717-U 50957-U 51406-U
100 x 2.1 50813-U 51658-U 51576-U 51608-U 584601-U 50863-U 51724-U 50959-U 51409-U
150 x 2.1 50814-U 51661-U 51577-U 51609-U 584602-U 50867-U 51725-U 50962-U 51418-U
250 x 2.1 on request on request on request on request 584603-U on request on request on request on request
30 x 3 50815-U 51663-U 51582-U 51611-U 584604-U 50869-U 51727-U 50963-U 51419-U
50 x 3 50816-U 51664-U 51583-U 51614-U 584605-U 50871-U 51728-U 50964-U 51421-U
75 x 3 50817-U 51672-U 51587-U 51616-U on request 50876-U 51729-U 50965-U 51424-U
100 x 3 50819-U 51673-U 51588-U 51617-U on request 50879-U 51732-U 50967-U 51428-U
150 x 3 50821-U 51674-U 51589-U 51618-U 584606-U 50881-U 51734-U 50968-U 51429-U
Guard 5 mm x 2.1 50822-U 51676-U 51594-U 51619-U 584607-U 50884-U 51736-U 50969-U 51430-U
Guard 5 mm x 3 on request 51679-U 51595-U 51623-U 584608-U 50886-U 51739-U 50973-U 51433-U
Ascentis® Express Guard Cartridge Holder 53500-U
Finger-tight Guard
Finger-tight Guard Cartridge Holder,
Titanium Hybrid
Cartridge Holder, Inlet Outlet
Ferrule
Provides easy Allows for replacement of
Adds durability for
replacement of guard guard cartridge without
multiple connections.
cartridge. removing the guard holder
from the flow path.
Auto-adjusting
Zero-Dead Volume
Ascentis® Express (ZDV) End Fitting
guard cartridge Ensures optimum,
All Ascentis® Express ZDV connection to the
phases available. analytical column.
Ascentis® Express Capillary columns (2.7 µm)

Length (mm) I.D. (mm) C18 Peptide ES-C18 C8
50 x 0.075 53982-U 53543-U 53983-U
50 x 0.100 53985-U 53544-U 53987-U
50 x 0.200 53989-U 53545-U 53991-U
50 x 0.300 53992-U 53546-U 53997-U
50 x 0.500 53998-U 53547-U 53999-U
50 x 1 53548-U
150 x 0.075 54219-U 53549-U 54229-U
150 x 0.100 54256-U 53552-U 54260-U
150 x 0.200 54261-U 53553-U 54262-U
150 x 0.300 54271-U 53554-U 54272-U
150 x 0.500 54273-U 53558-U 54275-U
150 x 1 53561-U
37
BIOshell™ HPLC and UHPLC Columns
Maximum Resolution for Glycan, Peptide, Protein, and IgG Separation
As the pharmaceutical and biotechnology industries • Bottom-up analysis (peptide mapping) of proteins for
continuously evolve into the development of “large primary structure confirmation
molecule” biotherapeutics to treat a myriad of diseases, • Middle-up analysis of mAb fragments (light and
both fast and high-resolution separations are required heavy chains)
in order to resolve the numerous structural variants
• High resolution separation of released N- and
that exist in these complex samples. The BIOshell™ line
O-linked glycans.
of superficially porous particle (SPP) packed columns
has been developed to aid research into understanding
the subtleties of the molecule that is being developed. Features and Benefits
• Application specific columns for bioseparations that
Highlighted Applications for these outperform fully porous particulate silica columns
columns include: • Significantly higher separation efficiency
• Top-down analysis of intact proteins, monoclonal • Offer better peak shape and peak capacity
antibodies (mAbs), antibody-drug conjugates (ADCs), • Breakthrough 1000 Å pore diameter particles for
and other large biomolecules. large molecule enablement
ColumntoSelection
Guide SelectingGuide for Biomolecule
BIOshell™ UHPLC andSeparations
HPLC Column Phases
Glycans Peptides Proteins IgGs & Proteins

< 2-3 kDa MW ≤ 15-20 kDa 2 kDa < MW < 500 kDa >100 kDa
Polar or
Released Peptides & Hydrophilic Hydrophobic
Membrane
Glycans Polypeptides Proteins Proteins
Peptides
BIOshell™ 160 Å BIOshell™ BIOshell™

BIOshell™ 90 Å BIOshell™ 160 Å BIOshell™ 160 Å BIOshell™ 400 Å BIOshell™ 400 Å BIOshell™
Peptide IgG 1000 Å IgG 1000 Å
Glycan Peptide C18 Peptide CN Protein C18 Protein C4 IgG 1000 Å C4
Phenyl-Hexyl C18 Diphenyl

Pore Size Mismatch Can Lead to Significant Losses in Efficiency
Column: BIOshell™ A160 Peptide C18,
15 cm x 2.1 mm I.D., 2.7 µm;
BIOshell™ A400 Protein C18,
15 cm x 2.1 mm I.D., 3.4 µm;
BIOshell™ IgG 1000 Å C18, 1. Ribonuclease A (13.8 kDa)
15 cm x 2.1 mm I.D., 2.7 µm;
2. Lysozyme (14.4 kDa)
Mobile phase: [A] Water (0.1% v/v trifluoroacetic acid);
3. SILu™ Lite SigmaMAb Antibody (~150 kDa)
[B] 20:80 Water:Acetonitrile (0.085% v/v
trifluoroacetic acid) 4. Enolase (46.7 kDa)
Gradient: 27% B to 60% B in 15 min

Flow rate: 0.4 mL/min 0.048
0.048
0.165 0.122
Column temp.: 60 °C 2.7 μm, 160Å
0.041 0.036 0.048
Detector: UV, 280 nm 0.069
3.4μm, 400Å
Injection: 4 µL 0.039 0.035 0.041
0.045
Sample: Proteins, varied concentration, 2.7μm, 1000Å
water (0.1% v/v trifluoroacetic acid) 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Time, min
Higher efficiencies and better sensitivity can be realized with proper
pore diameter selection. Here, the 1000 Å pore diameter is the only
one capable of providing good peak shape of the mAb (peak 3) analyte.
39
Molecule size Properties Applications Bonded Phase

Large (> 50 kDa) Largest pore diameter in mAbs; ADCs; Biosimilars; C4
the portfolio allowing for H/D Exchange;
unrestricted access of proteins mAb Fragments
and other large molecules.
Compatible for UHPLC, HPLC,
and mass spectrometry (MS).
C4, C18, and Diphenyl phase C18
chemistries provide different
selectivities. Resolution of very
large proteins with superior
peak shape and efficiency as
IgG
compared to separations on
FPP-packed columns.
Diphenyl
Large (2 kDa < MW Fast separation of biomolecules mAbs; ADCs; Biosimilars; C4

< 500 kDa) due to a more shallow shell. Proteins; mAb Fragments
Temperature stable up to
90 °C enabling high efficiency
separations perfect for UHPLC,
HPLC, and LC-MS assays. C4
Protein
and C18 chemistries provide C18

different selectivities for
hydrophobic and hydrophilic
proteins. Resolution of large
proteins with superior peak
shape and efficiency as
compared to separations on
FPP-packed columns.
Medium (0.1 kDa < Wide range of particle sizes for Tryptic Digests; Post- C18
MW < 15 kDa) both high efficiency separations Translational Modifications
and high throughput. Peak (PTMs); Polypeptides
capacities of columns
capable of resolving complex
peptide mixtures.
Peptide
Cyano
Phenyl-
Hexyl
Small (< 20 kDa*) Improved retention of polar Resolution of Proprietary

*for glycans, compounds and zwitterions oligosaccharides (released Ligand
glycopeptides, as compared to bare silica. and labeled glycans) via
Glycan
and glycoproteins Resolution of peaks unaffected hydrophilic interaction liquid

by slight changes in buffer chromatography (HILIC).
concentration. Capable of
resolving complex mixtures of
glycans (isobaric glycans with
different linkages).

USP Particle Sizes Carbon Load Surface Area Low pH /T High pH /T
Designation Bonding Chemistry (s) (µm) Pore Size (Å) (%) (m2/g) Limit Limit Endcapped
L26 Dimethylbutylsilane 2.7 1000 0.6 22 2/90 °C 9/40 °C Yes
L1 Diisobutyloctadecylsilane 2.7 1000 1.4 22 1/90 °C 8/40 °C Yes
L11 Diphenylmethylsilane 2.7 1000 1.0 22 2/90 °C 9/40 °C Yes
L26 Dimethylbutylsilane 3.4 400 0.4 15 2/90 °C 9/40 °C Yes
L1 Diisobutyloctadecylsilane 3.4 400 1.0 15 1/90 °C 8/40 °C Yes
L1 Diisobutyloctadecylsilane 2 160 4.0 65 1/90 °C 8/40 °C No
2.7 160 4.6 90 1/90 °C 8/40 °C No
5 160 4.0 60 1/90 °C 8/40 °C No
L10 Diisopropylcyanopropylsilane 2.7 160 2.2 90 1/90 °C 8/40 °C Yes
5 160 1.5 60 1/90 °C 8/40 °C Yes
L11 Dimethylphenyl-hexylsilane 2.7 160 4.7 90 2/90 °C 9/40 °C Yes
L95 Penta-hydroxy 2.7 90 3.2 135 2/65 °C 9/40 °C No
41
BIOshell™ IgG 1000 Å U/HPLC Columns:
Maximizing Pore Diameter to Minimize Size Exclusion
• A 1000 Å pore diameter allows • Post-translational modifications (PTMs)
unrestricted access of large biomolecules of expressed proteins can lead to subtle
into the particles. differences in molecular structure and
• Superficially Porous Particles (SPPs) function of the protein. These minor
Fused- Core ®
provide narrower peak widths and variants can be resolved with BIOshell™
improved resolution for characterization of IgG 1000 Å columns.
biomolecules in comparison to fully Porous • Three different phase chemistries (C4,
Particles (FPPs). C18 and Diphenyl) for optimal selectivity
1.7 µm
0.5 µm
2.7 µm
Effect of Phase Chemistry on Protein Selectivity

Column: BIOshell™ IgG 1000 Å C4, 15 cm x 2.1 mm I.D., 2.7 µm;
BIOshell™ IgG 1000 Å C18, 15 cm x 2.1 mm I.D., 2.7 µm; BIOshell™ IgG 1000 Å C4
BIOshell™ IgG 1000 Å Diphenyl, 15 cm x 2.1 mm I.D., 2.7 µm
BIOshell™ IgG 1000 Å C18
Mobile phase: [A] 2:10:88 n-propanol/acetonitrile/water (0.1%
v/v difluoroacetic acid); BIOshell™ IgG 1000 Å Diphenyl
[B] 70:20:10 n-propanol/acetonitrile/water
(0.1% v/v difluoroacetic acid) 40000
Absorbance μV
Gradient: 16% B to 26% B in 20 min 1

30000 2
Flow rate: 0.2 mL/min 34
20000
Column temp.: 80 °C 5
Detector: UV, 280 nm 10000

6
Injection: 2 µL 0
0
4 6 8 10 12 14 16
Sample: Denosumab, 2 mg/mL,
Time, min
water (0.1% v/v trifluoroacetic acid)
Monoclonal antibodies are unique molecules and therefore can

interact differently with different phase chemistries.
The numbered peaks correspond to IgG2 disulfide bond variants.
BIOshell™ A400 Protein U/HPLC Columns:

Minimizing Mass Transfer for Maximum Throughput
• A 0.2 µm-thin, porous shell with in comparison to fully Porous
400 Å pores leads to rapid and efficient Particles (FPPs).
separations of proteins. • Ability to tolerate high temperature
• Superficially Porous Particles applications (up to 90 °C) in acidic
Fused- Core ®
(SPPs) provide narrower peak mobile phases.
widths and improved resolution for • Two different phase chemistries (C4
characterization of biomolecules and C18) for rapid protein separation
3.0 µm
0.2 µm
3.4 µm

Rapid Protein Separations on BIOshell™ A400 Peak Name kDa (native)
Column: BIOshell™ A400 Protein C4, 1. lysozyme; chicken 14.3
5 cm x 2.1 mm I.D., 3.4 µm 2. haptoglobulin, phenotype 1-1; human 96
Mobile phase: [A] 75:25 (0.1% TFA in water):(0.1% TFA 3. glutamate dehydrogenase; bovine 332
in acetonitrile); [B] 25:75 (0.1% TFA in 4. ß-galactosidase; E. coli 465
water):(0.1% TFA in acetonitrile)
5. lactate dehydrogenase contaminant
Gradient: 12 to 100% B in 1 min; held at 100% 6. lactate dehydrogenase; rabbit 140
B for 1 min
6
4
2
Injection: 1 µL 3
Sample: Protein mix, varied concentration,

water (0.1% TFA)
1
5
Due to the shallow, porous shell, protein separations can be
performed in less than one minute with the BIOshell™ A400 column.
0.0
0.0 0.2 0.4 0.6 0.8 1.0
0.2 0.4 0.6 0.8 1.0 1.2
1.2
Retention Time (min)
BIOshell™ A160 Peptide U/HPLC Columns:

High Resolution Peptide Separations
• BIOshell™ A160 Peptide U/HPLC columns • Lower backpressure allows for columns to
offer a broad portfolio of particle sizes and be used in series to maximize resolution
phase chemistries to create a superior and peak capacity of complex proteomic
Fused- Core ®
solution for fast and efficient separations samples or tryptic digests.

of peptides up to 20 kDa. • Compatible with all UHPLC and 1.2 µm
• Higher resolutions and higher peak LC-MS instrumentation.
capacities of peptides at ~ 50%
0.4 µm
• Three different particle sizes and phase

backpressure of sub-2 µm Fully Porous
2.0 µm
chemistries (Phenyl-Hexyl, C18 and

Particle (FPP)-packed columns. Cyano) for fast peptide separations
Fused- Core ®
Rapid Tryptic Digest Analysis Using

BIOshell™ A160 Peptide C18 2 mL/min
126 bar (1827 psi)
Column: BIOshell™ A160 1.7 µm
mAU 215 nm
Peptide C18, 0.5 µm

15 cm x 4.6 mm I.D.,
2.7 µm
5.0 µm
Mobile [A] Water (0.1% TFA); [B]
phase: Acetonitrile (0.1% TFA) 0 5 10 15 20
Gradient: As indicated 0
0 5
5 10 15
10 15 20
20
Flow rate: As indicated 4 mL/min

mAU 215 nm
Fused- Core ®
Column 60 °C 250 bar (3626 psi)
temp.:
Injection: 15 µL
Sample: Tryptic digest, 1 mg/mL, 3.3 µm
water (0.1% TFA) 0

0 2 4 6 8 10
2 4 6 8 10 12
12
0.6 µm
Min
0 2 4 6 8 10 12
5 µm
BIOshell™ A160 Peptide columns can more than double sample throughput by allowing the analyst to operate at
higher flow rates without a concomitant drop in efficiency.
43
BIOshell™ Glycan U/HPLC Columns: High Resolution Glycan Separations
• BIOshell™ Glycan U/HPLC columns consist • Increased retention of acidic and zwitterionic
of a proprietary, pentahydroxy chemistry analytes than bare silica columns. Fused- Core ®
tethered to a 2.7 µm, 90 Å superficially • Each lot of BIOshell™ Glycan particles is

porous particle (SPP). tested for quality control by separation
• Appropriate for USP L95 methods. of a series of labeled glycans having 2 – 1.7 µm
• Main application is for the resolution of 25 glucose units (GU). 0.5 µm
2.7 µm
oligosaccharides (released and labeled
glycans) via hydrophilic interaction liquid
chromatography (HILIC).
HPLC Analysis of Procainamide-Labeled Human IgG Peak Glycan Peak Glycan

Glycans on BIOshell™ Glycan using HILIC-FLR 1
hIgG glycans
11
4
Column: BIOshell™ Glycan, 15 cm x 2.1 mm 2 12
I.D., 2.7 µm 3 13
2 8
Mobile phase: [A] 50 mM ammonium formate, pH 4.4 4 14
(50 mM ammonium hydroxide, adjusted to
5 15
pH 4.4 with formic acid); [B] acetonitrile
16
6
5 17
15
7
18
8
Temp.: 60 °C
19
9 6
Detector: FLR, ex 308 nm, em 359 nm 3 20
10
Injection vol: 10 μL 11
17
9 12 19 20
7
Sample: Released human IgG glycans 1 10 1314 16 18
Excellent resolution and peak shape of released glycans. 0 5 10 15 20 25 30 35 40 45 50 55

Min
Ordering Information
BIOshell™ IgG 1000 Å (2.7 µm) BIOshell™ A160 Peptide (2.7 µm)
Length (mm) I.D. (mm) C18 C4 Diphenyl Length (mm) I.D. (mm) C18 CN Phenyl-Hexyl
20 x 2.1 63281-U on request on request 20 x 2.1 on request on request 577523-U
30 x 2.1 63282-U on request on request 30 x 2.1 66901-U 66965-U 577524-U
50 x 2.1 581362-U 63283-U 577419-U 50 x 2.1 66902-U 66966-U 577525-U
100 x 2.1 582701-U 63288-U 577420-U 100 x 2.1 66904-U 66968-U 577527-U
150 x 2.1 582703-U 63289-U 577421-U 150 x 2.1 66905-U 66969-U 577528-U
250 x 2.1 582704-U on request 577422-U 20 x 3.0 on request on request 577529-U
30 x 3.0 582705-U 63307-U 577423-U 50 x 3.0 66907-U 66971-U 577531-U
50 x 3.0 582706-U 63308-U 577424-U 75 x 3.0 on request on request 577532-U
100 x 3.0 582707-U 63313-U 577425-U 150 x 3.0 66909-U 66973-U 577534-U
20 x 4.6 63322-U on request on request 30 x 4.6 on request on request 577536-U
30 x 4.6 582709-U 63324-U 577427-U 50 x 4.6 66913-U 66974-U 577537-U
100 x 4.6 582713-U 63328-U 577429-U 150 x 4.6 on request 66976-U 577540-U
150 x 4.6 581348-U 63329-U 577430-U Guard 5 x 2.1 66918-U 66977-U 577541-U
Guard 5 x 2.1 581349-U 63291-U 577431-U Guard 5 x 3.0 66919-U 66978-U 577542-U
Guard 5 x 3.0 581360-U 63315-U 577432-U Guard 5 x 4.6 66921-U 66979-U 577543-U
Guard 5 x 4.6 581361-U 63334-U 577433-U
HPLC Columns "on request" are available as Custom Product. Please see page 120/121 BIOshell Glycan

BIOshell™ A160 Peptide (2.0 µm) BIOshell™ A160 Peptide (5.0 µm)
Length (mm) I.D. (mm) C18 Length (mm) I.D. (mm) C18 CN
20 x 2.1 67234-U 30 x 2.1 67001-U 67061-U
30 x 2.1 67238-U 50 x 2.1 67002-U 67062-U
50 x 2.1 67239-U 75 x 2.1 67003-U 67063-U
75 x 2.1 67241-U 100 x 2.1 67004-U 67064-U
100 x 2.1 67242-U 150 x 2.1 67006-U 67065-U
150 x 2.1 67243-U 30 x 3.0 67007-U 67066-U
20 x 3.0 67257-U 50 x 3.0 67008-U 67067-U
30 x 3.0 67263-U 100 x 3.0 67011-U 67068-U
50 x 3.0 67267-U 150 x 3.0 67012-U 67069-U
75 x 3.0 67274-U 50 x 4.6 67013-U 67071-U
100 x 3.0 67275-U 100 x 4.6 67014-U 67080-U
150 x 3.0 67277-U 150 x 4.6 67015-U 67081-U
Guard 5 x 2.1 67281-U Guard 5 x 2.1 67016-U 67082-U
Guard 5 x 3.0 67282-U Guard 5 x 3.0 67017-U 67083-U
Guard 5 x 4.6 67018-U 67084-U
BIOshell™ A160 Peptide C18 (2.7 µm) BIOshell™ A160 Peptide C18 (5.0 µm)
Capillary Columns Capillary Columns
Length (mm) I.D. (mm) C18 CN Length (mm) I.D. (mm) C18 CN
50 x 0.075 67085-U 67150-U 50 x 0.075 67201-U 67305-U
150 x 0.075 67086-U 67152-U 150 x 0.075 67202-U 67307-U
50 x 0.1 67087-U 67153-U 50 x 0.1 67203-U 67311-U
150 x 0.1 67088-U 67155-U 150 x 0.1 67204-U 67312-U
50 x 0.2 67089-U 67157-U 50 x 0.2 67205-U 67314-U
150 x 0.2 67091-U 67158-U 150 x 0.2 67206-U 67315-U
50 x 0.3 67092-U 67159-U 50 x 0.3 67207-U 67321-U
150 x 0.3 67093-U 67160-U 150 x 0.3 67208-U 67324-U
50 x 0.5 67095-U 67161-U 50 x 0.5 67209-U 67325-U
100 x 0.5 67096-U on request 150 x 0.5 67212-U 67326-U
150 x 0.5 67097-U 67163-U 50 x 1.0 67215-U 67327-U
50 x 1.0 67098-U 67164-U 150 x 1.0 67219-U 67329-U
150 x 1.0 67099-U 67165-U
BIOshell™ A400 Protein C4 (3.4 µm)
BIOshell™ A400 Protein C4 (3.4 µm) Capillary Columns
Length (mm) I.D. (mm) C18 C4 Length (mm) I.D. (mm) C18 C4
20 x 2.1 67457-U on request 50 x 0.075 67489-U 67031-U
30 x 2.1 67458-U on request 150 x 0.075 67490-U 67032-U
50 x 2.1 67459-U 66824-U 50 x 0.1 67491-U 67033-U

75 x 2.1 67462-U on request 150 x 0.1 67493-U 67034-U
100 x 2.1 67463-U 66825-U 50 x 0.2 67494-U 67036-U

150 x 2.1 67469-U 66826-U 150 x 0.2 67495-U 67037-U
20 x 3.0 67471-U on request 50 x 0.3 67496-U 67038-U
30 x 3.0 67472-U on request 150 x 0.3 67497-U 67039-U
50 x 3.0 67473-U on request 50 x 0.5 67499-U 67040-U

75 x 3.0 67474-U on request 150 x 0.5 67502-U 67041-U
100 x 3.0 67475-U on request 50 x 1.0 67503-U 67042-U
150 x 3.0 67477-U on request 150 x 1.0 67504-U 67045-U

20 x 4.6 67478-U on request
30 x 4.6 67482-U on request BIOshell™ Glycan (2.7 µm)
50 x 4.6 67483-U 66827-U Length (mm) I.D. (mm) p/n
75 x 4.6 67485-U on request 50 x 2.1 50991-U
100 x 4.6 67487-U 66828-U 100 x 2.1 50993-U
150 x 4.6 67488-U 66829-U 150 x 2.1 50994-U
Guard 5 x 2.1 67505-U 66830-U 50 x 4.6 50997-U
Guard 5 x 3.0 67506-U on request 100 x 4.6 50998-U
Guard 5 x 4.6 67508-U 66831-U 150 x 4.6 50999-U
45
Monolithic Silica HPLC Columns
Revolutionary monolithic silica for rapid and robust separations
Thanks to their patented monolithic silica technology, The secret to the speed of Chromolith® columns
Chromolith® HPLC columns allow you to race through is their exceptionally low back pressure. Produced
separations with maximum robustness and selectivity— from a continuous piece of porous silica using a sol-
at minimal back pressure. gel process, Chromolith® columns possess a defined
bimodal pore structure with macro and mesopores in
Macropores the micro and nanometer range. The high permeability
and porosity of the silica skeleton, and the resulting low
To truly accelerate chromatographic separations, there back pressure, allow for more flexible flow rates than
is no better choice than Chromolith® HPLC columns. particle-packed columns. As a result, Chromolith® HPLC
Due to their revolutionary, monolithic technology, columns enable high-throughput analysis without loss
Chromolith® columns provide excellent and rapid of separation efficiency or peak capacity.
separations with extremely high robustness and matrix
tolerance compared to particulate columns. The revolutionary, bimodal pore structure of
Chromolith® columns provides a unique combination of
macropores and mesopores.
SEM image: Cross section of a silica monolith.

Total porosity > 80%.
Mesopores: Average pore size is Macropores: Average pore

13 nm for Chromolith®, 15 nm for size is 1.5 μm for Chromolith®
Chromolith®HR, and 30 nm for 2 mm I.D. , 1.15 μm for
Chromolith®WP 300. Chromolith® HR, and 2 μm for
Forms a fine porous structure with a all others.
large, uniform surface area on which Allows rapid flow of the mobile
adsorption takes place, thus enabling phase at low back pressure.
high-performance chromatographic
separation.
Chromolith® Pore Sizes

Mesopores Macropores
Chromolith® Performance 13 nm (130 Å) 2 µm
Chromolith® 2 mm ID 13 nm (130 Å) 1.5 µm
Chromolith® HighResolution 15 nm (150 Å) 2 µm
Chromolith® WP 300 30 nm (300 Å) 2 µm Monolithic Silica Technology

Chromolith® HPLC Columns
for small and large molecule separation
Several key benefits result directly from the Column Dimensions

bimodal pore structure of the silica gel: HighResolution RP-18e 2 and 4.6 mm I.D. and Capillary columns
HighResolution RP-8e 4.6 mm I.D. and Capillary columns
• Rapid separations at very low column back-pressure
RP-18e 2, 3, 4.6, 10, 25 mm I.D. and Capillary
• Standard HPLC instruments are fully compatible with columns
all Chromolith® columns and UHPLC instruments are RP-8e 4.6 mm I.D.
fully compatible with Chromolith® 2 mm I.D. columns. CN 4.6 mm I.D.
• Matrix-rich samples (such as food or biological Diol 4.6 mm I.D.
samples) can be analyzed without the need for NH2 4.6 mm I.D.
sophisticated and time-consuming sample preparation. Si 4.6, 10 and 25 mm I.D.
Guard column cartridges are also available.
• Cost-savings are achieved as the column lifetimes are Chromolith® WP 300 Columns for
much longer than for particulate HPLC columns, in Biomolecule separation
particular when analyzing matrix-rich samples.
Chromolith® columns have shown great potential for
• Complex, multi-component samples can be separated
the analysis of proteins, antibodies and large peptides
either by using Chromolith® HighResolution (HR)
where columns with good permeability, along with
columns or by using long, very high-efficiency
better mass transfer and selectivity are required.
columns formed by connecting two or more
Chromolith® columns remove back pressure as the
Chromolith® columns together. The low column
primary consideration in method development and
backpressure makes this possible.
allow flow rate flexibility for much higher throughput,
• Easy transfer of methods from a particulate column a choice of column lengths for superior resolution,
to a Chromolith® column. and more solvent options for optimum selectivity.
The columns are available with several surface With no individual particles to shift or break, column
modifications such as octadecyl (RP-18e) or octyl performance is consistent over a much longer lifetime,
(RP-8e) endcapped, Phenyl, CN (nitrile), Diol and making them ideal for matrix-rich sample analysis.
NH2 (amino) as well as an unmodified pure silica. The Column Dimensions
available column dimensions range from Capillary WP 300 RP-18 2 and 4.6 mm I.D.
(Nano) columns to preparative HPLC columns with WP 300 RP-8 4.6 mm I.D.
25 mm ID. WP 300 RP-4 4.6 mm I.D.
WP 300 Protein A 2 and 4.6 mm I.D.
WP 300 Epoxy 2 and 4.6 mm I.D.
Chromolith® HPLC and UHPLC Columns Chromolith® WP 300 HPLC Columns
47
Analysis speed
Chromolith® columns owe their rapid separation speed
to their unique bimodal pore structure of macro and
mesopores. The macropores reduce column back
1 mL/min
pressure and allow the use of faster flow rates, thereby
considerably reducing analysis time. The mesopores
form a fine porous structure, which creates a very large,
active surface area for high-efficiency separations.
200
Particulate column, 3 µm
Back pressure [bar]
100
9 mL/min
150
Particulate column, 5 µm
0 6 12 Retention time [min]
100
Chromolith® column,
50 100-4.6 mm Chromolith® Performance RP-18e 100-4.6 mm
Column: Chromolith® Performance RP-18 endcapped
0 100-4.6 mm
0 2 4 6 8
Mobile phase: Isocratic acetonitrile / 0.1 % trifluoroacetic
Flow rate [mL/min]
acid in water, 20/80 (v/v)
With Chromolith® columns, flow rates can now easily be Pressure: Total pressure (including HPLC system) 25 °C
varied from 1 mL up to 9 mL per minute with the same
Detection: UV 220 nm
high quality resolution. A mixture of five beta-blocker
drugs was analyzed to demonstrate the extreme time Injection volume: 5 µL
savings and high separation efficiency made possible Sample: Atenolol 63 μg/mL
with Chromolith® columns. Due to excellent mass Pindolol 29 μg/mL
transfer properties of the monolithic skeleton, high-speed Metoprolol 108 μg/mL
separation was possible, even at high flow rates. The Celiprolol 104 μg/mL
beta-blockers were well separated with excellent peak Bisoprolol 208 μg/mL
symmetry. At 9 mL/min, analysis time was less than
1 minute, and the column back pressure was only 153 bar.
High separation efficiency

The traditional plate-count method of measuring quality 30
shows that the separation efficiency of Chromolith®
25 Chromolith® HR 100 - 4.6
columns is better than standard 5 μm particulate
Chromolith® 100 - 4.6
columns, and just as good as 3.5 μm columns, but with
Chromolith® 100 - 3
the ability to continue up to 9 mL/min without reaching 20
Chromolith® 100 - 2
HETP
HPLC system pressure limits. The van Deemter plot

of the Chromolith® column clearly demonstrates that 15
separation efficiency does not decrease significantly
when flow rate is increased, as is the case with 10
particulate columns. It is therefore possible to operate
Chromolith® columns at high flow rates with minimal 5
loss of peak resolution. For complex separations, it is
still necessary to use long columns in order to provide 0
the separation efficiency required for resolution of all 0 1 2 3 4 5 6
compounds of interest. Chromolith® HPLC columns Flow rate (mL/min)
can be connected in series to produce a column with
high plate count at low back pressure. (Please see:
Chromolith® column coupler). With particulate columns,
further column length is prevented by excessive
back pressure.

Long-term stability
Besides lower back pressure and greater flow rate
flexibility, Chromolith® columns also achieve faster
equilibration after gradient elution than particle-packed
columns of similar dimensions. These features allow 300 Particulate column
high-throughput analysis without loss of separation
Column back-pressure (bar)

guard column
efficiency or peak capacity. 250 Chromolith® HR with
guard column
The significantly longer column lifetime of Chromolith ®
200
monolithic HPLC columns, in comparison to particulate
HPLC columns, is over-compensating the higher cost 150
of this column material in comparison to particulate
100
columns. In particular, if matrix-rich samples are
used with simplified sample preparation. Therefore, 50
the cost per sample can be significantly lower in
direct comparison under equal conditions and the 0
0 10000 20000 50000 60000
30000 40000
same sample.
Injection volume µL
Column robustness
Chromolith® columns offer excellent robustness and unsurpassed column lifetime. This trait not only ensures
maximum reliability and versatility, but also minimizes maintenance on the HPLC system. As a result, Chromolith®
columns reduce cost per analysis while enhancing data integrity.
When analyzing challenging, matrix-rich samples with traditional HPLC, the benefit of monolithic silica columns is
significant. Sample preparation is time-consuming and costly. The transfer from particulate columns to monolithic
columns can, therefore, save a substantial amount of cost and time.
Previous method New Chromolith® method

Cost breakdown (per analysis) Cost breakdown (per analysis)
CRM CRM
SPE cartridge Sample filtration
SPE reagents Vial
Sample filtration Mobile phase -60%
HPLC equiment
Vial
Mobile phase
HPLC equiment
Previous method New Chromolith® method

Time breakdown (per analysis) Time breakdown (per analysis)
Decarbonation
Acidification Chromatography
Report
-86%
Col. conditioning
Extraction Filtration
Elution
Col. wash
Chromatography
Report
Filtration
Time and cost per analysis for the determination of Iso-alpha-acids in Beer using a particulate and a monolithic column.
49
Chromolith® and Chromolith® HR HPLC Columns
Robust and rapid separation of small molecules
Chromolith® Performance HPLC columns provide the highest matrix-tolerance due to their large marcopores
(2 µm) and are available with several column chemistries for a broad selectivity range.
Chromolith® columns with 2 mm internal diameter are ideal for use with UHPLC or UPLC® instruments, thanks to
their very small internal volumes. A particular benefit is the very fast analysis, reduced sample-preparation and
the low column backpressure. Ultra-high performance and extremely low operating pressure make Chromolith®
2 mm columns truly unique.
Column: Chromolith® FastGradient RP-18 endcapped 50-2 mm

Mobile A: ACN + 0.1 % HCOOH
11 phase: B: Water + 0.1 % HCOOH
Gradient:
Time %A %B
Excellent, ultra-fast 1.0 2
0 15 85
results are obtained, 4.5 70 30
not only in UHPLC and 0.8
6 70 30
UPLC® instruments, Flow rate: 0.5 mL/min
but equally well in
1
Pressure: 55 – 85 bar
all standard HPLC 3
Intensity x105
0.6 12
7 Detection: MS; Ion Source: ESI; Ion Trap
systems with low dead Sample: 1. Metabolite of Fluoxymesterone 353 m/z
volume. Chromolith® 0.4 4 6 10 2. Metabolite of Stanozolol 345 m/z
2 mm columns 8 3. Metabolite of Danazol 343 m/z
5 9 4. Testosterone 289 m/z
have macropores of
5. Epitestosterone 289 m/z
1.5 μm in diameter, 0.2
6. Metabolite of Methyltestosterone 271 m/z
resulting in a column 7. Metabolite of Calusterone 285 m/z
efficiency that exceeds 0 8. Metabolite of Clostebol 305 m/z
100,000 plates/meter. 0 1 2 3 4 5 9. Boldenone-acetate 329 m/z
Retention time (min)
10. Testosterone-acetate 331 m/z
11. Nandrolone-17-Propionate 331 m/z
12. Testosterone-Propionate 345 m/z
Pore Surface
Phase USP Bonding Silica Macropore Mesopore volume Area Carbon pH Max
Bonding Designation Chemistry Type Size (µm) Size (Å) (mL/g) (m2/g) Load (%) Stability Temperature Endcapped
RP-18 L1 Octadecylsi- Monolithic 2 130 1 300 18 2 - 7.5 50 Yes
endcapped lane Type B [2 mm I.D.
silica Columns:
1.5 µm]
High L1 Octadecylsi- Monolithic 1.15 150 1 250 18 1.5 - 8 50 Yes
Resolution lane Type B
RP-18 silica
endcapped
RP-8 L7 Octylsilane Monolithic 2 130 1 300 11 2 - 7.5 50 Yes
endcapped Type B
silica
High L7 Octylsilane Monolithic 1.15 150 1 300 11 1.5 - 8 50 Yes
Resolution Type B
RP-8 silica
endcapped
CN L10 Cyanosilane Monolithic 2 130 1 300 6 2 - 7.5 50 No
Type B
silica
Diol L20 Diolsilane Monolithic 2 130 1 300 9 2 - 7.5 50 No
Type B
silica
NH2 L8 Aminopropyl- Monolithic 2 130 1 300 5 2 - 7.5 50 No
silane Type B
silica
Si L3 unbonded Monolithic 2 130 1 300 n.a. 2 - 7.5 50 No
Type B
silica
Chromolith HPLC Columns

Chromolith® HighResolution (HR) columns have around 50% higher 150,000
Number of theoretical
plates per meter
efficiency, which is approximately equivalent to 2.6 µm particulate columns,
whereas the backpressure is approximately equivalent to 5 µm columns, Back pressure
excellent peak symmetry and still more than 30 % longer lifetime compared (bar) at 100,000
1 mL/min flow
with particulate columns. Two Chromolith® HighResolution columns rate of
Column lifetime
compared to a
could be easily coupled in order to achieve even higher resolution. The Acetonitrile/ 50,000 particulate
Water
completely endcapped stationary phase enables peak-tailing free elution of (50/50, v/v)
column
basic compounds. 0
10
1x
Excellent batch-to-batch reproducibility 20
The batch-to-batch reproducibility of Chromolith® HPLC columns is strictly 30 2x
controlled and fulfills the requirements of QA and QC laboratories.

Chromolith® Chromolith® HighResolution
Chromolith® Performance RP-18e 100-4.6 mm

5
Mobile phase: Acetonitrile / water 60/40
4 Flow rate: 2 mL/min

2
3 Detection: UV 254 nm
N/m 101.300
Temperature: ambient
TUSP 1.49
1 Injection volume: 5 µL
Pressure 21 bar
Sample: 1. Urea
2. Biphenyl-2-ol
3. Progesterone
4. Hexanophenon
0 2 4 6 8 5. Anthracene
Time (min)
Chromolith® HR RP-18e 100-4.6 mm
4
N/m 165.900
2 3
TUSP 1.25
1 Pressure 71 bar
0 2 4 6 8
Time (min)
Chromolith® HighResolution RP-18e 100-4.6 mm

Column: Chromolith® HighResolution RP-18 endcapped
100-4.6 mm
600
Mobile phase: A: A
cetonitrile + 0.1 % TFA
B: Water + 0.1 % TFA
500
Gradient: 2 min 0% A
10 min 30% A
400 Flow rate: 1 mL/min

(mAU)
Temperature: 25°C
300
Injection volume: 2 µL
Sample: 1. Norepinephrine
200
2. Octopamine
3. Epinephrine tartrate
4. Dopamine
100
5. DOPA
Batch 1 6. Norephedrine
Batch 2 7. Ephedrine
0 Batch 3 8. N-Methylephedrine
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
Time (min)
51
Chromolith® Semi-preparative and Preparative HPLC Columns
Chromolith® SemiPrep 10 mm Chromolith® Prep columns

I.D. columns Preparative HPLC involves much higher sample volumes
Combine high separation speed with excellent than analytical chromatography. Consequently, greater
performance. These traits make them the perfect sample throughput and separation speed are essential
alternative to particulate columns of 10 mm I.D. (and for optimal productivity. These criteria are best fulfilled
even 21.2 mm I.D.). These columns have the same by Chromolith® Prep columns. The combination of
bimodal porous silica rod structure as Chromolith® macro and mesopores maximizes separation efficiency
analytical columns with an internal diameter of 4.6 mm. and flow rate, while minimizing resistance.
Their macropores are 2 μm in diameter and the
mesopores are 13 nm. This combination dramatically
reduces separation time while increasing efficiency.
Ready-to-use Chromolith® Prep column
Column dimension
Length I.D.
(mm) (mm) RP-18e HR RP-18e RP-8e HR RP-8e CN Diol NH2 Si
25 x 4.6 1.51463.0001 1.52020.0001 1.52046.0001 1.53170.0001 1.52026.0001
25 x 3 1.52003.0001
25 x 2 1.52014.0001 1.52320.0001
50 x 4.6 1.51450.0001 1.52021.0001 1.52047.0001 1.53171.0001 1.52027.0001
50 x 3 1.52002.0001
50 x 2 1.52007.0001 1.52321.0001
100 x 4.6 1.02129.0001 1.52022.0001 1.51468.0001 1.52064.0001 1.52048.0001 1.53172.0001 1.52028.0001 1.51465.0001
100 x 3 1.52001.0001
100 x 2 1.52006.0001 1.52322.0001
150 x 4.6 1.52023.0001
100 x 10 1.52016.0001 1.52015.0001
100 x 25 1.25252.0001 1.25251.0001
Validation Kits [3 Chromolith® HPLC cartridges from 3 different sorbent batches]

50 x 2 1.52062.0001
100 x 4.6 1.51466.0001 1.52019.0001
100 x 3 1.52063.0001
10 x 10 1.52036.0001 1.52035.0001
Chromolith® Guard cartridges [3 units]

5 x 4.6 1.51451.0001 1.52025.0001 1.52013.0001 1.52050.0001 1.53175.0001 1.52030.0001 1.52011.0001
10 x 4.6 1.51452.0001
5 x 3 1.52005.0001
5 x 2 1.52009.0001 1.52325.0001
Chromolith® Guard cartridges Set [1 starter kit with holder and 3 guard cartridges]
5 x 4.6 1.52008.0001
5 x 3 1.52004.0001
Validation kits are available

Chromolith® Guard Column Holder
Chromolith® HPLC guard cartridges are extremely Guard cartridge holders

easy to use. The guard cartridges are simply added
Depending on your needs, we offer several different
directly in front of the main column to protect it from
guard cartridge holders: made out of PEEK for 2 and
chemical or mechanical contamination. Due to the
3 mm I.D. cartridges; bioinert PEEK lined stainless
benefits of monolithic technology, and the convenience
steel holder and standard stainless steel holder for
of Chromolith® guard columns, they are also popular
4.6 mm I.D. cartridges and holders for 10 and 25 mm
for use with classical particulate columns. Moreover,
I.D. cartridges.
guard columns can be used as trap columns when
large sample volumes are to be injected. Guard
columns should be changed frequently in order to avoid
excessive accumulation of impurities.
a) b) c) d)
Guard cartridge Material holder is Guard cartridge Guard cartridge

holder type made of Max. back pressure How to tighten holder I.D. length
a) PEEK 200 bar (2,940 psi) Finger-tight 2, 3 mm 5 mm
b) PEEK lined SS 400 bar (5,880 psi) Finger-tight + tool (not included) 4.6 mm 5 mm
c) SS 400 bar (5,880 psi) Finger-tight + tool (not included) 4.6 mm 5 mm, 10 mm
d) PEEK / SS 150 bar (2,205 psi) Finger-tight + tool (not included) 10 mm 10 mm
Chromolith® Guard cartidge holder
for dimension Type Material Item No.
5 x 2 and 3 a PEEK 1.52004.0001
5 x 4.6 b Bioinert 1.52255.0001
5 x 4.6 c SST 1.52032.0001
10 x 4.6 c SST 1.52033.0001
10 x 10 d PEEK/SST 1.52037.0001
Chromolith® Column Coupler

Make your column longer for extra high resolution
The Chromolith® HPLC column coupler is designed
for linking several monolithic columns together in
order to further increase separation efficiency and Ordering Information
column performance. The combination results in a Chromolith® Column coupler
theoretical plate count that is significantly higher than
for analytical columns 1.51467.0001
any particulate column available. At the same time,
for 25 mm I.D. columns 1.25259.0001
pressure is kept well below the HPLC system limit.
53
Chromolith® WP 300
Monolithic HPLC Columns for Biomolecule Separation
Biotherapeutics, for example bio-engineered drugs and Features and Benefits
peptide therapeutics, represent the promise of new
• Completely bioinert column hardware
medical treatments for the future. Production costs
have been falling, leading to an extremely high demand • High biorecovery
for suitable analytical methods for process monitoring • Selectivity for a range of biomolecules
and quality control of these biomolecules. HPLC is the • Very low column backpressure
preferred method of analysis, and it is important to use
the right column for these larger molecules. • High-speed separation possible
• Longer column lifetime
Accurate analysis of proteins, antibodies and large
• High resistance to column blockage
peptides requires columns with good permeability,
along with better mass transfer and selectivity. In order • Cost savings from higher sample throughput and
for size-exclusion not to influence the separation, the column durability
pore size should be approximately ten-times larger than • Possibility to use flow gradients
the molecule being analyzed.
In contrast to conventional packed-particle columns, Fast chromatography with low column
wide pore (300 Å) monolithic silica columns are made back pressure
of a single continuous-bed rod of high purity porous
silica that is then bonded with C18, C8, C4, epoxy and
Protein A depending on the use of the column.
Monolithic columns remove backpressure as the
primary consideration in method development 1 mL/min
and allow:
• Flow rate flexibility for much higher throughput
• Choice of column lengths for superior resolution
• More solvent options for optimum selectivity
With no individual particles to shift or break, column
performance is consistent over a much longer lifetime,
making them ideal for relatively ”dirty or matrix-rich” 9 mL/min
sample analysis. High permeability also makes them
0 6 12 Retention time [min]
very forgiving of less rigorously prepared samples, in
addition to making it easier to aggressively flush out for
re-equilibration.
Owing to the very high porosity of the Chromolith®
WP 300 column, very high flow rates can be applied
with very low pressures as seen above.
A mixture of five compounds demonstrates the extreme
time savings and high separation efficiency made
possible with Chromolith® WP 300 columns. Due to
excellent mass transfer properties of the monolithic
skeleton, high-speed separation is possible even at high
flow rate.
Chromolith® WP 300

Chromolith® WP 300 Protein A –
Fast monoclonal antibody quantitation
Affinity chromatography is a selective technique which antibody for the optimal time for harvest of the
takes advantage of specific molecular interactions, for monoclonal antibody products. Chromolith® WP 300
example antigen and antibody. The Chromolith® WP Protein A column could be used for separation of all
300 Protein A HPLC column is designed to monitor IgGs (except class 3). Columns provide extremely
monoclonal antibody titer and yield determination fast separations and could be used longer minimizing
from cell-culture supernatants. The analytical scale overall analysis costs.
procedure helps to monitor the titer of monoclonal
Monoclonal Antibody Analysis Linearity

5000
Eluent A 100 mM sodium phosphate pH 7.4
4500
Eluent B 100 mM sodium phosphate pH 2.5 200 μg
4000
Flow rate 2.0 mL/min 100 μg
Absorption (mAU)
75 μg
3500
Detection 280 nm 50 μg
3000 40 μg
Temperature 25 °C
25 μg
Injection volume 10 µL 2500 20 μg
18.75 μg
Gradient Time %A %B 2000
12.5 μg
0.00 100 0 1500 10 μg
0.25 100 0 1000

6.25 μg
5 μg
0.26 0 100
500 2.5 μg
1.25 0 100 1.25 μg
1.26 100 0
0.5 0.75 1 1.25 1.5 1.75 2
2.50 100 0 Time (min)
160
140
Area (mAu x min)
120
100 R2 = 0.9991
80
60
40
20
0
0 50 100 150 200 250
0.00 0.50 1.00 1.50 2.00 2.50 Injected mass IgG (µg)
Constant binding efficiency at any flow rate

• High-speed separation at high flow rate due
to excellent mass transfer properties of the
monolithic skeleton
• Separation of IgG demonstrates the extreme time
savings and high separation efficiency made possible
with Chromolith® Protein A columns.
• IgG was well separated with excellent peak symmetry
• At 5 mL/min, the total analysis time is less than
1 minute and the net column backpressure is only 21 bar
Parameter RSD
• Antibody binding is not affected by flow rate
Retention time < 0.1%
• As depicted at right, antibody capture and release is
very reproducible across 50 injections Peak Height < 0.9%
Peak area < 0.5%
55
Chromolith® WP 300 Epoxy
Create your own column selectivity on demand
Chromolith® WP 300 Epoxy columns are specially Potential applications: Attach trypsin to obtain HPLC
designed for the user-specific immobilization of ligands column-protein digestion reactor, attach a protein for
and their later application in HPLC. The unique bimodal protein-protein interaction analysis, attach any chiral
pore structure of silica monoliths allows efficient selector to obtain a chiral column, or attach any affinity
coupling independent of molecule size. The wider ligand for a custom made affinity column, among
mesopores also enable the use of both proteins and other options.
antibodies as ligands immobilized on the column, and
The Chromolith® WP 300 Epoxy column is shipped in
as analytes separated by an immobilized column.
100% 2-Propanol. The column has to be washed with
20 CV deionized water before immobilization.
Immobilization via epoxide functions

Step I & III Pump
Step I - Equilibration
Column equilibration with 50 mL 50 mM sodium
phosphate + 1.9 M ammonium sulfate pH 8.0
2.0 mL/min flow rate at room temperature
Step II - Immobilization Chromolith® Epoxy column
Dissolving of ligand in 25 mL 50 mM sodium phosphate
Waste
+ 1.9 M ammonium sulfate pH 8.0
Connection of ligand solution to pump
Step II Pump
Immobilization in cycles with 0.2 mL/min flow rate at
room temperature for 4 – 24 h
Step III - Quenching & washing
Quenching of remaining epoxide functions with 150 mM
phosphoric acid or 1 M glycine (optional) Chromolith® Epoxy column
Washing of the immobilized column with 100 mM

sodium phosphate pH 7.4
After immobilization, the column is ready to use for (DMSO). However pure DMSO can be used as solvent
the desired purpose of the immobilized ligand. The for samples. Buffers, organic modifiers and ion
type of required solvent or buffers depends on the pair reagents present no problems as long as the
type of ligand used. Chromolith® WP Epoxy columns appropriate pH range is not exceeded. Nevertheless, be
can be used with all commonly used HPLC grade careful not to expose the column to conditions which
organic solvents, with the following restrictions. The could cause denaturation of the ligand.
mobile phase should NOT contain more than 50%
Tetrahydrofuran (THF), 5% Chlorinated solvent
(e.g. Dichloromethane) or 5% Dimethylsulfoxide
Chromolith® WP 300 Epoxy

Affinity Column created by immobilization of concanavalin A onto Chromolith® Epoxy
Preparing the Column:

Immobilization according to Epoxy method using a Chromolith® WP 300 Epoxy 100-4.6 mm column with 50 mg
concanavalin A from Jack bean dissolved in 25 mL 50 mM disodium hydrogen phosphate, 1 mM calcium chloride
+ 1.9 M ammonium sulfate, pH 8.0. Immobilization for 4 hours at 0.2 mL/min and quenching of remaining
epoxide functions with glycine.
Chromatographic conditions
Eluent A: 50 mM sodium acetate, 200 mM sodium

chloride, 1 mM calcium chloride pH 5.3
Ribonuclease
Eluent B: Eluent A + 100 mM Methyl-
Horseradish Peroxidase (HRP) α-D-mannopyranoside
Detection: 214 nm
Temperature: 25 °C
Gradient: Time %A %B
0 100 0
0 1 2 3 4 5 1 100 0
1.25 0 100
Time (min) 3.5 0 100
3.6 100 0
5 100 0
Protein G Chromatography 300
Eluent A: 100 mM sodium phosphate pH 7.4
R2 = 0.99935
250
Eluent B: 100 mM sodium phosphate pH 2.5
Peak area (mAU x min)
200
150
Detection: 280 nm
Rabbit IgG 100
Temperature: 25 °C
Human IgG 50
0
0 100 200 300 400
Gradient: Time %A %B
IgG load (µg) 0 100 0
0.5 0 100
0.6 0 100
1.2 100 0
0 1 2
Time (min)
Separation of racemic thalidomide
Column: Chromolith® Epoxy immobilized with vancomycin

Eluent: 10 mM disodium hydrogen phosphate, pH 7.0
Flow: 1.0 mL/min
Temperature: 25 °C
Detection: 214 nm
0 1 2 3 4 5
Time [min] Injection: 1.0 µL 1 mM thalidomide in acetonitrile
57
Chromolith® WP 300 HPLC Columns
Chromolith® WP 300 RP-18, RP-8 and RP-4: reversed-phase HPLC columns for bioapplications
Reversed-phase chromatography is often used for protein and peptide separations. The longer octadecyl (C18)
chains can efficiently separate complex peptide mixtures; shorter C8 modified columns are used for small, less
hydrophobic proteins; C4 is mainly applied for separation of hydrophobic proteins.
Separation of five peptides using Chromolith® WP 300 RP-18

1. GLY-TYR
1 2. VAL-TYR-VAL
3. MET-ENKEPHALIN
Column: Chromolith® WP 300 RP18 100 x 4.6 mm 4. LEU-ENKEPHALIN
Mobile phase: A: Water 0.1% TFA 5. Angiotensin II
B: Acetonitrile 0.1% TFA
2
Flow rate: 1.0 mL/min 3 5
4
Detection: 220 nm
Temperature: 60 °C
0 2 4 6 8 10 12 14
Injection volume: 1.0 µL
Time [min]
Sample: HPLC peptide standard mixture (Sigma, H2016)
Chromolith® WP 300 Specifications

Pore Surface
Phase USP Bonding Macropore Mesopore volume Area Carbon pH Max
Bonding Designation Chemistry Silica Type Size (µm) Size (Å) (mL/g) (m2/g) Load (%) Stability Temperature Endcapped
RP-18 L1 Octadecylsilane Monolithic 2 130 1 300 8-9 2 - 7.5 60 No
Type B silica
RP-8 L7 Octylsilane Monolithic 2 130 1 300 4-5 2 - 7.5 60 No
Type B silica
RP-4 L26 Butylsilane Monolithic 2 130 1 300 3-4 2 - 7.5 60 No
Type B silica
Protein A Protein A Monolithic 2 130 1 300 n.a. 2 - 7.5 45 No
Type B silica
Epoxy Glycidoxipropylsilane Monolithic 2 130 1 300 5-6 2 - 7.5 60 No
Type B silica
Column dimension
Length (mm) I.D. (mm) RP-18 RP-8 RP-4 Protein A Epoxy
Chromolith® WP 300 HPLC Column [1 unit]
25 x 4.6 1.52258.0001 1.52252.0001
25 x 2 1.52358.0001 1.52352.0001
50 x 4.6 1.52271.0001 1.52266.0001 1.52261.0001 1.52251.0001
50 x 2 1.52371.0001 1.52351.0001
100 x 4.6 1.52270.0001 1.52265.0001 1.52260.0001 1.52250.0001
100 x 2 1.52370.0001 1.52350.0001
Chromolith Guard cartridges [3 units]
®
5 x 4.6 1.52273.0001 1.52268.0001 1.52263.0001 1.52254.0001

5 x 2 1.52372.0001 1.52353.0001
10 x 4.6 1.52272.0001 1.52267.0001 1.52262.0001 1.52253.0001
Chromolith® Guard cartridges Holder
5 x 4.6 1.52032.0001
10 x 4.6 1.52033.0001
Chromolith® Column coupler
1.51467.0001

Chromolith® CapRod® Capillary Columns
A Chromolith® CapRod® capillary column combines the with various nano or capillary-LC systems. This trait
speed of monolithic silica technology with the sensitivity provides the highest efficiency and performance when
of nano-LC. These traits enable superior productivity coupled to mass spectrometers, both on-line (ESI,
for high throughput, highly sensitive proteomics- nanospray) and off-line (MALDI). Compared to classical
LC applications. Compared to particulate capillary micro-particulate sorbents, Chromolith® CapRod®
columns, Chromolith® CapRod® capillaries demonstrate columns can be operated at higher flow rates—without
better performance with optimal resolution (narrow loss of performance, resolution, or limitations due to
peak widths), increased productivity (higher column back pressure. Separations can be achieved
sample throughput), and extended column lifetime. at 1–3 μL/min, compared to 200–400 nL/min for
Furthermore, column length is less limited than with conventional media on a standard 100 μm LC capillary
any other type of column. The capillaries can even be column. For more complex, biological samples, a
slightly bent to fit any LC configuration or instrument. trapping capillary can be used to protect the separation
Chromolith® CapRod® columns are designed to work column and optimize separation efficiency.
Recommended use and flow rate ranges

RP-18e RP-18e
RP-18e RP-8e 50 x RP-18e RP-18e RP-18e 50 x RP-18e RP-18e
Recommended 150 x 150 x 0.1 mm 150 x 300 x 150 x 0.2 mm 150 x 150 x
use 0.05 mm 0.1 mm Trap 0.1 mm 0.1 mm 0.1 mm HR Trap 0.2 mm 0.2 mm HR
Separation of
small molecules
– of peptides
– of proteins
Micro ESI
Nano ESI
High Resolution
Flow rates
0.2 – 0.8 0.4 – 3 1 – 10 0.4 – 3 0.2 – 1.5 0.1 – 0.4 10 – 50 5 – 20 0.5 – 2
(μL/min)
Max back
200 200 200 200 200 218 218 218 218
pressure (bar)
Column dimension
Length (mm) I.D. (mm) HR RP-18e RP-18e RP-8e
Chromolith®CapRod® HPLC capillary columns [1 unit]
50 x 0.1 Trap 1.50426.0001
50 x 0.2 Trap 1.50409.0001
150 x 0.05 1.50403.0001
150 x 0.1 1.50404.0001 1.50402.0001 1.50400.0001
150 x 0.2 1.50407.0001 1.50405.0001
300 x 0.1 1.50424.0001
59
Fully Porous Particulate Silica (FPP) Columns
High Loadability and scalable from Nano LC to Preparative LC
Fully porous silica particles (FPP) are well established in the chromatographic community over the past several
decades. These columns are in use in thousands of methods and ensure reliable results over the complete range
of use, particle sizes and column dimensions in
• Nano-LC (Capillary columns)
• UHPLC
• Analytical HPLC
• Semipreparative LC
• Preparative LC
Fully porous silica particles provide the full loadability of the stationary phase due to its fully porous physical
characteristics. This trait ensures high sensitivities because the peak broadening effect of overloading the
stationary phase is minimized.
Traditional fully porous silica particles such as LiChrosorb®, LiChrospher®, Superspher® and SUPELCOSIL™ are
based on Type A silica which is produced from sodium waterglass.
The more modern, high purity Type B silica was introduced in the early 1990´s. Type B silica particles are
produced from tetraalkoxysilane in a sol-gel process. This metal-free stationary phase base material can be used
for the analysis of acidic, basic, and chelating compounds providing excellent peak symmetries with less need for
strong buffer concentrations.
Therefore, this type of stationary phase base material is the preferred FPP option for method development,
method improvement and LC-MS use.

FPP Type B stationary phases (RP and HILIC):
UHPLC (pressure stability of 1000 bar): • Discovery® BIO

• Purospher™ STAR – 5 µm particle size
– 2 µm particle size – 300 A pores
– superior peak symmetry – Available modifications: C18, C8, C5
– extended pH stability • SeQuant® ZIC®
– Available modifications: RP-18e (C18), RP-8e (C8), – 3/3.5 µm and 5 µm particle size
Phenyl, Si
– superior separation of polar compounds
• Titan™ – Available modifications: Sulfobetaine (ZIC®-HILIC),
– 1.9 µm particle size Phosphorylcholine (ZIC®-cHILIC)
– Monodisperse silica particles providing very – Additional: polymeric particle (5 µm) with
high efficiency Sulfobetaine (ZIC®-pHILIC) for extended pH stability
– Available modifications: C18
Semi-Preparative/Preparative LC:
Analytical HPLC: • Ascentis® Discovery® and Discovery® BIO
• Purospher™ STAR – 10 µm particle size
– 3 µm and 5 µm particle size – Column dimensions up to 21.2 mm I.D.
– superior peak symmetry • SeQuant® ZIC®
– extended pH stability – 5 µm particle size
– Available modifications: RP-18e (C18), RP-8e (C8), – Column dimensions up to 21.2 mm I.D.
Phenyl, NH2, Si
• Discovery® and Ascentis® Nano LC:
– 3 µm and 5 µm particle size • SeQuant® ZIC®

– broad range of selectivities • Discovery® BIO
– Available modifications: C18, C8, Phenyl,
RP-Amide; F5 (PFP), CN, Si
Validation kits
The success of an HPLC method depends strongly
on the consistent quality of the stationary phase.
Long-term reproducibility is a key factor in achieving
reliable results. Supelco® validation kits consists of
three HPLC columns, packed with three different
sorbent lots to confirm the reliability of HPLC
methods and their robustness.
61
Purospher™ STAR HPLC and UHPLC Columns
Accuracy and precision made simple
1 2
Purospher™ STAR HPLC and UHPLC columns are based
on 99.999% ultra-pure fully porous silica (Type B).
These columns are designed for universal use and allow OH Batch 10
the separation of basic, neutral, and metal chelating NH Batch 9

compounds with simple mobile phases and excellent CH3
CH3 Batch 8
peak symmetry. These columns offer an outstanding
stability from pH 1.5 to 10.5 over a wide temperature Ephedrine (1)
Batch 7
range, and suitability in up to 100% aqueous mobile Batch 6

phases. Plus, the columns demonstrate best all around
Batch 5 HO O
retention characteristics, as proven by the Tanaka
O
test. Thanks to these features, Purospher™ STAR Batch 4
H3C
HPLC columns allow high-throughput applications Batch 3 N
and allow maximum flexibility for use with the best H
Batch 2
chromatographic conditions for any separation including HS
reversed phase. Batch 1 Captopril (2)

0 10 20 min30
Features and Benefits

• Ultra-pure silica (99.999%) for excellent Consistent results
peak symmetry
The success of any method depends on the quality of
• High separation efficiency the stationary phase. Precise, long-term reproducibility
• Reproducible results from run-to-run and is a key factor in achieving reliable results. The base
batch-to-batch silica of Purospher™ STAR columns is 99.999% pure.
• Best overall performance according to Tanaka test Furthermore, meticulous care is given to quality control
all aspects of silica structure and chemistry. These
• Outstanding pH stability from pH 1.5 to 10.5
factors ensure that the columns will always perform
• No column dewetting when using highly aqueous consistently, resulting in method reproducibility you
mobile phases can trust.
• LC-MS compatible
Phase USP Bonding

Purospher™ STAR Bonding Designation Chemistry Chromatographic Properties / Use
O
CH2 CH2 CH2 CH2 CH2 CH2 RP-18 L1 Octadecylsilane Best in class C18 column for excellent peak symmetry, performance and pH
endcapped with polymeric stability. Accurate results with excellent peak shape for all types of analytes •
Si O Si CH2 CH2 CH2 CH2 CH2 CH2 C6H13
OH O
Si O Si CH3
endcapping Outstanding resolution due to high separation efficiency • Proven reliability and
reproducibility from run to run and batch to batch • Universal compatibility with
OH O
CH2 CH2 CH2 CH2 CH2 CH2
Si O Si CH2 CH2 CH2 CH2 CH2 CH2 C6H13
Si O Si
O
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
C6H13
best all around performance according to Tanaka test • Maximum flexibility in
O
method development and choice of mobile phase • pH stability from pH 1.5 –
10.5 • Suitable in up to 100% aqueous mobile phases • Highest sensitivity and
suitability for LC-MS applications
Si O
O
Si CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH24 RP-8 L7 Octylsilane Less hydrophobic compounds, faster retention of very hydrophobic compounds.
OH O
endcapped Excellent peak symmetry for acidic, basic and chelating compounds; Excellent
Si O Si CH3
resolution due to high separation efficiency; Excellent stability from pH 1.5 to
10.5; Enhanced selectivity for positional isomers
OH O
CH2 CH2 CH2 CH2
Si O Si CH2 CH2 CH2 CH2
O
CH2 CH2 CH2 CH21
Si O Si CH2 CH2 CH2 CH2
Phenyl L11 Phenylsilane Enhanced selectivitiy for separation of aromatic compounds due to π-π
interactions. • Low silanol activity • Excellent pH stability from 1.5 to 10.5 •
Suitable in up to 100% aqueous mobile phases
NH2 L8 Amino Separation of carbohydrates and polar compounds with normal-phase or HILIC
NH2 chromatography. Very high separation efficiency as measured by the plate count
• Absence of metal impurities, thus giving consistently symmetrical peaks •
Extended column lifetime
Si L3 unbonded Separation of polar compounds with normal-phase or HILIC chromatography
Si Very high separation efficiency as measured by the plate count • Absence
of metal impurities, thus giving consistently symmetrical peaks • Extended
column lifetime
Purospher™ STAR HPLC and UHPLC Columns

Outstanding stability Efficiency
140000
The combination of extremely high purity 120000
silica, best overall retention characteristics, 100000
outstanding pH stability up to pH 10.5 and 80000
suitability for use with 100 % aqueous mobile 60000
phases makes Purospher™ STAR RP-18 40000
endcapped an all-round top performance 20000
column, almost universal in its range of 0
applications. The present stability test shows 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Amitriptyline Valerophenone o-Nitrophenol No. of injections
the suitability with 100 % aqueous mobile
phase at high temperature at 60 °C.
Retention
12
Column: Purospher™ STAR RP-18 endcapped, 10
3 μm LiChroCART® 55-4
8
Mobile phase: 0.1 v/v% H3PO4 in Water
6
4
Temperature: 60 °C
2
Sample: Amitriptyline 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Valerophenone
Amitriptyline Valerophenone o-Nitrophenol No. of injections
o-Nitrophenol
Particle Pore Size Pore volume Surface Carbon Surface coverage pH Max Shipping
Size (µm) (Å) mL/g Area (m2/g) Load (%) µmol/m2 Stability Temperature Endcapped solvent
2, 3 and 5 120 1.1 330 17 3 1.5 - 10.5 65 °C Yes Acetonitrile/
Water
2, 3 and 5 120 1.1 330 11.2 3 1.5 - 10.5 65 °C Yes Acetonitrile/

Water
2, 3 and 5 120 1.1 330 12.5 3 1.5 - 10.5 65 °C Yes Acetonitrile/

Water
5 120 1.1 330 3.5 3 2 - 7.5 60 °C No Acetonitrile/

Water
5 120 1.1 330 n.a. n.a. 2 - 7.5 65 °C N.A. Acetonitrile/

Water
63
Purospher™ STAR (5 µm)

Column dimension
Length (mm) I.D. (mm) RP-18 endcapped RP-8 endcapped Phenyl NH2 Si
LiChroCART® HPLC Cartridge [1 unit]
100 x 2 1.50623.0001 on request on request on request on request
100 x 4.6 1.50627.0001 on request on request on request on request
125 x 2 1.50255.0001 1.50274.0001 on request on request on request
125 x 4 1.50251.0001 1.50271.0001 on request 1.50244.0001 1.50268.0001
150 x 4.6 1.50358.0001 1.50031.0001 1.51922.0001 1.50247.0001 1.50356.0001
250 x 4 1.50252.0001 1.50272.0001 on request 1.50245.0001 1.50269.0001
250 x 4.6 1.50359.0001 1.50032.0001 1.51921.0001 1.50248.0001 1.50357.0001
Validation Kits [3 LiChroCART® HPLC cartridges from 3 different sorbent batches]
150 x 4.6 1.50358.1003 1.50031.1003 1.51922.1003 on request on request
250 x 4.6 1.50359.1003 1.50032.1003 1.51921.1003 on request on request
Guard cartridges LiChroCART® [10 units]
4 x 4 1.50250.0001 1.50270.0001 on request 1.50267.0001 1.50249.0001
The LiChroCART® columns (75, 100, 125, 150 and 250 mm length) in the list above (2, 3, 4 and 4.6 mm I.D.) require
part number 1.51486.0001 manu-CART® cartridge column holder, which can be used to hold one cartridge column
with or without a 4-4 mm guard column. LiChroCART® columns 250-10 mm require part number 1.51419.0001 manu-
CART® 10. Additional dimensions and validation kit available as customized packings see page 120.
Hibar® RT HPLC Column [1 unit]

100 x 4.6 1.50622.0001 1.51917.0001 on request on request on request
150 x 2 on request on request on request on request on request
150 x 3 1.50617.0001 1.50644.0001 1.51920.0001 on request on request
250 x 4.6 1.51456.0001 1.51454.0001 1.51918.0001 1.51913.0001 1.51911.0001
250 x 10 on request on request on request 1.51912.0001
Validation Kits [3 Hibar® RT HPLC Columns from 3 different sorbent batches]
125 x 4 1.50036.1003 1.50033.1003 on request
150 x 3 1.50617.1003 1.50644.1003 1.51920.1003
150 x 4.6 1.51455.1003 1.51453.1003 1.51919.1003
250 x 4 1.50037.1003 1.50035.1003 on request
240 x 4.6 1.51456.1003 1.51454.1003 1.51918.1003
The Hibar® columns are complete with endittings. When using a guard column with a Hibar® column, we recommend
part number 1.51487.0001 guard column cartridge holder for 4-4 mm guard column cartridges LiChroCART®.
Additional dimensions available as customized packings see page 120.

Purospher™ STAR (3 µm)
Column dimension
Length (mm) I.D. (mm) RP-18 endcapped RP-8 endcapped
LiChroCART® HPLC Cartridge [1 unit; * 3 units] [** One set contains: 1 LiChroCART®
cartridge and one holder]
30 x 2 on request on request
30 x 4 1.50225.0001* on request
55 x 2 1.50241.0001* on request
55 x 2; Set** 1.50240.0001 on request
55 x 4 1.50231.0001* on request
55 x 4; Set** 1.50242.0001 on request
75 x 4 1.51460.0001 on request
The LiChroCART® columns (75, 100, 125, 150 and 250 mm length) in the list above (2, 3, 4
and 4.6 mm I.D.) require part number 1.51486.0001 manu-CART® cartridge column holder,
which can be used to hold one cartridge column with or without a 4-4 mm guard column.
LiChroCART® columns 250-10 mm require part number 1.51419.0001 manu-CART® 10.
Additional dimensions and validation kit available as customized packings see page 120.

50 x 3 1.50393.0001 on request
50 x 4 1.50428.0001 on request
100 x 3 1.50398.0001 on request
100 x 4.6 1.50469.0001 on request
125 x 3 1.50413.0001 on request
125 x 4 1.50431.0001 on request
150 x 3 1.50414.0001 1.50750.0001
150 x 4.6 1.50470.0001 on request
250 x 3 1.50427.0001 on request
250 x 4 1.50468.0001 on request
250 x 4.6 1.50471.0001 on request
125 x 4 1.50431.1003 on request
150 x 3 1.50414.1003 1.50750.1003
150 x 4.6 1.50470.1003 on request
250 x 3 1.50427.1003 on request
250 x 4 1.50468.1003 on request
250 x 4.6 1.50471.1003 on request
The Hibar® columns are complete with endittings. When using a guard column with a Hibar®
column, we recommend part number 1.51487.0001 guard column cartridge holder for 4-4 mm
guard column cartridges LiChroCART®. Additional dimensions available as customized packings
see page 120.
Other Purospher™ Columns (5 µm)

Purospher™ HPLC columns can easily be replaced by the advanced Purospher™ STAR HPLC columns for many
applications. Purospher™ STAR HPLC columns provide an extended pH stability and outstanding peak symmetry for
basic and chelating comounds.
Column dimension
Length (mm) I.D. (mm) RP-18 RP-18 endcapped
LiChroCART® HPLC Cartridge [1 unit]
125 x 3 on request 1.50798.0001
125 x 4 1.50142.0001 1.50168.0001
250 x 3 on request 1.51384.0001
250 x 4 1.50144.0001 1.50169.0001
Guard cartridges LiChroCART [10 units]
®
4 x 4 1.50141.0001 1.50167.0001
The LiChroCART® columns (75, 100, 125, 150 and 250 mm length) in the list above (2, 3, 4 and 4.6 mm I.D.) require
part number 1.51486.0001 manu-CART® cartridge column holder, which can be used to hold one cartridge column with or
without a 4-4 mm guard column. LiChroCART® columns 250-10 mm require part number 1.51419.0001 manu-CART® 10.
Additional dimensions and validation kit available as customized packings see page 120.
Validation kits are available HPLC Columns "on request" are available as Custom Product. Please see page 120/121
65
Perfect peak shape
Accurate results rely on two important chromatographic compounds. This makes Purospher™ STAR RP-18
properties of the stationary phase: resolution and peak endcapped and RP-8 endcapped columns the optimal
shape. With Purospher™ STAR columns, high efficiency choice for pharmaceutical applications, USP methods as
and bonded phase surface coverage produce sharp, well as for general method development.
symmetrical peaks for acidic, basic and chelating
Purospher™ STAR RP-18 endcapped Column L
Intensity [mV]
8.88
Intensity [mV]
35 35
Symmetry factor Symmetry factor
30 Quinizarin 1.07
12.64
30 Quinizarin 1.46
1.55
1.60
9.81
25 25
4.14
4.50
20 20
5.30
14.52
5.70
15 15
10 10
5 5
0 0
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Retention time [min] Retention time [min]
Column X Column I
Intensity [mV] Intensity [mV]

35 Symmetry factor 35 Symmetry factor
Quinizarin 1.36 Quinizarin 1.41
30 30
6.59
Moblie phase: Methanol/Buffer

1.65
25 25 pH 7.0 80/20
1.54

3.38
13.13
7.51
20 20
4.08
5.17
Temprature: 22 °C
14.59
6.46
15 15 Sample: 1. Uracil;
2. Toluene;
10 10 3. Ethylbenzene;
4. Quinizarin;
5 5 5. Amitryptyline
0 0
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Retention time [min] Retention time [min]

Purospher™ STAR UHPLC columns
High resolution at lower column backpressure
Although UHPLC is typically performed with a particle

size smaller than 2 μm, we employ 2 μm particles due
to two important factors. Firstly, column efficiency Efficiency N/m Anthracene Column backpressure (bar)
and backpressure depends on the particle size of the

column material. Secondly, column efficiency is also 225 bar
highly influenced by instrument effects. When UHPLC 209 bar
174080 N/m 175 bar
columns with 1.7 μm, 1.8 μm, 1.9 μm and 2 μm
187620 N/m
particles are compared on the same instrument and 172840 N/m 173260 N/m
under the same conditions, results show no significant
130 bar
difference in efficiency. However, column pressure
varies substantially among the different particle size
materials. For example, a 1.7 μm particulate material
Purospher™ STAR Column W Column R Column A
significantly higher column backpressure, compared to RP-18 (2 µm) (1.7 µm) (1.9 µm) (1.8 µm)
a 2 μm material.
Column dimension: 50-2.1 mm
Mobile phase: Acetonitrile/Water 60/40
Injection: 0.2 µL
Sample: Thiourea; Biphenyl-2-ol; Progesterone;
Hexanophenone; Anthracene
Column temperature: 40°C
Purospher™ STAR RP-18e (5 µm) 1. Acetanilide
Eluents: A. Water, B. Acetonitrile LiChroCART® 150-4.6 2. Acetophenone
Purospher™ STAR RP-18e (2 µm) 3. Propiophenone
UV: 247 nm
Hibar® HR 50-2.1 4. Butyrophenone
Injection volume: 10 µL 5. Benzophenone
6. Valerophenone
Green: Purospher™ STAR RP-18e (5 µm)
7. Hexanophenone
LiChroCART® 150-4.6 mAU
8. Heptanophenone
Gradient: 0 min 45 % B, 800 1
9. Octanophenone
from 45 to 95 % B in 15 min,
from 15.1 to 20 min
reequilibration with 45 % B 700 2

600
Pressure: 105 bar
500 3
Total run time: 20 min 5
Purple: Purospher™ STAR RP-18e (2 µm) 400 4
Hibar® HR 50-2.1 6
300 7
Gradient: 0 min 45 % B, 8 9
from 55 to 100 % B in 0.8 min
from 0.9 to 2 min 200
reequilibration with 55 % B
Flow rate: 1.1 mL/min 100
Pressure: 505 bar
0
Total run time: 2 min
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
minutes
67
Excellent for LC-MS
Intensity
x=10 5
In order to obtain sensitive results with LC-MS, it is Base peak intensity:
essential to avoid trace impurities in the column and 0.8 < 6 x 103
solvents. Purospher™ STAR HPLC and UHPLC columns

0.6
are highly suitably for LC-MS. To ensure low and
stable background signals, it is recommended to wash 0.4
columns with an eluent of isopropanol and 0.1% formic
acid. Displayed here is an extracted ion chromatogram 0.2 Source: ESI (+)
Bruker esquire 3000
of NSAIDs in negative ion mode separated on 301.1
163.2 229.2 269.2
Purospher™ STAR RP-18 endcapped

0.0
100 200 300 400 500 600 700 800 900 1000
[m/z]
Extracted ion chromatograms of NSAIDs in negative ion mode separated on Purospher™ STAR
RP-18 endcapped
Intensity Chromatographic conditions
[x=10 7]
Column: Purospher™ STAR RP-18 endcapped,
6 2 3 µm, LiChroCART® 55-2
5 1
Mobile phase A: 0.1 % Acetic acid in Acetonitrile
4 Mobile phase B: 0.1 % Acetic acid in Water
3
3 Gradient: From 25 % A to 50 % A in 3 min,
then isocratic
2
Flow Rate: 300 µL, without split
1
0
Detection: UV 220 nm, Ion Trap MS
0 2 4 6 8 10 12 Temperature: ambient
Time [min] Injection volume: 1 µL
EIC 209; 253; 507 – All MS
EIC 197; 241; 483 – All MS Sample: 1. Ketoprofen 0.1 µg/µL
EIC 161; 205; 411 – All MS 2. Fenoprofen 0.1 µg/µL
3. Ibuprofen 0.1 µg/µL
Intensity [x=10 7] MS conditions
-MS, 6.4-7.2 min, background subtracted Ionization: ESI(-)

2 1. Ketoprofen
M-H 2M-H
M=254 M-H-CO2 253.0 506.9 Nebulizer: 36 psi
1 209.0
2M-2H+Na Dry gas: 8.5 L/min
529.0
0
2. Fenoprofen -MS, 7.5-8.2 min, background subtracted Dry temperature: 330 °C
2
M=206 197.1 241.0
Smart mode optimization: Target mass 205
1 482.9
505.0 Ion charge control: Target 50,000, max 50 ms
0 -MS, 8.5-9.3 min, background subtracted
3. Ibuprofen Scan mode: Standard/Normal
2 M=206 205.1
Scan range: 50 – 600 m/z
1
161.2 411.0433.2
0
0 100 200 300 400 500 600
[m/z]
Ketoprofen, Fenoprofen and Ibuprofen (100 ng) give ghost-peak-free MS spectra using
LiChrosolv® Acetonitrile hypergrade and Purospher™ STAR RP-18 endcapped columns.

Purospher™ STAR Hibar® HR UHPLC Columns [2 µm and 3 µm particle size]
Column dimension
Length (mm) I.D. (mm) RP-18 e (2 µm) RP-18 e (3 µm) RP-8 e (2 µm) RP-8 e (3 µm) Phenyl (2 µm) Phenyl (3 µm)
Hibar® HR UHPLC Column [1 unit]
30 x 2.1 1.50645.0001 1.50650.0001 on request on request on request on request
50 x 2.1 1.50646.0001 1.50651.0001 1.50630.0001 1.50674.0001 1.51013.0001 on request
100 x 2.1 1.50648.0001 1.50653.0001 1.50629.0001 1.50675.0001 1.51014.0001 1.50673.0001
150 x 2.1 1.50649.0001 1.50654.0001 on request on request on request on request
250 x 2.1 on request 1.50655.0001 on request on request on request on request
Validation Kits [3 Hibar® HR UHPLC Columns from 3 different sorbent batches]
100 x 2.1 1.50648.1003 1.50653.1003 1.50629.1003 1.50675.1003 on request on request
Hibar® HR UHPLC columns are designed for use in UHPLC instruments. The pressure stability is set at 1000 bar.
69
Discovery® HPLC Columns
Alternative selectivities for straightforward method development
The Discovery® HPLC columns are available with a k’ Butylbenzene (60:40 Acetonitrile: Water, 30 0C)
broad range of modifications providing the best suitable
selectivity for many applications. Although designed 0 1 2 3 4 5 6 7
to meet the exacting requirements of pharmaceutical Discovery® C18
analysis and purification, Discovery® columns are
Discovery® RP-AmideC16
also ideal for all application segments requiring
reversed-phase HPLC Discovery® C8
Method development scientists often choose a single Discovery® HS F5
stationary phase for development. If the chosen phase
is not the best chemistry to yield a given separation, Discovery® Cyano
many hours may be spent studying mobile phase
compositions that may or may not yield a suitable Hydrophobic Retention Ranking of Discovery® Reversed-Phases
separation. Screening several stationary phase

chemistries up front during method development
and choosing the best phase for further optimization
can save many precious hours. In addition, the use
of a more effective stationary phase chemistry often
eliminates the need for mobile phase additives that can
greatly complicate separation conditions.
Phase USP
Bonding Designation Bonding Chemistry Chromatographic Properties / Use
C18 L1 Octadecyl Classic reversed-phase selectivity and retention with excellent

peak shape for all compounds. Very high stability and no-bleed
properties for LC-MS applications

HS C18 L1 Octadecyl Non-polar, reversed-phase column with excellent, no bleed LC-

MS performance. Higher hydrophobicity for better resolution of
difficult analytes.

C8 L7 Octyl Less hydrophobic reversed-phase selectivity and retention with

excellent peak shape for all compounds. Very high stability and no-
bleed properties for LC-MS applications

Cyano L10 Cyanopropyl Cyanopropyl reversed-phase column with lower hydrophobicity than

C18 or C8 and unique selectivity. Excellent peak shape, significantly
less retention than C18 (typically requires lower % organic mobile

phase) and high stability with mobile phase). Low-bleed for
LC-MS separations

RP-Amide L60 Palmitamidopropyl Polar-embedded, palmitamidopropyl reversed-phase column

C16 with unique retention and selectivity. Excellent peak shape and

efficiency. Due to the nature of the bonded phase, we do not
recommend the RP-AmideC16 be used for LC-MS applications

HS F5 L43 Pentafluorophenylpropyl Pentafluorophenyl terminated reversed-phase column with unique

(PFP) retention and selectivity (e.g. basic & halogenated compounds).

The Discovery® HS F5 bonded phase provides reversed-phase
separations that are distinctly different from C18 columns.
However, compounds will generally elute within the same retention
time window, making most C18 methods easily transferable.

Excellent peak shape and stable, low-bleed LC-MS separations

As a visual representation of how the
Discovery® C18
different phase chemistries give different
selectivity, the chart at right shows the Discovery® RP-AmideC16
k’ of various analytes relative to toluene on
Discovery® HS F5
Discovery® columns.
The polar functional group-containing solutes Discovery® Cyano
- aniline, phenol, N,N-dimethylaniline 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9
(N,N-DMA) and ethylbenzoate - clearly k’ compound / k’ toluene
illustrates the very different selectivities of
Aniline
the functionalized reversed-phases vs. C18.
Phenol
The colors representing solutes containing
Toluene
polar groups dramatically change positions
N,N-Dimethylaniline
from phase to phase. Also, observe the
Ethylbenzoate
changing hydrophobic selectivity by looking at
Ethylbenzene
the ethylbenzene bar.
Mobile phase: 45:55, 25mM Potassium Phosphate (pH 7.0): MeOH

Flow Rate: 1.0 mL/min
Particle Pore Surface Area Carbon Load Surface Coverage Max Shipping
Size (µm) Size (Å) (m2/g) (%) (µmol/m2) pH Stability Temperature Endcapped Eluent
5 180 200 12 3 2-8 70 °C No 34% Water:
66%
Methanol
3, 5, 10 120 300 20 3.8 2-8 70 °C Yes 30% Water:

70%
Methanol
5 180 200 7.5 3.4 2-8 70 °C Yes 100%

Methanol
5 180 200 4.5 3.5 2-8 70 °C Yes Acetonitrile
5 180 200 12 2.6 2-8 70 °C Yes Acetonitrile
3, 5, 10 120 300 12 4 2-8 70 °C Yes Acetonitrile
71
Discovery® (5 µm)
Column dimension
Length (mm) I.D. (mm) HS C18 C18 C8 RP-Amide C16 HS F5 (PFP) Cyano
20 x 2.1 on request 577507-U 577501-U on request on request on request
30 x 2.1 on request on request on request on request on request on request
50 x 2.1 568500-U 50494721 on request on request 567508-U on request
100 x 2.1 568501-U 569220-U on request 569320-U 567510-U on request
125 x 2.1 on request 569229-U on request on request on request on request
150 x 2.1 568502-U 50495521 59353-U21 50501321 567511-U on request
250 x 2.1 568503-U on request on request on request 567512-U on request
20 x 3 on request on request on request on request on request on request
100 x 3 on request on request on request on request on request 569522-U
50 x 4 568510-U on request on request on request on request on request
100 x 4 on request 569222-U on request on request on request on request
125 x 4 on request 569231-U 569426-U on request on request 569526-U
150 x 4 568512-U on request on request on request 567535-U on request
250 x 4 568513-U on request on request on request 567536-U on request
50 x 4.6 568520-U 504947 59352-U 505005 567513-U on request
100 x 4.6 568521-U 569223-U on request on request 567515-U on request
125 x 4.6 on request 569232-U 569427-U on request on request on request
150 x 4.6 568522-U 504955 59353-U 505013 567516-U 59356-U
250 x 4.6 568523-U 504971 59354-U 505064 567517-U 59357-U
250 x 10 568533-U 569224-U on request on request 567520-U on request
250 x 21.2 568543-U 569226-U on request on request 567523-U on request
Discovery® Validation Packs (3 columns, each from a different lof of bonded phase)
Discovery® Supelguard™ Guard Cartridge (2 pack)
20 x 2.1 on request 505188 59588-U on request 567574-U on request
20 x 3 on request 59576-U on request 59578-U on request on request
20 x 4 568572-U 505137 on request 505099 567576-U 59586-U
20 x 2.1 on request 505161 on request on request 567575-U on request
20 x 3 on request 59575-U on request on request on request on request
20 x 4 568573-U 505129 59589-U 505080 567577-U on request
on request on request on request on request on request on request

Discovery® (3 µm) Discovery® (10 µm)
Column dimension Column dimension
Length (mm) I.D. (mm) HS C18 HS F5 (PFP) Length (mm) I.D. (mm) HS C18
30 x 2.1 on request 567501-U 50 x 10 on request
50 x 2.1 569253-U 567500-U 100 x 10 on request
75 x 2.1 569254-U on request 150 x 10 on request
100 x 2.1 on request 567502-U 250 x 10 on request
150 x 2.1 569255-U 567503-U 50 x 21.2 on request
30 x 3 on request on request 100 x 21.2 on request
150 x 3 on request 567542-U 150 x 21.2 on request
50 x 4 on request 567530-U 250 x 21.2 568643-U
75 x 4 on request on request
100 x 4 on request 567531-U
150 x 4 on request 567532-U
50 x 4.6 569250-U 567504-U
75 x 4.6 569251-U
100 x 4.6 on request 567506-U
150 x 4.6 569252-U 567507-U
20 x 2.1 569276-U 567570-U
20 x 4 569274-U 567572-U
Discovery® Supelguard™ Guard Kit
(Guard cartridge, stand-alone holder, tubing, 2 nuts and ferrules)
20 x 2.1 on request 567571-U
20 x 4 569275-U 567573-U
73
Discovery® BIO
Reversed-Phase Solutions to Protein and Peptide Separation Challenges
Discovery® BIO Wide Pore reversed-phase columns Features and Benefits

satisfy the need for efficiency, selectivity, LC-MS
• Better protein and peptide resolution compared to
sensitivity, stability, scalability, and reproducibility for
leading RP-HPLC FPP columns
reversed-phase HPLC analyses of proteins, peptides,
and small biomolecules. Three phase chemistries, • High efficiency for peptide mapping
C18, C8, and C5, give unmatched selectivity and • Complementary selectivity choices with C5, C8, and
performance. Separations are completely scalable C18 phase chemistries
from analytical to preparative column dimensions. The • C5 has enhanced stability and lifetime compared to
low-bleed, inert surface chemistry make them ideal for conventional C4 phases
proteomics and LC-MS applications.
• Excellent LC-MS properties
• Reliable reproducibility run-to-run, column-to column,
batch-to-batch
Choosing a Discovery® BIO Wide Pore Reversed Phase for Sample and Separation Modes
Sample or Usage Separation Mode Discovery® BIO Product
Peptide Mapping / Proteolytic Digests Reversed-phase Discovery® BIO Wide Pore C18
Discovery® BIO Wide Pore C8
Hydrophobic Peptides Reversed-phase Discovery® BIO Wide Pore C5
Proteins Reversed-phase Discovery® BIO Wide Pore C5
Discovery® BIO Wide Pore HPLC columns are packed with C5, C8, or C18 ligands bonded to 3, 5, or 10 μm,
spherical, high purity silica particles containing 300 Å pores. All Discovery® BIO Wide Pore products provide stable,
efficient, and reproducible separations of proteins and peptides. The low-bleed character and excellent peak shape
without TFA in the mobile phase makes these columns ideal for proteomics and other LC-MS applications and
preparative purifications.
Phase Particle Size Pore Surface Area Carbon Surface Coverage pH Max
Bonding (µm) Size (Å) (m2/g) Load (%) (µmol/m2) Stability Temperature Endcapped Shipping Eluent
C18 3, 5, and 10 300 100 9 3.6 2 to 8 70 °C Yes Acetonitrile/Water
C8 5 and 10 300 100 5 4 2 to 8 70 °C Yes Acetonitrile/Water
C5 3, 5, and 10 300 100 3.5 4.5 2 to 8 70 °C Yes Acetonitrile/Water

Tryptic Digest of Carboxymethylated Apohemoglobin on a
Discovery® Wide Pore C18 versus Competitive Columns
Discovery® BIO Wide Pore C18 Stability at pH 11.5
Columns: ( A) Discovery® BIO Wide Pore C18, 15 cm x 4.6 mm, 5 μm
(568222-U), (B) and (C) Competitive protein and peptide
Column: Discovery® BIO Wide Pore C18, C18, 15 cm x 4.6 mm, 300 Å, 5 μm;
5 cm x 2.1 mm I.D., 3 μm (567200-U) Mobile phase: (A) 95:5, (0.1% (v/v) TFA in water):(0.1% (v/v) TFA in
acetonitrile); (B) 50:50, (0.1% (v/v) TFA in water):(0.1%
Mobile phase: 65:35, 50 mM pyrrolidine HCl, (v/v) TFA in acetonitrile)
pH 11.5: acetonitrile
Gradient: 0-100% B in 65 min
Column temp.: 35 °C Flow rate: 1.0 mL/min
Injection: 50 μL
Sample: Carboxymethylated apohemoglobin tryptic digest, varied

concentration, 50 mM aqueous ammonium bicarbonate
(A) Discovery® BIO

Stable retention on Discovery BIO Wide Pore C18 Wide Pore C18
after 40,000 column volumes at pH 11.5.
74 peptides
resolved
0 20 40 60
Min
(B) Competitor A
Note: Stability was measured using small molecule probes 61 peptides

because they are generally more sensitive to changes in the resolved
silica and bonded phase chemistry than peptides and proteins. If
the retention and selectivity for the small molecule probes does
not change, it is very likely that the retention and selectivity for
proteins and peptides will be stable as well. 0 20 40 60
Min
(C) Competitor B
68 peptides
resolved
0 20 40 60
Min
Ordering Information Note: The absolute number of peptides detected depends on the detector settings.
In this comparison, the relative number of detected peptides is important, not
the absolute number. The Discovery® BIO Wide Pore C18 column detected more
Discovery® BIO (3.0 µm) peptides relative to the competitive columns under the same conditions.
Length (mm) I.D. (mm) C18 C5
50 x 1.0 on request 65511-U
Discovery® BIO (5.0 µm)
50 x 2.1 567200-U 567226-U
Length (mm) I.D. (mm) C18 C8 C5
100 x 2.1 567201-U 567227-U
50 x 2.1 on request on request on request
150 x 2.1 567202-U 567228-U
50 x 4.6 on request 567229-U 100 x 2.1 on request on request on request
100 x 4.6 567204-U 567230-U 150 x 2.1 on request on request 568402-U
150 x 4.6 567205-U 567231-U 250 x 2.1 568203-U on request on request
Guard 2 pk x 2.1 567270-U 567278-U 250 x 4.0 568213-U on request on request
Guard 2 pk x 4.0 on request 567280-U 50 x 4.6 on request on request 568420-U
Guard Kit x 2.1 567271-U on request 100 x 4.6 on request on request 568421-U
Guard Kit x 4.0 on request 567281-U 150 x 4.6 568222-U on request 568422-U
250 x 4.6 568223-U 568323-U 568423-U
Discovery BIO (10.0 µm)

® 250 x 10.0 568230-U on request 568430-U
250 x 21.2 on request 567225-U on request
Length (mm) I.D. (mm) C18 C8 C5
Guard 2 pk x 2.1 on request on request on request
250 x 4.6 on request on request 567232-U
Guard 2 pk x 4.0 568272-U on request 568472-U
50 x 10.0 567207-U on request on request
Guard Kit x 2.1 568271-U on request on request
150 x 10.0 567208-U on request 567234-U
Guard Kit x 4.0 568273-U on request on request
250 x 10.0 567209-U on request 567235-U
150 x 21.2 567211-U on request on request
250 x 21.2 567212-U 567225-U on request
75
Ascentis® HPLC Columns
Ascentis® HPLC Columns are optimized to the three terms of the resolution equation: efficiency, retention and
selectivity. Ascentis® bonded phases have a wide range of selectivities. It is likely that one or more Ascentis®
phase will accomplish any small molecule HPLC separation. Packed in micro- to preparative hardware dimensions,
Ascentis® products cover all HPLC application areas, including the most sensitive trace-level analyses.
The general features of the Ascentis® family include:

• High purity, type B silica for inertness, reproducibility and stability
• Modern bonding processes that optimize bonded phase coverage and maximize stability, while minimizing bleed
and unwanted secondary interactions
• Wide selection of bonded phase chemistries and bare silica
• Phases with enhanced polar compound retention
• Compatible with LC-MS and all of today’s sensitive instruments and methods
• Scalable selectivity from analytical to preparative
• High surface area silica for high preparative loading capacity
Phase USP Bonding

Bonding Designation Chemistry Chromatographic Properties / Use
C18 L1 Octadecyl The classic reversed-phase column suitable for any method that specifies a C18-type column. Its
high surface area gives Ascentis® C18 strong hydrophobic retention and high loading capacity
for preparative applications. Ascentis® C18 is low-bleed for clean ESI and APCI traces. The high
retentivity means that the mobile phase can contain high levels of organic modifier.
C8 L7 Octyl Ascentis® C8 is suitable for any method that specifies a C8-type column. Although C8 columns
often show similar selectivity to C18 columns, shorter alkyl chains sometimes show different
selectivity toward polar compounds because they can solvate differently with the mobile phase and
interact differently due to the size and shape of certain molecules. Also, C8 reagents are smaller
than C18 reagents and have improved primary phase coverage, thereby requiring less endcapping.
Ascentis® C8 has excellent peak shape and very high phase stability.
Cyano L10 Cyanopropyl Useful for selectivity in the reversed-phase mode, including π-π and dipole-dipole interacting
compounds. Can also be used in HILIC mode and normal phase chromatography.
RP-Amide L60 Palmitamidopropyl Ascentis® RP-Amide can be used for many of the same separations as a C18 while avoiding some
of the disadvantages of C18 such as poor wettability in high aqueous mobile phases. In addition,
it is much more retentive for those molecules that can interact by hydrophobic interactions and
also by H-bonding with the amide group. Compared to alkyl only phases, Ascentis® RP-Amide
has enhanced retention and selectivity for phenols, organic acids and other polar solutes due to
strong H-bonding between the amide carbonyl (H-bond acceptor) and H-bond donors, like phenols
and acids. Compared to other embedded polar group (EPG) phases, like carbamates, ureas,
sulfonamides and ethers, Ascentis® RP-Amide gives retention comparable to C18 and C8 for easy
column comparison without the need to change mobile phase conditions.
Phenyl L11 Phenyl ring Phenyl phases are π-basic (electron donating) and are similar in overall retention to alkyl and EPG
with short phases for easy column screening. The alternate selectivity of phenyl phases is often explained
butyl spacer by the π-π interactions available through the phenyl ring. They provide low-bleed for MS or
UV gradient applications due to the use of trifunctional bonding reagent, outstanding phenyl
selectivity due to high phase loading and short butyl spacer and 100% aqueous-compatible for
highly-polar compounds
Silica L3 unbonded The classic use of silica columns is for normal phase HPLC. The rigid structure of the silica surface,
as opposed to the flexible nature of bonded phases, allows it to distinguish between molecules with
different molecular shape that may have the same hydrophobicity. Ascentis® Si columns provide a
high-loading capacity and operates in both normal-phase and HILIC modes.
*Under certain conditions such as low temperature, the column can be operated in the extended pH range from pH 1.5 - 10.

Phosphate buffers are often preferred in HPLC applications, but they are aggressive at high pH and can cause
dissolution of silica, stripping of phase and voiding in columns and usually should be avoided at high pH conditions.
Ascentis® Columns have a pH stability of pH 2-8. Under special conditions, especially at lower temperature,
Ascentis® C18, C8 and RP-Amide can be used with mobile phase conditions at pH 1.5 and pH 10, even with
aggressive mobile phase conditions with phosphate and methanol.
Column: Ascentis® C18, Ascentis® C8 or Ascentis®

RP-Amide, 5 cm x 3.0 mm I.D., 5 µm particles 18
Mobile phase: 40:60, 10 mM ammonium phosphate 15
(pH 10):methanol
12
Flow rate: 0.9 mL/min.
9
K'
Temp.: 25 °C
Et.: UV at 220 nm 6
Stability test Uracil was used as a void marker. 3

mix: Dimethylaniline (DMA) was used as a polar basic 0
analyte that is sensitive to phase loss and silanol 0 10000 20000 30000 40000
activity. As end-capping is lost, peak shape and
efficiency should suffer.
Column Voids
Toluene provided neutral k’, efficiency and
asymmetry marker.
C18 DMA C18 p-Xylene C8 DMA
p-Xylene is a highly retained neutral efficiency
marker that is very sensitive to loss of main phase C8 p-Xylene RPA DMA RPA p-Xylene
due to its high k’
Particle Size Pore Size Surface Area Carbon Load Surface Coverage Max Shipping
(µm) (Å) (m2/g) (%) (µmol/m2) pH Stability Temperature Endcapped Eluent
3, 5 100 450 25 3.7 2 - 8* 70 °C Yes 40% Water:
60%
Acetonitrile
3, 5 100 450 15 4.0 2 - 8* 70 °C Yes 30% Water:

70%
Methanol
5 100 450 10 2.5 1-8 70 °C Yes Acetonitrile
3, 5 100 450 19.5 2.7 2 - 8* 70 °C Yes Acetonitrile
3, 5 100 450 19 5.2 2 - 8* 70 °C Yes 35% Water:

65%
Methanol
3, 5 100 450 n.a. n.a. 2-6 70 °C n.a. Ethanol
77
Ascentis® (5 µm)
Column dimension
Length (mm) I.D. (mm) C18 C8 Phenyl RP-Amide ES Cyano Si
50 x 2.1 on request on request on request 565303-U on request on request
100 x 2.1 581326-U on request on request 565304-U 577301-U 581500-U
150 x 2.1 581304-U on request on request 565305-U on request 581509-U
250 x 2.1 581305-U on request on request 565306-U on request 581510-U
250 x 4 on request on request on request 565327-U on request on request
50 x 4.6 581323-U on request 581615-U 565323-U on request on request
150 x 4.6 581324-U 581424-U 581616-U 565324-U 577306-U 581512-U
250 x 4.6 581325-U 581425-U 581617-U 565325-U 577307-U 581513-U
250 x 10 581343-U on request 581618-U 565344-U on request 581514-U
150 x 21.2 581346-U on request on request 565347-U on request on request
250 x 21.2 581347-U 581442-U 581619-U 565348-U on request 581515-U
Ascentis® Validation Packs (3 columns, each from a different lof of bonded phase)
Ascentis® Supelguard™ Guard Cartridge (2 pack)
20 x 2.1 581370-U on request on request 565372-U on request on request
20 x 4 581372-U 581426-U 581620-U 565370-U on request 581518-U
Ascentis® Supelguard™ Guard Kit (Guard cartridge, stand-alone holder, tubing, 2 nuts and ferrules)
20 x 4 581373-U 581427-U 581621-U 565371-U on request 581519-U
Ascentis® (3 µm)
Column dimension
Length (mm) I.D. (mm) C18 C8 RP-Amide Si
20 x 2.1 on request on request 565313-U on request
30 x 2.1 on request 581414-U on request 581522-U
50 x 2.1 581300-U 581400-U 565300-U 581500-U
75 x 2.1 on request on request on request on request
100 x 2.1 581301-U 581401-U 565301-U on request
150 x 2.1 581302-U 581402-U 565302-U 581502-U
20 x 3 on request on request on request on request
100 x 3 581308-U on request 565312-U 581503-U
20 x 4.6 on request on request on request on request
50 x 4.6 581320-U on request 565320-U on request
100 x 4.6 581321-U 581407-U 565321-U on request
150 x 4.6 581322-U 581408-U 565322-U on request
Ascentis® Supelguard™ Guard Cartridge (2 pack)
20 x 2.1 581377-U on request on request on request
Ascentis® Supelguard™ Guard Kit (Guard cartridge, stand-alone holder, tubing, 2 nuts and ferrules)
20 x 2.1 581376-U on request on request on request
20 x 4 581379-U on request on request on request

All Supelco® HPLC columns including the complete range of
Fully Porous Particles (FPP), Superficially Porous Particles
(SPP) and Monolithic Columns (Chromolith®) perfectly fit to
every HPLC, UHPLC and UPLC®® instrument independent of
the instrument supplier.
79
Titan™ UHPLC Columns
Titan™ UHPLC columns, based on 1.9 µm fully porous The performance for a Titan™ column is improved
monodisperse silica, outperform other UHPLC columns. compared to smaller, Porous Particles that have
These UHPLC columns provide the performance broader size distributions. Lower values for reduced
scientists expect (> 250,000 N/m). plate height (h), which is plate height (H) divided by
particle diameter, is very significant for Titan™ columns
The Titan™ particle is based on spherical, fully porous
because it means that higher column efficiency is
silica, with an ultra-narrow particle size distribution
observed with larger particles which create lower
for optimum efficiency. Research findings support
pressure drop.
observations that porous-layer particles with very
narrow particle size distribution demonstrate superior The efficiency advantage for Titan™ over commercial
efficiency and kinetic performance. Uniform Titan™ 1.8 µm and 1.7 µm Porous Particles confirms that
particles are packed into rugged column beds that Titan™ 1.9 µm column pressure is lower than 1.7 µm
are stable over a range of UHPLC flow and pressure or 1.8 µm particle columns and is actually closer to a
conditions. Excellent batch reproducibility and 2.5 µm particle column.
robustness is also noted.
14 14000
Titan™ (1.9 µm) Titan™ 1.9 µm
12 Vendor A (1.8 µm) 12000 Competitor C18 1.8 µm
Vendor B (1.7 µm)
Incremental Number (%)
10 Pressure, PSI 10000 Competitor C18, 1.7 µm
8 8000
6 6000
4
4000
2
2000
0
0
0 1 2 3 4 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Particle Size (µm) Flow, mL/min
Comparison of Particle Size Distribution for Different Independent Evaluation of Titan™ Pressure Drop
Sub-2 µm Porous Particles
Titan™ Porous Silica Characteristics

Particle Pore Surface Pore Pressure
Size* μm Diameter Å Area m2/g Volume cc/g psi (bar)
1.9 80 410 0.76 18000 (1241)
*Very narrow distribution: D (90/10) < 1.15

3 Titan™ Outperforms Other Fully Porous UHPLC
2
Columns
1 5
Titan™ C18, 1.9 µm 4
Diphenhydramine
N= 14,710 Diazepam
N/m = 294,200 Naphthalene
h= 1.75 Vendor A
σ= 1.05 C18
Pressure*= 4,100psi 1.8 µm
1.0 2.0
Min Vendor B
C18 Titan™ UHPLC columns
3 1.7 µm deliver highest number of
plates per unit pressure.
Competitor A C18, 1.8 µm 2
N= 9,260 Titan™
N/m= 185,200 1 4 5 C18
h= 3.18 1.9 µm
σ= 1.02
Pressure*= 4,650psi 0.00 1.00 2.00 3.00 4.00
Efficiency per Unit Pressure
1.0 2.0
Min
3
Competitor B C18, 1.7 µm 2
N= 9,783
N/m= 195,700 1 5
4
h= 2.84
σ= 1.16
Pressure*= 4,900psi
1.0 2.0
Min
* Total System Pressure
Titan™ C18 Performance Comparison vs. Competitor UHPLC Columns
Length (mm) I.D. (mm) Qty. Cat. No.
Titan™ C18 Columns, 1.9 µm
20 x 2.1 1 577120-U
30 x 2.1 1 577121-U
50 x 2.1 1 577122-U
75 x 2.1 1 577123-U
100 x 2.1 1 577124-U
30 x 3.0 1 577125-U
50 x 3.0 1 577126-U
Titan™ C18 Guard Cartridges, 1.9 µm
5 x 2.1 3 577127-U
5 x 3.0 3 577128-U
Titan™ Guard Cartridge Holder 1 577133-U
(cartridge not included)
81
SeQuant® HILIC HPLC and Capillary Columns
ZIC®-HILIC, ZIC®-cHILIC and ZIC®-pHILIC are the ideal
columns for all classes of polar and hydrophilic compounds
SeQuant® HILIC (hydrophilic interaction liquid Features and Benefits

chromatography) HPLC columns constitute a range of
• High-performance HPLC and LC-MS separations of
high-performance tools for separating polar, hydrophilic
polar hydrophilic compounds
compounds. All columns carry densely bonded, truly
zwitterionic functional groups with a charge balance of • Zwitterionic stationary phase ensures
1:1. Separation is achieved by hydrophilic partitioning reproducible retention
combined with weak ionic interactions for maximum • Two complementary phase chemistries
selectivity, high loadability and easy optimization • Maximum LC-MS compatibility with minimized
of methods. column bleed
• Excellent reproducibility and robustness
• Available in a variety of lengths, particle sizes, and
pore dimensions
Choose your SeQuant® HILIC selectivity
SeQuant® Column Functional Group Features Base Particle Particle Size Pore Size Column Types
SeQuant® ZIC®- High-performance selectivity 3.5 µm, analytical, capillary,

silica 100 Å, 200 Å
HILIC and robustness 5 µm semi-prep, guards
Sulfobetaine
Complementary selectivity
SeQuant® ZIC®- analytical, capillary,
with favorable LC-MS silica 3 µm 100 Å
cHILIC guards
Phosphoryl-choline performance
SeQuant® ZIC®- High pH-stability and low

polymer 5 µm analytical, guards
pHILIC noise with ion detectors
Sulfobetaine
Your ideal choice for separation of all types of polar and protein digests and oligonucleotides can therefore be
hydrophilic compounds are the SeQuant® HILIC HPLC achieved with a selectivity complementary to reversed-
columns. Reproducible retention for compounds that phase columns. Enhanced LC-MS sensitivity is an
have proved difficult to separate on reversed-phase additional benefit of using these columns.
HPLC columns is ensured by the high-performance
Columns are available in a wide range of formats from
zwitterionic sorbents in these columns.
capillary to semi-preparative dimensions, and with
Straightforward separation of compounds such as several different particles sizes and pore sizes.
acids and bases, anions and cations, carbohydrates,
metabolites, metal complexes, amino acids, peptides,
SeQuant® HILIC Analytical and Capillary HPLC Columns

SeQuant® ZIC®-HILIC and ZIC®-cHILIC SeQuant® ZIC®-HILIC
The ZIC -HILIC Column is designed to retain and
®
separate all types of polar and hydrophilic compounds

and for robust chromatography with high selectivity
and reproducibility. The silica-based ZIC®-HILIC
sorbent has a bonded stationary phase consisting
of a highly polar, permanent zwitterion. Separation
selectivity is favored by the 1:1 zwitterion charge
Sulfobetaine (SB)
balance, which makes the ZIC®-HILIC Column overall
neutral, with weak, but important, ionic interactions.
Tuning of the selectivity on the ZIC®-HILIC Column
during method development is facilitated by the pH-
inpendent, permanent zwitterion, ensuring that only SeQuant® ZIC®-cHILIC
the analytes (and not the column) is affected during
eluent optimization.
SeQuant® ZIC®-cHILIC is designed for excellent HPLC
and LC-MS of polar, hydrophilic compounds. This new,
zwitterionic stationary phase with phosphorylcholine
functional group provides you with complementary Phosphorylcholine (PC)
selectivity for easier method development for analytes
that have been difficult to separate by previous types
of HPLC columns operated in reversed-phase or
HILIC mode.
Isocratic separations of the positively charged benzyltrimethylamine (BTMA, peaks indicated with arrows) and
the neutral toluene (void marker), uracil and cytosine on ZIC®-cHILIC (left) and ZIC®-HILIC (right) illustrating
differences and similarities in selectivity caused by the different charge orientation of the zwitterionic functional
groups (see illustrations).
SeQuant® ZIC® - cHILIC SeQuant® ZIC® - HILIC Chromatographic conditions

Column dimensions were 100 x 4.6 mm, particles size 3 or 3.5 µm,
and pore size 100 Å. Eluent was 80 : 20 acetonitrile/ 25 mM aqueous
ammonium acetate pH 6.8 pumped at 0.5 mL/min at 23 °C. Detection
by UV absorption at 254 nm.
BTMA
BTMA
BTMA
0 2 4 6 8 10 0 2 4 6 8 10
Retention Time (min) Retention Time (min)
SeQuant® ZIC®-pHILIC
The ZIC®-pHILIC stationary phase has the same highly polar, bonded, permanent zwitterion, functional group as
the silica-based ZIC®-HILIC Column. The user can therefore expect the same selectivity, however, with a trade-off
in flow-rate range, and separation efficiency, common with polymeric materials. The more durable support allows
for use in an extended pH range, which can be beneficial for certain applications.
The application example below
illustrates how the selectivity of O O
the ZIC®-pHILIC material can OH
OH
HO
be enhanced by performing the pH 6.8 pH 9.6 HO
HO OH
separation at elevated pH. The O
chromatograms show isocratic OH
HO
O
OH
separations of gentisic acid, HO
OH HO O O
O O
protocatechuic acid and isophthalic HO OH OH
acid on a ZIC®-pHILIC Column. The OH OH
increase in pH also results in higher

retention and improved peak shape 0 2.5 5 7.5 10
for these analytes. 0 2.5 5 7.5 10
83
Outstanding suitability for LC-MS Glycomics and Glyco- proteomics
Thanks to their favorable retention and robust SeQuant® ZIC® is the ideal sorbent for the separation
performance, SeQuant® HILIC columns have become and extraction of glycans and glycopeptides. A material
extremely popular in numerous laboratories around the pore size of 200 Å is recommended to avoid size-
world. For example, the US FDA (United States Food exclusion effects from the relatively large hydrodynamic
and Drug Administration) recommended SeQuant® volume of the hydrated glycan structures. Small I.D.
ZIC®-HILIC for analysis of melamine and related HPLC columns are suitable for LC-MS methods, whereas
substances. SeQuant® HILIC columns excel in stability larger conventional column dimensions are more
and low bleed. These features make them particularly appropriate for separations of labeled glycan structures
suitable for LC-MS applications where high background detected with other techniques.
can lead to signal suppression and interference with
quantitative measurements while also increasing
instrument wear. The column's high stability makes
these high-performance HILIC columns also attractive
for traditional HPLC applications.
Column bleed (Mcps)
Column
SeQuant® HILIC columns have been used in numerous methods for
analysis of a wide variety of polar hydrophilic compounds such as acids
and bases, anions and cations, carbohydrates, metabolites, metal
complexes, amino acids, peptides, and protein digests.

Peptide Mapping with High-Sequence Coverage
SeQuant® ZIC® HPLC columns are suitable for peptide mapping, especially so when exploring information
from hydrophilic peptide fragments. Thanks to their advantageous combination of reversed phase
chromatography and HILIC technology, the columns provide complementary information and may be
coupled to an online 2-D peptide mapping approach.
Column dimension
ZIC®-HILIC ZIC®-HILIC ZIC®-HILIC ZIC®-cHILIC ZIC®-pHILIC,
Length (mm) I.D. (mm)
200A, 5 µm 100 A, 3.5 µm 200A, 3.5 µm 100A, 3 µm 5 µm
50 x 2.1 1.50450.0001 1.50440.0001 1.50445.0001 1.50656.0001 1.50459.0001
100 x 2.1 1.50452.0001 1.50441.0001 1.50447.0001 1.50657.0001 1.50462.0001
150 x 2.1 1.50454.0001 1.50442.0001 1.50448.0001 1.50658.0001 1.50460.0001
250 x 2.1 1.50457.0001 1.50443.0001
50 x 4.6 1.50451.0001 1.50446.0001 1.50659.0001 1.50463.0001
100 x 4.6 1.50453.0001 1.50660.0001 1.50464.0001
150 x 4.6 1.50455.0001 1.50444.0001 1.50449.0001 1.50661.0001 1.50461.0001
250 x 4.6 1.50458.0001 1.50662.0001
150 x 10 1.50493.0001
250 x 10 1.50494.0001
50 x 21.2 1.50496.0001
150 x 21.2 1.50497.0001
SeQuant® Capillary columns
30 x 0.3 1.50489.0001
30 x 1 1.50478.0001
150 x 0.075 1.50465.0001
150 x 0.3 1.50481.0001 1.50479.0001 1.50669.0001
150 x 1 1.50482.0001 1.50487.0001 1.50480.0001 1.50670.0001
SeQuant® Guard (1 piece; * 3 Pieces; **5 pieces)
5 x 0.3 1.50492.0001** 1.50765.0001*
5 x 1 1.50490.0001** 1.50766.0001*
20 x 2.1 1.50435.0001 1.50439.0001 1.50437.0001
SeQuant® Guard Kit (3 guard columns including column coupler)
20 x 2.1 1.50764.0001 1.50438.0001
20 x 1 1.50436.0001
Guard Fitting x 14x1 1.50434.0001
85
LiChrospher® HPLC columns
Classical silica carrier for consistent results
LiChrospher® is the name given to reliable and versatile, and 2 mm I.D. narrow bore cartridges for HPLC save
traditionally produced spherical silica carriers (Type A). costs by reducing solvent consumption and allow
LiChrospher® silica carriers are available in a number the handling of very small quantities with excellent
of different modifications. The polar modified phases sensitivity and resolution. LiChroCART® cartridges
LiChrospher® CN, LiChrospher® NH2 and LiChrospher® 4.6 mm, 4 mm I.D., 3 mm I.D. and 2 mm I.D. are
DIOL as well as LiChrospher® Si with no modification compatible with manu-CART® “4”. This trait facilitates
are best for normal-phase HPLC. The non-polar modified faster and more flexible method adaptation to smaller
phases LiChrospher® RP-8, RP-8 endcapped, RP-select bore columns. LiChroCART® cartridges 10 mm I.D. have
B, RP-18, RP-18 endcapped are made for reversed- to be used with manu-CART® “10”.
phase separations. Furthermore, LiChrospher® PAH is
For improvements in resolution, column efficiency
highly efficient and selective for the separation of PAHs.
and peak symmetry as well as for fast and UHPLC
LiChrospher® packing materials are available as Hibar® applications, we recommend alternatives based
RT columns and as LiChroCART® cartridges of various on more advanced column technology such as
lengths and internal diameters (10 mm, 4.6 mm, Type B silica, Superficially Porous Particles or
4 mm, 3 mm and 2 mm). LiChroCART® 3 mm I.D. monolithic columns.
Particle Pore Pore Surface Carbon Surface

Phase USP Bonding Size Size volume Area Load Coverage pH Max Shipping
Bonding Designation Chemistry (µm) (Å) (mL/g) (m2/g) (%) (µmol/m2) Stability Temperature Endcapped Eluent
RP-18 L1 Octadecylsilane 5, 10 100 1.25 350 21 3.61 2 - 7.5 65°C No Acetonitrile/
Water
(80:20)
RP-18 L1 Octadecylsilane 5, 10 100 1.25 350 21.6 4.09 2 - 7.5 65°C Yes Acetonitrile
endcapped with / Water
endcapping (80:20)
PAH L1 Octadecylsilane 5 150 200 2 - 7.5 65°C No Acetonitrile
/ Water
(80:20)
RP-8 L7 Octylsilane 5, 10 100 1.25 350 12.5 4.04 2 - 7.5 65°C No Acetonitrile
/ Water
(80:20)
RP-8 L7 Octylsilane 5, 10 100 1.25 350 13.0 4.44 2 - 7.5 65°C Yes Acetonitrile
endcapped with / Water
endcapping (80:20)
RP- L7 Octylsilane 5, 10 60 0.9 360 11.5 3.55 2 - 7.5 65°C No Acetonitrile
selectB deactivated for / Water
the separation (80:20)
of basic
compounds
Diol L20 Diol 5, 10 100 1.25 350 8.0 3.87 2 - 7.5 65°C No n-Heptane
CN L10 Cyanosilane 5, 10 100 1.25 350 6.6 3.52 2 - 7.5 65°C No n-Heptane
NH2 L8 Aminosilane 5, 10 100 1.25 350 4.6 4.10 2 - 7.5 65°C No n-Heptane
Si L3 unbonded 5, 10 60 0.85 700 n.a. n.a. 2 - 7.5 65°C N.A. n-Heptane

100 1.25 400

LiChrospher® (5µm)
RP-18 RP-8
Length I.D. RP-select B
RP-18 (5 µm) endcapped PAH RP-8 (5 µm) endcapped Diol (5 µm) CN (5 µm) NH2 (5 µm) Si 60 (5 µm) Si 100 (5 µm)
(mm) (mm) (5 µm)
(5 µm) (5µm)
LiChroCART® HPLC Cartridge [1 unit; * 3 units]
25 x 4 1.50931.0001* 1.50936.0001* on request 1.50930.0001* on request 1.50937.0001* on request on request on request on request on request
75 x 4 1.50987.0001* on request on request on request on request 1.50993.0001* on request on request on request on request on request
100 x 4.6 1.50600.0001 1.50603.0001 on request 1.50634.0001 on request 1.50640.0001 on request on request on request on request on request
125 x 3 1.50159.0001 on request on request on request on request 1.50158.0001 on request on request on request on request on request
125 x 4 1.50823.0001 1.50828.0001 on request 1.50822.0001 1.50827.0001 1.50829.0001 1.50826.0001 1.50825.0001 1.50824.0001 1.50820.0001 on request
125 x 4 1.50943.0001* 1.50734.0001* on request 1.50942.0001* on request 1.50981.0001* on request on request on request on request on request
125 x 4.6 on request 1.51908.0001 on request on request on request on request on request on request on request on request on request
150 x 4.6 1.50601.0001 1.50604.0001 on request 1.50635.0001 1.50638.0001 1.50641.0001 on request on request on request on request on request
250 x 3 1.50154.0001 on request 1.50156.0001 on request on request 1.50155.0001 on request on request on request on request on request
250 x 4 1.50833.0001 1.50838.0001 1.50149.0001 1.50832.0001 1.50837.0001 1.50839.0001 1.50836.0001 1.50892.0001 1.50834.0001 1.50830.0001 on request
250 x 4 1.50983.0001* 1.50995.0001* on request on request on request 1.50984.0001* on request on request on request on request on request
250 x 4.6 1.50602.0001 on request on request 1.50636.0001 1.50639.0001 on request on request on request on request on request on request
100 x 4.6 1.50600.1003 on request on request 1.50634.1003 on request 1.50640.1003 on request on request on request on request on request
125 x 3 1.50159.1003 on request on request on request on request on request on request on request on request on request on request
125 x 4 1.50823.1003 on request on request 1.50822.1003 on request 1.50981.1003 on request on request on request on request on request
150 x 4.6 on request 1.50604.1003 on request on request on request 1.50641.1003 on request on request on request on request on request
250 x 3 1.50154.1003 on request on request on request on request 1.50155.1003 on request on request on request on request on request
250 x 4 1.50833.1003 1.50838.1003 on request 1.50832.1003 on request 1.50839.1003 on request on request on request on request on request
250 x 4.6 1.50602.1003 1.50605.1003 on request 1.50636.1003 on request 1.50642.1003 on request on request on request on request on request
4 x 4 1.50957.0001 1.50962.0001 1.50148.0001 1.50956.0001 1.50961.0001 1.50963.0001 1.50960.0001 1.50959.0001 1.50958.0001 1.50955.0001
The LiChroCART® columns (75, 100, 125, 150 and 250 mm length) in the list above (2, 3, 4 and 4.6 mm I.D.) require part number 1.51486.0001 manu-CART® cartridge column holder, which
can be used to hold one cartridge column with or without a 4-4 mm guard column. LiChroCART® columns 250-10 mm require part number 1.51419.0001 manu-CART® 10. Additional dimensions
and validation kit available as customized packings see page 120.
100 x 4.6 1.50545.0001 on request on request 1.50578.0001 1.50581.0001 1.50573.0001 on request on request on request on request on request
125 x 2 on request 1.51907.0001 on request on request on request on request on request on request on request on request on request
125 x 4.6 on request on request on request on request on request on request on request on request on request on request on request
150 x 4.6 1.50546.0001 1.50549.0001 on request 1.50579.0001 1.50582.0001 1.50574.0001 on request on request 1.51905.0001 on request on request
250 x 4 1.50377.0001 on request on request 1.50329.0001 on request on request on request on request on request on request 1.50316.0001
250 x 4.6 1.50547.0001 1.50550.0001 on request 1.50580.0001 1.50583.0001 1.50575.0001 on request on request 1.51904.0001 on request on request
100 x 4.6 1.50545.1003 on request on request 1.50578.1003 on request 1.50573.1003 on request on request on request on request on request
150 x 4.6 on request 1.50549.1003 on request on request on request 1.50574.1003 on request on request on request on request on request
250 x 4 1.50377.1003 on request on request 1.50329.1003 on request on request on request on request on request on request on request
240 x 4.6 on request 1.50550.1003 on request on request on request on request on request on request on request on request on request
The Hibar® columns are complete with endittings. When using a guard column with a Hibar® column, we recommend part number 1.51487.0001 guard column cartridge holder for 4-4 mm guard
column cartridges LiChroCART®. Additional dimensions available as customized packings see page 120.
Stainless steel ready to use HPLC Column SIAL
150 x 3.2 54775

250 x 3.2 54777 54785
125 x 4 50141-U 50146-U
250 x 4 50137-U
125 x 4.6 50140-U
150 x 4.6 54774 54778 54782 54790-U
250 x 4.6 54776 54780 54788 54792
SupelGuard™ Column SIAL
10 x 4.6 54794 54798 54797-U
LiChrospher® - Bulk Sorbents

10 g Sorbent in
1.16177.0010 1.19637.0010 1.16129.0010 1.19636.0010 1.19641.0010 1.16152.0010 1.19638.0010 1.19640.0010
glass bottle
87
Superspher® HPLC Columns
Classical silica carrier for highly efficient separations
Superspher®, a spherical silica carrier with a mean particle size of 4 µm, providing higher separation performance
in HPLC Applications. The number of theoretical plates for Superspher® is approx. 100,000 N/m. Thus,
Superspher® HPLC columns are a good choice when complex mixtures demand high peak capacity. A broad
range of modifications on Superspher® is available: non-polar derivatives (RP-8, RP-8 endcapped, RP-18, RP-18
endcapped and RP-select B) and polar derivatives (Si 60).
Pore Surface Surface

Phase USP Bonding Particle Pore volume Area Carbon Coverage pH Max
Bonding Designation Chemistry Size (µm) Size (Å) (mL/g) (m2/g) Load (%) (µmol/m2) Stability Temperature Endcapped
RP-18 L1 Octadecylsilane 4 100 1.25 350 21.0 3.61 2 - 7.5 65 °C No
RP-18 L1 Octadecylsilane 4 100 1.25 350 21.6 4.09 2 - 7.5 65 °C Yes
endcapped
RP-8 L7 Octylsilane 4 60 1.25 350 4.04 2 - 7.5 65 °C No
RP-8 L7 Octylsilane 4 60 1.25 350 13.0 4.44 2 - 7.5 65 °C Yes
endcapped
RP-select L7 Octylsilane 4 60 0.9 3.55 2 - 7.5 65 °C No
B deactivated for
the separation
of basic
compounds
Si L3 unbonded 4 60 0.85 700 n.a. n.a. 2 - 7.5 65°C N.A.
Superspher® packing materials are available as LiChroCART® cartridges in various lengths and internal diameters (4.6 mm,
4 mm, 3 mm and 2 mm). LiChroCART® 3 mm I.D. and 2 mm I.D. narrow bore cartridges for HPLC save costs by reducing solvent
consumption and allow the handling of very small quantities with excellent sensitivity and resolution. LiChroCART® cartridges
4.6 mm, 4 mm I.D., 3.9 mm I.D., 3 mm I.D. and 2 mm I.D. are compatible with manu-CART® “4”. This facilitates faster and
more flexible method adaptation to smaller bore columns.
Superspher® (4 µm)
Column dimension
Length (mm) I.D. (mm) RP-18 RP-18 endcapped RP-8 RP-8 endcapped RP-select B Si
LiChroCART® HPLC Cartridge [1 unit; * 3 units]
25 x 4 1.16039.0001* 1.16869.0001* on request on request on request on request
75 x 4 1.50980.0001* on request on request on request 1.50974.0001* on request
125 x 2 on request 1.50198.0001 on request on request 1.50197.0001 on request
125 x 3 1.50792.0001 1.51909.0001 on request on request 1.50791.0001 on request
125 x 4 1.16051.0001 1.16855.0001 1.16052.0001 1.16854.0001 1.50975.0001 1.16054.0001
250 x 2 on request 1.50193.0001 on request on request 1.51308.0001 on request
250 x 3 1.51299.0001 1.51910.0001 on request on request 1.51288.0001 on request
250 x 4 1.16056.0001 1.16858.0001 1.16010.0001 1.16857.0001 1.50973.0001 1.16009.0001
125 x 4 1.16051.1003 on request on request on request on request on request
250 x 3 1.51299.1003 on request on request on request on request on request
Superspher® - Bulk Sorbents
10 g Sorbent in glass bottle 1.19613.0010 1.19612.0010 1.19643.0010
The LiChroCART columns (75, 125, 150 and 250 mm length) in the list on the left (2, 3, 4 and 4.6 mm I.D.) require part
®
number 1.51486.0001 manu-CART® cartridge column holder, which can be used to hold one cartridge column with or without a
4-4 mm guard column. Additional dimensions available as customized packings see page 120. As guard column we recommend
LiChroCART® 4-4 LiChrospher® guard cartridges.

LiChrosorb® HPLC Columns
Irregular shaped silica sorbent
LiChrosorb® is one of the most successful and reliable packing materials, used in HPLC for decades and
documented in the literature in the form of several thousand applications. The totally porous, irregular particles
are linely graded in the 5 and 10 µm range.
LiChrosorb® packing materials offer the complete program of non-polar (RP-8, RP-18,
RP-select B) and polar modifications (Si 60 and Si 100). In addition to the analytical cartridges and columns, such as
LiChroCART® 250-4 or Hibar® RT 250-4, we offer semi-preparative cartridges LiChroCART® 250-10 as well as Hibar®
RT columns 250-10, packed on request with various LiChrosorb® packing materials.
LiChrosorb® HPLC column can be easily replaced with LiChrospher® spherical fully porous particulate columns.
For further improvements in resolution, column efficiency and peak symmetry as well as for fast and UHPLC
applications, we recommend alternatives based on more advanced Type B silica column technology.
LiChrosorb®
Column dimension
I.D.
Length (mm) (mm) RP-18 (5 µm) RP-18 (10 µm) RP-8 (5 µm) RP-8 (10 µm) Si 60 Si 100
LiChroCART HPLC Cartridge [1 unit]
®
125 x 4 1.51349.0001 on request 1.51345.0001 on request on request 1.51343.0001

250 x 4 1.51355.0001 1.51356.0001 1.51353.0001 1.51354.0001 on request 1.51351.0001
The LiChroCART® columns (125, and 250 mm length) in the list above (2, 3, 4 and 4.6 mm I.D.) require part number
1.51486.0001 manu-CART® cartridge column holder, which can be used to hold one cartridge column with or without a
4-4 mm guard column. LiChroCART® columns 250-10 mm require part number 1.51419.0001 manu-CART® 10. Additional
dimensions and validation kit available as customized packings see page 120.
125 x 4 1.50433.0001 on request 1.50432.0001 on request on request on request
125 x 4.6 on request on request 1.50012.7057 on request on request on request
250 x 4 1.50333.0001 1.50334.0001 1.50332.0001 1.50318.0001 1.50388.0001 on request
250 x 4.6 1.51902.0001 on request 1.51903.0001 on request on request on request
The Hibar® columns are complete with endittings. When using a guard column with a Hibar® column, we recommend part number 1.51487.0001 guard
column cartridge holder for 4-4 mm guard column cartridges LiChroCART®. Additional dimensions available as customized packings see page 120.
LiChrosorb® - Bulk Sorbents
10 g Sorbent in glass bottle 1.09333.0010 1.09318.0010 1.09309.0010
Stainless steel ready to use HPLC Column SIAL
150 x 3.2 54952
125 x 4 57484-U
100 x 4.6 50124-U
150 x 4.6 54951 54955-U
200 x 4.6 50125-U
250 x 4.6 54949 54953-U
SupelGuard™ SIAL
10 x 4.6 54965-U 54966
89
SUPELCOSIL™ HPLC Columns
Our SUPELCOSIL™ silica-based HPLC column line includes many phase chemistries, in a range of particle sizes and
column configurations from microbore to preparative scale. This product line is the original high quality Supelco®
product line referenced in many USP methods.
For improvements in resolution, column efficiency and peak symmetry as well as for fast and UHPLC applications,
we recommend alternatives based on more advanced column technology such as Type B silica, Superficially
Porous Particles or monolithic columns.
USP
Phase Bonding Designation Bonding Chemistry Chromatographic Properties / Use
LC-18 L1 Octadecyl General-purpose hydrophobic alkyl phase suitable for a variety of compounds.
LC-18-DB L1 Octadecyl C18-DB phases are specially deactivated for the separation of basic compounds
providing improved peak shape.
LC-18-T L1 Octadecyl C-18-T columns feature an octadecylsilane bonded phase and a special surface
treatment for efficient separations of nucleotides.
LC-18-S L1 Octadecyl C-18-S columns are designed for reliable separations of deoxyribonucleosides
and ribonucleosides.
LC-8 L7 Octyl C8 phases are less hydrophobic than C18 and povides less retention of both polar
and non-polar compounds than C18.
LC-8-DB L7 Octyl C8-DB phases are specially deactivated for basic compounds providing improved
peak shape.
LC-1 Methyl L13 Methyl Due to a mixed retention mechanism, selectivity differences for polar groups are
more pronounced than on C8 and C18 columns.
LC-DP Diphenyl L11 Diphenyl Diphenyl bonded phase, which gives greater selectivity for aromatic groups
compared to alkyl-type bonded phases.
LC-ABZ L60 Alkylamide This deactivated phase provides enhenced reversed-phase performance for basic
compounds, as well as those that are acidic, polar neutral, and non-polar.
ABZ+Plus L60 Alkylamide SUPELCOSIL™ ABZ+Plus HPLC columns offer both high deactivation and unique
selectivity allowing the use of low ionic strength buffers without having to add an
ion-suppressing modifier.
Suplex™ L68 Alkylamide Suplex pKb-100 columns are not end-capped, but feature the same bonded phase
pKb-100 functionality as SUPELCOSIL™ LC-ABZ columns. The absence of end-capping
reagent results in better performance for the strongest basic compounds, while LC-
ABZ is preferred when the sample also contains acids and zwitterions.
LC-CN L10 Cyano The LC-CN phases are suitable for operation under reversed-phase conditions
(HILIC) as well as under normal phase conditions.
LC-Diol L20 Diol LC-Diol columns can be used to separate proteins by gel filtration chromatography.
They are suitable for operation under reversed-phase conditions (HILIC) as well as
under normal phase conditions.
LC-NH2 L8 Amino The amino column is most often employed for the separation of mono- and
disaccharides. As a normal-phase application, amino columns are used in the
petroleum industry
LC-Si L3 Silica Non-polar compounds elute first on a normal phase silica column, while polar
compounds elute late. LC-Si columns can operate in normal phase mode as well as
in HILIC mode.
LC-SCX L52 Sulfonic acid; strong The LC-SCX cation-exchange columns have strongly acidic propylsulfonic acid
cation exchanger groups and are used for separating cations.
SAX1 L14 propyltrimethylammonium SAX1 HPLC Column is typically employed as an anion exchange column with
phase strongly basic quaternary aminopropyl phase and is used for separating anions.

Particle Size Surface Area Carbon Load Surface Coverage Max
(µm) Pore Size (Å) (m2/g) (%) (µmol/m2) pH Stability Temperature Endcapped
3, 5 120 170 11 3.1 2 - 7.5 70 yes
3, 5 120 170 11 3.1 2 - 7.5 70 yes
3, 5 120 170 12.3 3.1 2 - 7.5 70 yes
5 120 170 11 3.1 2 - 7.5 70 yes
3, 5 120 170 6 3.2 2 - 7.5 70 yes
3, 5 120 170 6 3.2 2 - 7.5 70 yes
5 120 170 2 3.4 2 - 7.5 70 yes
5 120 170 6 2.4 2 - 7.5 70 yes
5 120 170 12 3.4 2 - 7.5 70 yes
3, 5 120 170 12 3.4 2 - 7.5 70 yes
5 120 170 12.5 3.4 2 - 7.5 70 Yes
3, 5 120 170 4 3.5 2 - 7.5 70 yes
5 120 170 3.5 3.8 2 - 7.5 70 no
3, 5 120 170 3 5.1 2 - 7.5 70 no
3, 5 120 170 n.a. n.a. 2 - 7.5 70 n.a.
5 120 170 n.a. n.a. 2 - 7.5 70 n.a.
5 120 170 12 n.a. 2 - 7.5 70 n.a.
91
SUPELCOSIL™ (5 µm)
Length LC-1
(mm) I.D. (mm) LC-18 LC-18-DB LC-18-T LC-18-S LC-PAH LC-8 LC-8-DB Methyl
50 x 2.1 on request on request on request on request on request on request on request on request
150 x 2.1 57934 on request on request on request on request on request on request on request
250 x 2.1 57935 57940 on request 57939 on request on request on request on request
50 x 3 on request on request on request on request on request on request on request on request
150 x 3 58230C30 on request on request on request on request on request on request on request
250 x 3 58298C30 on request on request on request 59187 on request on request on request
50 x 4 58239C40 on request on request on request on request on request on request on request
150 x 4 58230C40 58348C40 on request on request on request 58220C40 58347C40 on request
250 x 4 58298C40 on request on request on request on request on request 58354C40 on request
300 x 4 59165 59164 on request on request on request on request on request on request
50 x 4.6 58239 58345 on request on request on request 58238 58344 on request
150 x 4.6 58230-U 58348 on request 58931 58318 58220-U 58347 on request
250 x 4.6 58298 58355-U 58971 58928-U 58229 58297 58354 58296
250 x 10 58368 58358 on request on request on request 58367 on request on request
SUPELCOSIL™ Supelguard™ Guard Cartridge (2 pack)
20 x 2.1 59613 59617 on request 59162 59613 59615 on request on request

20 x 3 59564C30 on request 59621C30 on request 59564C30 on request 59563C30 on request
20 x 4 59564 59565 59621 59630 59564 59562 59563 59561
SUPELCOSIL™ Supelguard™ Guard Kit (Guard cartridge, stand-alone holder, tubing, 2 nuts and ferrules)
20 x 4 59554 59555 59620 59629 59554 59552 59553 on request
SUPELCOSIL™ (3 µm)
Length I.D. ABZ+Plus
(mm) (mm) LC-18 LC-18-DB LC-18-T LC-8 LC-8-DB Alkylamide LC-CN LC-NH2 LC-Si
33 x 2.1 on request 57943 on request on request 58149-U on request on request on request on request
100 x 2.1 on request on request on request on request on request 57917 on request on request on request
250 x 2.1 57942 57943 on request on request on request on request on request on request on request
33 x 3 on request 58978C30 on request 58975C30 on request on request 58979C30 on request on request
50 x 3 on request on request on request on request on request on request on request on request on request
75 x 3 on request on request on request 58982C30 58990C30 on request 58986C30 on request on request
150 x 3 58985C30 58993C30 58970C30 on request on request 59194C30 on request 58989C30 58981C30
75 x 4 58984C40 on request on request on request on request on request on request on request on request
150 x 4 58985C40 on request on request on request 58991C40 on request on request on request on request
33 x 4.6 58977 58978 on request 58975 58976 on request 58979 on request on request
50 x 4.6 58973 on request on request on request on request on request on request on request on request
75 x 4.6 58984 58992 on request 58982 58990-U on request 58986 58988 on request
150 x 4.6 58985 58993 58970-U 58983 58991 59194 on request 58989 58981

Suplex™
LC-DP LC-ABZ ABZ+Plus pKb-100
Diphenyl Alkylamide Alkylamide Alkylamide LC-CN LC-Diol LC-NH2 LC-Si LC-SCX SAX1
on request on request on request on request on request on request on request on request on request on request
on request on request on request on request on request on request on request 57930-U on request on request
on request 59142C30 59197C30 58934C30 on request 58201C30 58338C30 on request 58997C30 on request
59167 on request on request on request on request on request on request on request on request on request
on request 59142C40 59197C40 58934C40 on request 58201C40 58338C40 on request on request on request
on request 59141 on request on request on request on request on request on request on request on request
59211 on request on request on request on request on request on request on request on request on request
59150-U 59140-U 59196 58932 58221-U on request on request 58200-U on request on request
58842 59142 59197 58934 58231 58201 58338 on request 58997 59138
on request on request 59179 59172 on request on request on request on request on request on request
on request on request 54855 on request on request on request on request on request on request on request
on request 59611 59605 on request on request on request on request on request on request on request
on request on request on request on request on request on request 59568C30 on request on request on request
59566 59545-U 59535-U 59541-U 59567 59569 59568 on request 59519 on request
59556 59544-U 59534-U 59531-U 59557 59559 59558 on request 59509 on request
93
Chiral HPLC Columns
HPLC Chiral Stationary Phases
(-)
Macrocyclic Glycopeptides Astec® CYCLOBOND™ and ChiraDex®
(Astec® CHIROBIOTIC®) (+)
0 2 4 6 8 10 12 14 16 18
Min
Polysaccharides
(Astec® Cellulose DMP)
Proteins
(e.g. Chiral-ACP)
Chiral Ligand Exchange

(e.g. Astec® CLC-D)
Chirality belongs to the discipline of stereochemistry, activity by the unwanted enantiomer. In many cases,
which is the study of the three-dimensional structure the unwanted enantiomer will have different biological
of molecules. Chiral compounds are optically active, activity (e.g. toxic or harmful) and will interfere with
that means they rotate polarized light to the left or to the performance of the intended enantiomer. (3) Time
the right depending on their configuration. The word savings in testing. If the product contains more than
comes from the Greek stem “chir-” meaning hand, for one enantiomer, the biological activity of each isomer
handedness. Chiral molecules are like left and right plus the racemate needs to be checked. This is three
hands – they are mirror images. With no amount of times the work than testing the pure enantiomer!
rotation can you make the two images or molecules These arguments are true for other industries besides
overlap. A chiral compound will rotate the plane of pharmaceutical, for example agrochemicals. This aspect
polarized light; the degree to which it does this is called has environmental implications as it can affect the total
the specific rotation or optical rotation. amount of chemical applied to the crop.
Besides the fact that one enantiomer is often safer and The Supelco® line of chiral columns offers a broad
more efficacious than the other enantiomer, there are portfolio of columns that can be used in reversed-
other arguments for having optically pure compounds. phase mode, normal phase mode, polar organic mode,
(1) Dosing is lower. If the product contains unwanted and polar ionic mode. In addition, these columns
or inactive enantiomer, then they need to dose twice are economically priced compared to other vendors’
as much than they would if clinicians had only the pure chiral columns and we offer complete scalability from
active enantiomer. (2) No interference of the desired analytical to prep.

CHIROBIOTIC® Chiral HPLC Columns
CHIROBIOTIC® phases are based on covalently For CHIROBIOTIC® V2, changes to the linkage
bonding macrocyclic glycoproteins to a high purity chemistry and silica offer improvements for preparative
5 µm silica gel in such a way as to establish its LC and for more demanding chiral separations.
stability while retaining essential components for CHIROBIOTIC® T, T2, and TAG are based on bonding
chiral recognition. CHIROBIOTIC® V and V2 are based the amphoteric glycopeptide, Teicoplanin, which
on bonding Vancomycin, which contains 18 chiral contains 23 chiral centers surrounding four pockets
centers surrounding three pockets or cavities. Five or cavities. For CHIROBIOTIC® T2, changes to the
aromatic ring structures bridge these strategic cavities. linkage chemistry and silica offer improvements
Hydrogen donor acceptor sites are readily available for preparative LC and for more demanding chiral
close to the ring structures. CHIROBIOTIC® V has separations. CHIROBIOTIC® TAG has the sugars
demonstrated selectivity similar to glycoprotein phases removed from the macrocyclic glycopeptide to produce
except it is stable from 0-100% organic modifier and an aglycone structure as a variant of CHIROBIOTIC® T.
exhibits high sample capacity. CHIROBIOTIC® R is based on bonding Ristocetin A to
high purity 5 µm silica.
LC-MS of Fluoxetine Enantiomers on CHIROBIOTIC® V2: LC-MS of Fluoxetine Enantiomers on CHIROBIOTIC® V2: Use
Fast, Sensitive Analysis on Short, Narrow I.D. Columns Column Dimensions and Flow Rate to Enhance Sensitivity
without Loss of Resolution
Column: CHIROBIOTIC® V2, 25 cm x 4.6 mm or
10 cm x 2.1 mm I.D. 5 µm Column: CHIROBIOTIC® V2, 25 cm x 4.6 mm or
10 cm x 2.1 mm I.D., 5 µm
Mobile Phase: 10 mM ammonium formate in 10:90 v/v
water:methanol Mobile Phase: 10 mM ammonium formate in 10:90 v/v
water:methanol
Flow Rate: 1.0 or 0.2 mL/min
Flow Rate: 1.0 or 0.1 mL/min
Detection: +ESI, m/z 310
Detection: +ESI, m/z 310
Injection: 2 µL
Injection: 2 µL
Sample: 5 µg/mL in methanol
Sample: 5 µg/mL in methanol
10 cm x 2.1 mm I.D. column at

10 cm x 2.1 mm I.D. column 0.1 mL/min.
100 100
% %
25 cm x 4.6 mm I.D. column 25 cm x 4.6 mm I.D.

column at 1 mL/min.
0
2 4 6 8 10 12 14 0
2 4 6 8 10 12 14
CHIROBIOTIC® (5 µm)
Length (mm) I.D. (mm) V V2 T T2 TAG R
100 X 2.1 11018AST 15018AST 12018AST 16018AST 14018AST on request
150 x 2.1 11019AST 15019AST 12019AST 16019AST 14019AST 13019AST
250 x 2.1 11020AST 15020AST 12020AST on request 14020AST 13020AST
100 x 3.0 on request on request 12010AST on request on request on request
50 x 4.6 on request on request 12021AST on request on request on request
100 x 4.6 11022AST 15022AST 12022AST on request 14022AST 13022AST
250 x 10 11034AST on request 12034AST on request 14034AST on request
250 x 21.2 11044AST 15044AST on request on request 14044AST on request
Guard 20 x 1.0 11101AST 15101AST 12101AST on request on request 13101AST
Guard 20 x 4.0 11100AST 15100AST 12100AST on request 14100AST on request
Guard Holder x 21150AST
95
CYCLOBOND™ Chiral HPLC Columns range of chiral separation phases with guaranteed
batch to batch reproducibility, greater stability and
CYCLOBOND™ is the name given to Supelco® improved selectivity and resolution. Based on the
technology for bonding cyclodextrins to a high purity original CYCLOBOND™ I (β-cyclodextrin) columns, the
silica gel through a stable ether linkage. Introduced in CYCLOBOND™ I 2000 series are second-generation
1983, this patented stationary phase has the ability products. The native β-cyclodextrin and eight
to form inclusion complexes for a wide variety of β-cyclodextrin derivatives are in the CYCLOBOND™ I
organic molecules into the cyclodextrin cavities leading 2000 series.
to numerous chiral separations. CYCLOBOND™ I are
bonded β-cyclodextrins and CYCLOBOND™ II are CYCLOBOND™ II series columns are excellent chiral
bonded γ-cyclodextrins. selectors for multi-ring structures such as those
based on anthracene, chrysene or pyrene. These are
CYCLOBOND™ I 2000 series of HPLC columns is γ-cyclodextrin bonded phases, and consist of eight
specially formulated to meet today’s stringent glucopyranose units arranged in the same truncated
requirements for analysis in the pharmaceutical cone shape. Applications include steroids, porphorins,
industry, and for small analytes of general interest in and FMOC amino acids.
chemical and environmental areas. We have focused
on the need to accurately and reproducibly separate
enantiomers. The result is a high performance
Positional Isomers (Xylenes) on CYCLOBOND™ l 2000 Norgestrel on CYCLOBOND™ ll
Column: CYCLOBOND™ I 2000, 25 cm x 4.6 mm I.D., Column: CYCLOBOND™ II, 25 cm x 4.6 mm I.D., 5 µm
5 µm (20024AST) (41020AST)
Mobile phase A: acetonitrile B: water Mobile phase A: water B: acetonitrile
Mobile phase ratio: 15:85 (A:B) Mobile phase 70:30 (A:B)
ratio:
Temp.: 45 °C
Temp.: 42 °C
Det.: UV, 230 nm
Det.: UV, 254 nm
Injection: 3 µL
Injection: 1 µL
Sample: each compound, 0.1 mg/mL in
acetonitrile:water (50:50) Sample: norgestrel, 1 mg/mL in methanol
Elution order: m-, o-, p-xylene
0
0 4
4 8
8 12 16
12 16
Min
Min
0 2 4 6 8 10 12
min
CYCLOBOND™ (5.0 µm)

Length (mm) I.D. (mm) I 2000 I 2000 AC I 2000 SP I 2000 RSP I 2000 HP RSP I 2000 DMP II
100 x 2.1 20018AST on request on request on request on request on request on request
150 x 2.1 20019AST on request on request on request on request on request on request
100 x 4.6 on request on request on request on request on request 20722AST on request
150 x 4.6 on request 20123AST on request on request 24023AST on request 46023AST
250 x 4.6 20024AST 20124AST 20224AST 20324AST 24024AST 20724AST 41020AST
250 x 10.0 20034AST on request on request on request on request 20734AST on request
250 x 21.2 on request on request on request on request on request 20744AST on request
Guard 20 x 4.0 on request on request on request 21103AST on request on request on request
Guard Holder 21150AST

Cellulose DMP Chiral HPLC Columns Copper Ligand Exchange (5.0 µm)
Length (mm) I.D. (mm) CLC-D CLC-L
Cellulose DMP is a chiral stationary phase (CSP)
comprising spherical, high-purity porous silica coated 150 x 4.6 53023AST 53123AST
with DMPC (3,5-dimethylphenyl carbamate)-derivatized
cellulose, and packed in analytical to preparative size Protein-Based Chiral HPLC Columns
HPLC columns. It separates a wide range of chiral Hermansson described the use of natural proteins
compounds under normal phase, polar organic, and immobilized onto a silica support for chiral separations
SFC conditions, with high efficiency, high loading in 1983. Proteins contain a large number of chiral
capacity, and excellent column lifetime. Performance is centers of one configuration, and many other sites
comparable to other DMPC-derivatized cellulose CSPs. that contribute to the general retention process. We
offer three CSPs with proteins as the chiral selectors,
Key Features and Application Areas:. CHIRALPAK® AGP (α1-acid glycoprotein), CHIRALPAK®
• Classic DMPC-cellulose chiral selectivity CBH (cellobiohydrolase) and CHIRALPAK® HSA (human
serum albumin). All are manufactured by DAICEL
• Efficient, rugged, reproducible, and scalable Corporation. These columns are typically used in
• Low backpressure reversed-phase mode, and perform a wide variety of
• Ideal for chiral analysis in the pharmaceutical chiral separations. CHIRALPAK® HSA is also used for
industry and for small analytes in chemical and drug-binding studies. Solutes are retained by three
environmental areas types of interactions: ionic (for charged solutes),
hydrophobic and hydrogen bonding. The relative
• Routine chiral column method development
contribution of the different forces to solute retention
screening protocols
depends on the nature of the analyte.
• Approximately half of the cost of most other
DMPC-cellulose columns • CHIRALPAK® AGP: Extremely broad applicability. First
choice when developing methods on protein-CSPs.
Cellulose DMP is complementary to the other CSPs,
• CHIRALPAK® HSA: Analytes are typically very
including CHIROBIOTIC® and CYCLOBOND™ product
hydrophilic acids.
lines, and a must-have for every chiral HPLC or SFC
screening protocol. • CHIRALPAK® CBH: Analytes are typically very
hydrophilic amines and amino alcohols.
Cellulose DMP (5.0 µm)
Length (mm) I.D. (mm) SKU
150 x 2.1 51100AST 2.85 min
100 x 4.6 on request

N S
150 x 4.6 51098AST
250 x 4.6 51099AST
S N OH
Guard 20 x 2.1 51104AST HO
Guard 20 x 4.0 on request
O
Copper Ligand Exchange (CLC) Chiral

HPLC Columns 7.68 min
The CLC phases are based on coupling an enantiomeric
form of an amine to a proprietary derivative to create
an appropriate distance for copper coupling. Using the
copper ligand concept, this phase resolves hydroxy
acids like lactic, malic, tartaric and mandelic. This
phase can also resolve amino acids and other amines
by the same mechanism. It has been reported that, in
addition to amino acids, other bifunctional racemates
like amino alcohols can be resolved. In theory, any
analyte that can complete the coordination with the
Column: CHIRALPAK® AGP, 10 cm x 4 mm I.D., 5 µm
copper ion can be resolved. For the CLC-D column, (58150AST)
the L enantiomer generally elutes before D with the
column temp.: 25 °C
exception of tartaric acid where the D elutes first. The
CLC-L column has the opposite elution order and the D mobile phase: 10 mM sodium phosphate, pH 6.0
enantiomer elutes before L. flow rate: 0.9 mL/min
Sample: luciferin
97
CHIRALPAK® (5.0 µm)
Length (mm) I.D. (mm) AGP CBH HSA Length (mm) I.D. (mm) AGP CBH HSA
50 x 2.0 58129AST on request on request 50 x 4.0 on request 58549AST 58449AST
100 x 2.0 58130AST 58530AST 58430AST 100 x 4.0 58150AST 58550AST 58450AST
150 x 2.0 58131AST 58531AST on request 150 x 4.0 58151AST 58551AST 58451AST
50 x 3.0 58169AST 58569AST 58469AST Coupler for Legacy Guard Column Holder: 54986
100 x 3.0 58170AST 58570AST 58470AST Guard Column Holder: 58159AST
150 x 3.0 58171AST 58571AST on request
ES Industries ChromegaChiral™ U/HPLC Columns

ChromegaChiral™ Columns, manufactured by ES Industries, are innovative chiral stationary phases
designed for unique chiral solutions. ES Industries is a recognized supplier of chiral HPLC columns providing
highly efficient columns with superior reproducibility. These columns are available in 3, 5, and 10 µm
analytical and preparative sizes. Abbreviations: CCA = tris-(3,5-dimethylphenyl) carbamoyl amylose;
CCC = 3-chloro-4 methylphenylcarbamate and 3,5-dichloro- phenylcarbamate on cellulose; CCJ = cellulose
4-methylbenzoate; CCO = tris-(3,5-di-methylphenyl) carbamoyl cellulose; CCO F2 = 2-Fluoro 5-methylphenyl
cellulose; CCO F4 = 4-Fluoro 3-methylphenyl cellulose; CCS = amylose tris [(S)-α-methylbenzylcarbamate]; CC2
= cellulose 3-chloro-4 methylphenyl-carbamate; CC3 = amylose tris(5-chloro-2-methylphenylcarbamate; CC4 =
cellulose tris(4-chloro-3-methyl-phenylcarbamate
ChromegaChiral™ (3.0 µm)
Length
(mm) I.D. (mm) CCA CCC CCJ CCO CCO F2 CCO F4 CCS CC2 CC3 CC4
50 x 2.1 ES5540 ES5573
100 x 2.1 ES5395 ES5441 ES5474 ES5507 ES5543 ES5576 ES5606 ES5639 ES5672 ES5704
250 x 2.1 ES5401 ES5447 ES5480 ES5513 ES5612 ES5645 ES5677 ES5710
50 x 3.0 ES5541 ES5574
100 x 3.0 ES5396 ES5442 ES5475 ES5508 ES5544 ES5577 ES5607 ES5640 ES5705
50 x 4.6 ES5542 ES5575

Length
(mm) I.D. (mm) CCA CCA F4 CCC CCJ CCO CCO F2 CCO F4 CCS CC2 CC3 CC4
100 x 2.1 ES5404 ES5428 ES5450 ES5483 ES5516 ES5549 ES5582 ES5615 ES5648 ES5680 ES5713

Length
(mm) I.D. (mm) CCA CCC CCJ CCO CCO F2 CCO F4 CCS CC2 CC3 CC4
ChiraDex® HPLC Columns

ChiraDex® can be used for the separation of enantiomers of numerous different compounds. ChiraDex® is based
on a beta-cyclodextrin covalently linked to spherical particles of silica and is well suited for chiral separations of
hydrocarbons, steroids, phenyl esters, aromatic amines, heterocycles with 5-membered ring to 7-membered ring.
Simply composed reversed phase-eluents can be used in most separations.
ChiraDex® (5.0 µm)

Length (mm) I.D. (mm) ChiraDex® ChiraDex® HR
4 x 4 1.50117 on request
250 x 4 1.51333 on request
250 x 4 on request 1.51000
99
Biomacromolecule Characterization:
A Multipronged Separation Challenge
Antibody Screening Protein Engineering Biophysical Pre-Clinical Scale up &
& Validation & Expression Characterization Assessment production
BIOLOGY FOCUS ANALYTICAL FOCUS BIOPROCESS
Titer Antibody Drug

Primary Charge Aggregation or Glycosylation
Determination Conjugate
Structure Analysis Variant Analysis Fragment Analysis Characterization
(Affinity) (ADC) Analysis
Hydrophilic
Ion Size Hydrophobic
Reversed Phase Interaction Protein A
Exchange (IEX) Exclusion (SEC) Interaction (HIC)
(HILIC)
BIOshell™ BIOshell™
Sepax (global) Sepax (global) Chromolith® WP Sepax (global)
Chromolith® WP Glycan, SeQuant®
TOSOH Columns* TOSOH Columns* Protein A TOSOH Columns*
Discovery® Bio ZIC®-HILIC
* Tosoh Bioscience columns are available in select countries. For a list, please go to the page 107 of this brochure.
Biomacromolecules (in particular monoclonal

antibodies (mAbs)) have seen a renewed interest
in the pharmaceutical and biotechnology industry. IgG1
Va
The reason for this high level of interest resides in

r
ia
n le
L)
R Va n
bl VL
io iab
(V
i
the number of benefits these biological molecules
e )
ha
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(
eg r
R
ig n R
e
have for patients including, but not limited to, (C nt t C

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n ta h
Ch Con ion
high efficacy in treating an illness, high specificity

io ns ig
on
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Va
ai sta (CL
for a target receptor or antigen, wide therapeutic

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(V e R
ia
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Fa Ch riab (V
bl
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eg o
range, and limited, undesirable side effects.

) gi
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However, due to the fact that these molecules are

H
n
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on
ea Fa
complex and are often produced in host cell lines,

vy b
av n
C
H egio
bacteria, or fermentation reactors, these potential

on
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y
(C t
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an H1
st
b
ha
H Re
therapeutics exhibit significant heterogeneity which

an
st (C
1) g
e
in
needs to be evaluated and characterized using

on
i on
various analytical techniques. The complexity of

C
such biomacromolecules can be easily illustrated Hinge Region

N-Linked
by examining the structure of a typical mAb as Glycan N-Linked Glycan
depicted on the right. (CH2) (CH2)
Constant Regions
Constant Regions
Heavy Chain
Heavy Chain
mAbs are large, tetrameric immunoglobulin G (IgG)

molecules with a molecular weight of approximately Fc
150 kDa (150,000 g/mol). These molecules form
a Y-shape composed of four peptide chains: two
identical light (L) chains, with a molecular weight
of approximately 25 kDa each, and two identical (CH3) (CH3)
heavy (H) chains, with a molecular weight of
approximately 50 kDa each. To form the Y-shape,
these four polypeptide chains associate with each
other through the creation of inter- and intra-chain
disulfide bonds.

Due to the inherent complexity of biomacromolecules, Ion Exchange Chromatography
multiple, orthogonal modes of chromatography to
reversed-phase chromatography and HILIC are Ion Exchange Chromatography (IEX) is a mode of
required to fully characterize these molecules. chromatography that separates analytes by charge.
Proteins and peptides are amphoteric, which means
that they exhibit both acidic and basic functionalities.
Size Exclusion Chromatography The acidic portions of a protein are provided by
Size Exclusion Chromatography (SEC) is a mode aspartic acid, glutamic acid, cysteine, tyrosine, and the
of chromatography that separates molecules by α-carboxylate on the C-terminus. The basic portions
their size (i.e. hydrodynamic radius). This mode of of a protein are provided by arginine, histidine, lysine,
chromatography does not rely on the interaction of and the α-amine on the N-terminus. Charge variants
the analytes with a stationary phase ligand; it is an of a biotherapeutic, another critical quality attribute
entropic process meaning that it relies on the random (CQA) that regulatory bodies require manufacturers
flow of the analytes through the stationary phase to monitor, can be detected and resolved by IEX.
particles. For most practical purposes, this can be These charge variants can arise from mistranslation
envisioned as analytes with a higher molecular weight of messenger RNA (mRNA) transcripts and/or post-
will elute earlier in the run, since these analytes translational modifications such as deamidation,
are fully or partially excluded from the pores of the oxidation, or glycosylation, among others.
stationary phase particles, while lower molecular
weight analytes will elute later in the run, since these Affinity Chromatography
analytes will spend more time navigating the tortuous
path through the particle. Affinity chromatography is a mode of chromatography
that relies on a specific interaction between the analyte
of interest and the stationary phase ligand. Ideally, no
Hydrophobic Interaction Chromatography other component of the sample would interact with the
Hydrophobic Interaction Chromatography (HIC) is a ligand, thus only the analyte of interest interacts with
mode of chromatography that separates analytes based the stationary phase. Afterwards, a different mobile
on the degree of interaction between hydrophobic phase is passed through the column that breaks this
moieties on the analyte and hydrophobic ligands interaction, thus eluting the analyte.
on the stationary phase. Under conditions of high
concentrations of salt, the hydration layer around a Reversed Phase Chromatography
protein may be disrupted enough such that it becomes
entropically favorable for hydrophobic regions of Reversed-Phase Chromatography (RPC) is a mode
the protein’s surface to interface with the non-polar of chromatography that separates analytes based
stationary phase. This phenomenon is not unlike the primarily on hydrophobicity. Unlike HIC, RPC employs a
classical biochemical technique of protein salting out, water/organic mixture for the mobile phase. Typically,
but in HIC’s case, the interactions are between protein- this mobile phase combination is supplemented with
stationary phase ligand, not between different protein an ion pairing reagent like trifluoroacetic acid (TFA),
molecules. Due to the lower molecular weight and lower formic acid, or difluoroacetic acid (DFA) to mask the
propensity for folding, HIC is usually not employed for secondary interactions between exposed silanols on
separating peptides. Salt selection in HIC is dictated the silica stationary phase and H-bonding donor groups
by the Hofmeister series, which classifies cations and on analytes. Common applications for characterizing
anions in terms of their ability to disrupt the hydration biomolecules by RPC include peptide mapping, where a
layer around a protein (chaotropic) or promote the protease, like trypsin, cleaves a protein at defined sites
formation of a hydration layer (kosmotropic). Typical into characteristic peptide fragments, and middle-up
salts in HIC are ammonium sulfate, potassium sulfate, analyses, which include reducing a protein into larger
and sodium sulfate. Elution is achieved by gradients fragments for easier characterization.
with decreasing salt concentration.
101
Sepax Technologies: HPLC Columns for Bioanalysis
Size Exclusion Chromatography (SEC)
Unix™ SEC-200 and SEC-300 UHPLC Columns SRT® and SRT®-C HPLC Columns
Utilizing proprietary surface technologies, Unix™ SRT® size exclusion columns are prepared from high
SEC-200 and SEC-300 phases are made of uniform, purity, porous silica that is treated to provide the
hydrophilic, and neutral nanometer thick films mechanical stability required for high performance
chemically bonded to high purity and mechanically HPLC analysis of biopolymers. The particles are
stabilized silica with the particle size of 1.8 µm. derivatized with a uniform, hydrophilic bonded layer
The combination of small particle size and large to minimize interaction of sample molecules with the
pore volume of Unix™ SEC-300 renders the highest silica surface. The proprietary surface technology
separation efficiency and resolution of analytes. The results in excellent column-to-column reproducibility,
well-controlled surface chemistry results in excellent while the composition of the bonded phase and the
lot-to-lot reproducibility. The unique bonding chemistry, maximized bonding density impart excellent chemical
coupled with the maximized bonding density, allows stability and negligible non-specific interactions. In
Unix™ SEC-300 to provide high stability and negligible addition to the very high pore volume per unit column
non-specific interactions. Typical applications for Unix™ volume, SRT® columns are available in six different
SEC-300 columns include separation and analysis of pore sizes from 100 Å to 2000 Å, allowing the user to
biological molecules and water-soluble polymers. select the pore size that best matches the dynamic
radius of the biopolymer, be it a peptide, protein, virus,
Unix™ (1.8 µm) or vaccine. SRT®-C SEC columns are packed with high
Length (mm) I.D. (mm) SEC-200 SEC-300
purity and mechanically stable, 5 µm, silica particles
which are derivatized with a uniform, chemically
150 x 4.6 Z777303 Z777300
neutral, hydrophilic bonded phase that effectively
300 x 4.6 Z777304 Z777302 shields the sample from interacting with the underlying
Guard 10 x 4.0 Z777305 Z777301 silica. Since SRT®-C columns can be operated up to
3500 psi pressure, they allow faster analysis and higher
throughput than competitor columns. SRT®-C columns
are the preferred choice for relatively hydrophobic
sample types such as insulin, membrane proteins and
derivatized monoclonal antibodies.
SRT® (5.0 µm)

Length (mm) I.D. (mm) Column Hardware SEC-100 SEC-150 SEC-300 SEC-500 SEC-1000 SEC-2000
300 x 4.6 Stainless Steel Z777037 Z777043 Z777049 Z777055 Z777061 Z777067
300 x 4.6 PEEK Z777041 Z777047 Z777053 Z777059 Z777065 Z777071
Guard 50 x 4.6 Stainless Steel Z777036 Z777042 Z777048 Z777054 Z777060 Z777066
Guard 50 x 4.6 PEEK Z777040 Z777046 Z777052 Z777058 Z777064 Z777070
SRT®-C (5.0 µm)

Length (mm) I.D. (mm) Column Hardware SEC-100 SEC-150 SEC-300 SEC-500 SEC-1000 SEC-2000
300 x 4.6 PEEK Z777100 Z777106 Z777112 Z777118 Z777124 Z777130
Guard 50 x 4.6 PEEK Z777099 Z777105 Z777111 Z777117 Z777123 Z777129

Zenix™ and Zenix™-C HPLC Columns
Use Zenix™ SEC high performance gel filtration sample throughput. Zenix™-C SEC columns are the
columns to analyze hydrophilic polymers including preferred gel filtration columns to analyze relatively
proteins and other water soluble polymers. Prepared hydrophobic sample types such as insulin, membrane
from spherical 3 μm silica particles, Zenix™ SEC proteins and derivatized monoclonal antibodies.
columns represent a breakthrough technology for Zenix™-C SEC columns are packed with high purity
high performance size exclusion chromatography of and mechanically stable, 3 µm, silica particles that are
biopolymers. The combination of 3 μm silica particle chemically modified with a uniform, chemically neutral,
size and a proprietary surface technology provides hydrophilic bonded phase that effectively shields the
the highest separation efficiency and resolution for sample from interacting with the underlying silica. Since
biological molecules and water soluble polymers. Zenix™-C columns can be operated up to 3500 psi
Zenix™ columns are available in 100, 150 and 300 Å pressure, they allow fast analysis and high sample
pore sizes, and are packed in stainless steel or PEEK throughput. Zenix™-C columns are available in 100,
hardware. Since Zenix™ columns can be operated up 150 and 300 Å pore sizes, and are packed in stainless
to 3500 psi pressure, they allow fast analysis and high steel or PEEK hardware.
Zenix™ (3.0 µm)

Length (mm) I.D. (mm) Column Hardware SEC-100 SEC-150 SEC-300
300 x 1.0 Stainless Steel Z777002 Z777012 Z777022
150 x 4.6 Stainless Steel Z777026
300 x 4.6 PEEK Z777010 Z777020 Z777035
Guard 50 x 1.0 Stainless Steel Z777001 Z777011 Z777021
Guard 50 x 4.6 PEEK Z777009 Z777019 Z777034
Zenix™-C (3.0 µm)

Length (mm) I.D. (mm) Column Hardware SEC-100 SEC-150 SEC-300
300 x 4.6 PEEK Z777077 Z777083 Z777094
Guard 50 x 4.6 PEEK Z777076 Z777082 Z777093
103
Ion Exchange Chromatography
Antibodix™ U/HPLC Columns with 1.7, 3, 5 or 10 μm particles that can withstand

pressures varying from 4,000 psi (10 μm) up to
Antibodix™ columns are specially designed for 10,000 psi (1.7 μm). The hydrophobic PS/DVB resin
high resolution, high efficiency and high recovery surface is shielded by a hydrophilic, neutral polymer to
separations of antibodies. These columns are packed which a dense layer of weak cation-exchange functional
with spherical, non-porous, particles that consist groups are attached, thus combining the benefits of
of highly cross-linked poly(styrene divinylbenzene) minimal secondary interaction of antibodies with the
(PS/DVB). Antibodix™ columns are available packed base matrix and high dynamic binding capacity.
A. Antibodix™ WCX-NP10
25 cm × 4.6 mm I.D., 10 µm (non-porous)
B. Vendor D WCX Column: Antibodix™ WCX-NP10, 25 cm x 4.6 mm I.D., 10 µm

25 cm × 4.0 mm I.D., 10 µm (non-porous) (Z777272)
Column 25 °C
1. Cytochrome c (12.2 kDa) temp.:
2. Lysozyme (14.3 kDa)
3. Ribonuclease A (13.7 kDa) 3 Mobile phase: (A) 10 mM sodium phosphate buffer, 6.0; (B) A +
1 2 1.0 M sodium chloride
Gradient: 0 to 100% B in 42 min
A Flow rate: 1.0 mL/min

3 Sample: 1 mg/mL each
Injection: 5 µL
2
B 1 Detector: UV, 214 nm
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Min
Antibodix™ (1.7 µm) Antibodix™ (3.0 µm)

Length (mm) I.D. (mm) Column Hardware SKU Length (mm) I.D. (mm) Column Hardware SKU
30 x 2.1 Stainless Steel Z777278 30 x 2.1 Stainless Steel Z777282
Guard 10 x 2.0 Stainless Steel Z777277 50 x 2.1 PEEK Z777287
Guard 10 x 4.0 Stainless Steel Z777279 50 x 4.6 Stainless Steel Z777285
50 x 4.6 PEEK Z777288
Antibodix™ (5.0 µm)
Length (mm) I.D. (mm) Column Hardware SKU
Guard 10 x 2.0 Stainless Steel Z777281
50 x 2.1 PEEK Z777295
50 x 4.6 Stainless Steel Z777292 Antibodix™ (10.0 µm)
50 x 4.6 PEEK Z777296 Length (mm) I.D. (mm) Column Hardware SKU
250 x 4.6 Stainless Steel Z777294 50 x 2.1 PEEK Z777273
250 x 4.6 PEEK Z777297 250 x 2.1 Stainless Steel Z777269
Guard 10 x 2.0 Stainless Steel Z777289 250 x 2.1 PEEK Z777274
Guard 10 x 4.0 Stainless Steel Z777291 50 x 4.6 Stainless Steel Z777271
50 x 4.6 PEEK Z777275
250 x 4.6 PEEK Z777276

Proteomix® U/HPLC Columns to eliminate non-specific binding. Using proprietary
coupling chemistry, weak (WAX and WCX) and strong
Based on Non-Porous Particles, the Proteomix ion- ®
(SAX and SCX) anion and cation exchange functional
exchange column line from Sepax Technologies groups are attached to the hydrophilic bonded phase to
was designed to achieve high recovery of peptides, obtain a high capacity ion-exchange layer. Proteomix®
proteins, oligonucleotides, polysaccharides, cell columns are available packed with 1.7, 3, 5 or 10 μm
lysates, nanoparticles and nanotubes when analyzed particles that can withstand pressures varying from
under HPLC or UHPLC conditions. Proteomix® columns 4,000 psi (10 μm) up to 10,000 psi (1.7 μm). Non-
are packed with spherical, non-porous, poly(styrene porous Proteomix® ion-exchange columns are unique
divinylbenzene) (PS/DVB) particles that are as they combine increased ion exchange capacity with
encapsulated with a hydrophilic, neutral polymer layer high efficiency, recovery and throughput.
Proteomix® Anion Exchange (1.7 µm) Proteomix® Anion Exchange (3.0 µm)
Length I.D. Column Quaternary Tertiary Length I.D. Column Quaternary Tertiary
(mm) (mm) Hardware Ammonium Amine (mm) (mm) Hardware Ammonium Amine
30 x 2.1 Stainless Steel Z777210 Z777244 30 x 2.1 Stainless Steel Z777216 Z777250
50 x 2.1 Stainless Steel Z777211 Z777245 50 x 2.1 PEEK Z777221 Z777255
Guard 10 x 2.0 Stainless Steel Z777209 Z777243 50 x 4.6 PEEK Z777222 Z777256
Guard 10 x 4.0 Stainless Steel Z777212 Z777246 150 x 4.6 Stainless Steel Z777220 Z777254
Guard 10 x 2.0 Stainless Steel Z777215 Z777249
Proteomix® Anion Exchange (5.0 µm)
Length I.D. Column Quaternary Tertiary
(mm) (mm) Hardware Ammonium Amine
Proteomix® Anion Exchange (10.0 µm)
50 x 2.1 Stainless Steel Z777224 Z777258
Length I.D. Column Quaternary Tertiary
50 x 2.1 PEEK Z777230 Z777264 (mm) (mm) Hardware Ammonium Amine
50 x 4.6 PEEK Z777231 Z777265 250 x 2.1 Stainless Steel Z777201 Z777235
250 x 4.6 PEEK Z777232 Z777266 50 x 4.6 PEEK Z777207 Z777241
105
Proteomix® Cation Exchange (1.7 µm) Proteomix® Cation Exchange (3.0 µm)
Length I.D. Column Length I.D. Column
(mm) (mm) Hardware Sulfonate Carboxylate (mm) (mm) Hardware Sulfonate Carboxylate
Proteomix® Cation Exchange (5.0 µm)
Length I.D. Column
(mm) (mm) Hardware Sulfonate Carboxylate
Proteomix® Cation Exchange (10.0 µm)
50 x 2.1 Stainless Steel Z777156 Z777190
Length I.D. Column
50 x 2.1 PEEK Z777162 Z777196 (mm) (mm) Hardware Sulfonate Carboxylate
50 x 4.6 Stainless Steel Z777159 Z777193 50 x 2.1 PEEK Z777171
50 x 4.6 PEEK Z777163 Z777197 250 x 2.1 Stainless Steel Z777133 Z777167
250 x 4.6 PEEK Z777164 Z777198 50 x 4.6 PEEK Z777139 Z777173
Hydrophobic Interaction Chromatography (HIC)
Proteomix® HIC U/HPLC Columns 3

1. Polar Variant 1
2. Polar Variant 2
Proteomix® HIC columns are specially designed for
3. Native NIST mAb
high resolution and high efficiency separations of
proteins and oligonucleotides. Utilizing proprietary
surface technologies, Proteomix® HIC-NP resin is made
of non-porous polystyrenedivinylbenzene (PS/DVB)
beads with narrow-dispersed particle size distribution. 2
The PS/DVB bead is modified with alkyl groups or
aryl group that provides hydrophobic interaction with 1
analytes. Proteomix® HIC-NP resin is highly rigid and
mechanically stable. In comparison to silica-based
HIC phase media, Proteomix® HIC-NP phases have 5 10 15 20
advantages for biomolecule separations with wide pH Min
range (2-12) and high thermal stability. The nonporous
Column: Proteomix® HIC Butyl-NP5, 10 cm x 4.6 mm I.D., 5 µm
structure and narrow particle size distribution offer
special selectivity, high resolution separation of proteins Mobile [A] 2 M ammonium sulfate in 100 mM sodium
phase: phosphate, pH 7.0; [B] 100 mM sodium phosphate,
such as mAb (monoclonal antibody), ADC (antibody pH 7.0
drug conjugate) and related protein fragments, DNA
and oligonucleotides. Proteomix® HIC-NP media is
applicable at laboratory discovery, laboratory-scale Flow rate : 0.5 mL/min
purification and process chromatography for the Column 30 °C
production of a few mgs to kilogram of proteins. temp.:
Injection: 2.0 µL
Sample: NIST mAb, 10 mg/mL, 12.5 mM histidine, pH 6.0

Proteomix® HIC (5.0 µm)
Length I.D. Length I.D.
(mm) (mm) Butyl Ethyl Phenyl Propyl (mm) (mm) Butyl Ethyl Phenyl Propyl
50 x 2.1 61862-U 150 x 4.6 61866-U
35 x 4.6 61869-U 61874-U 61881-U 50 x 7.8 61867-U 61871-U 61878-U 61884-U
100 x 4.6 61865-U 61870-U 61876-U 61883-U Guard 10 x 4.0 61863-U 61868-U 61873-U 61879-U
Tosoh* Bioscience: HPLC Columns for Bioanalysis

Size Exclusion Chromatography (SEC)
TSKgel® UP-SW2000 and UP-SW3000 TSKgel® SWXL Type and QC-PAK

UHPLC Columns HPLC Columns
TSKgel® UP-SW3000 and UP-SW2000, the new 2 µm TSKgel® SWxl columns, introduced in 1987, represent
UHPLC columns with 25 nm and 12.5 nm pore sizes, the second generation of high performance Gel
respectively, are the latest addition to the renowned Filtration columns that have become synonymous
TSKgel® SW series. TSKgel® UP-SW represents the fifth with analyzing protein molecular weights in the
generation of high performance gel filtration columns. emerging field of biotechnology. TSKgel® SWxl-type
These columns feature the same pore size as the well- columns contain smaller particles than TSKgel® SW-
established TSKgel® G3000SWXL and SuperSW2000 type columns; 5 and 8 μm versus 10 and 13 μm.
columns and facilitate method transfer from As the TSKgel® SW-type columns, TSKgel® SWxl
conventional gel filtration to UHPLC technology. The columns feature highly porous silica particles with a
15 cm column is used to shorten analysis time while surface functionalized with diol groups to prevent any
maintaining resolution. The 30 cm column delivers interaction with proteins. TSKgel® SWxl-type columns
dramatically increased resolution between fragments, distinguish themselves from other silica- or polymer-
monomers, and aggregates. based high performance size exclusion columns by
virtue of their large pore volumes. These columns are
TSKgel® UP-SW (2.0 µm) commonly used in the quality control of monoclonal
Length (mm) I.D. (mm) SW3000 SW2000
antibodies and other biopharma products. TSKgel®
BIOassist columns are packed in PEEK hardware.
150 x 4.6 80023449 823515
300 x 4.6 80023448 823514
Direct Connect Guard 20 x 4.6 80023451 823517
Guard 20 x 4.6 80023450 823516
TSKgel® SWXL (5.0 µm)

Length (mm) I.D. (mm) G2000 G3000 G4000 QC-PAK 200 QC-PAK 300 SWXL-125 SWXL-250 SWXL-450
150 x 7.8 816215 816049
300 x 7.8 808540 808541 820027 820026

Guard 40 x 6.0 818008 808543

300 x 7.8 808542 820025
*Tosoh Bioscience columns are available in the following countries: Argentina, Albania, Andorra, Armenia, Austria, Azerbaijan, Afghanistan,
Algeria, Albania, Armenia, Azerbaijan, Bolivia, Brazil, Belarus, Belgium, Bosnia and Herzegovina, Bulgaria, Bahrain, Bosnia-Herzegovina, Botswana,
Burkina-Faso, Chile, Colombia, Costa Rica, Cuba, Caribbean Islands, Canada, Croatia, Cyprus, Czechia, Cameron, Central Afr. Rep, Chad, Congo,
Congo dem. Rep., Croatia, Denmark, Dominican Republic, Djibouti, Ecuador, El Salvador, Egypt, Eritrea, Ethiopia, Estonia, French Guiana, Finland,
France, Georgia, Germany, Greece, Gabon, Georgia, Ghana, Guatemala, Guyana, Haiti, Honduras, Hungary, Iceland, Ireland, Italy, Iraq, Israel,
Kazakhstan, Kosovo, Kenya, Kosovo, Kuwait, Kyrgyzstan, Lebanon, Libya, Latvia, Liechtenstein, Lithuania, Luxembourg, Malta, Moldova, Monaco,
Montenegro, Macedonia, Madagascar, Malawi, Mauretania, Mauritius, Moldavia, Mongolia, Morocco, Mozambique, Mexico, Nicaragua, Nigeria,
Netherlands, North Macedonia (formerly Macedonia), Norway, Oman, Poland, Portugal, Panama, Paraguay, Peru, Puerto Rico, Qatar, Rwanda,
Romania, Russia, San Marino, Serbia, Slovakia, Slovenia, Spain, Sweden, Suriname Switzerland, Saudi Arabia, Senegal, Serbia-Montenegro, South
Africa, Tadzhikistan, Tanzania, Togo, Tunisia, Turkey, Turkmenistan, Ukraine, United Kingdom (UK), Uruguay, United States of America, Uganda,
Utd.Arab.Emir., Uzbekistan, Vatican City, Venezuela, Yemen, Zambia, Zimbabwe, Belize
107
TSKgel® SuperSW, SuperSW mAb and
Detector Response (mAU)

UltraSW Aggregate HPLC Columns 30
monomer
TSKgel SuperSW columns, introduced in 1997,

®
dimer
20
represent the third generation of high performance Gel
Filtration columns that have become synonymous with Rs (3mer/2mer) = 1.40
10 trimer Rs (2mer/monomer) = 2.89
analyzing protein molecular weights in the emerging
aggregate
field of biotechnology. TSKgel® SuperSW-type columns
contain smaller particles than TSKgel® SWxl-type 0
3 5 7 9 11 13 15
columns: 4 µm versus 5 µm. In addition, the column
internal diameter has been reduced from 7.8 mm
I.D. to 4.6 mm I.D. to provide higher sensitivity in Column: TSKgel® UltraSW Aggregate, 30 cm x 7.8 mm I.D.,
sample-limited cases and to cut down on solvent use; 3 µm particles
in addition to 4.6 mm I.D., TSKgel® SuperSW3000 Mobile phase: 0.2 M phosphate buffer, pH 6.7 + 0.05% sodium azide
columns are also available in 2 mm and 1 mm I.D.
column formats. It is important to employ an HPLC
system that is optimized with regards to extra-column Sample: Monoclonal antibody 2
(mouse-human chimeric IfG, Erbitux)
band broadening to take full advantage of the high
column efficiency that can be obtained on TSKgel® Injection: 10 µL
SuperSW columns. The TSKgel® SW mAb product Detector: UV, 280 nm
line consist of three specialized columns designed for
the separation and analysis of monoclonal antibodies
(mAb). While mAbs can be analyzed using many
TSKgel® SuperSW (4.0 µm) and UltraSW (3.0 µm)
different modes of HPLC, size exclusion is best to
Length I.D. mAb- mAb- UltraSW-
determine aggregation, dimer, and fragmentation,
(mm) (mm) HR HTP 2000 3000 Aggregate
making it the best method for heterogeneity studies.
300 x 1.0 821845
The TSKgel® SW mAb columns include:
300 x 2.0 821485
• TSKgel® SuperSW mAb HR column is best suited for
150 x 4.6 822855
high resolution analysis of mAb monomer and dimer
300 x 4.6 818674 818675
• TSKgel® SuperSW mAb HTP column is optimized for
high throughput analysis of mAb monomer and dimer 300 x 7.8 822854 822856
(UHPLC compatible) Guard 30 x 3.0 822858
TSKgel® UltraSW Aggregate column provides the best Guard 35 x 4.6 818762
solution for the analysis of mAb aggregates. Compared Guard 40 x 6.0 822857 822859
to competitive columns, these stainless steel, silica-
based TSKgel® columns offer reduced lot-to-lot
variation, long column life, reduction of unspecified
adsorption, and superior recovery of aggregates. TSKgel® SW Type HPLC Columns
The TSKgel® SW mAb columns utilize a unique pore- TSKgel® SW columns, introduced in 1977, were the
controlled technology, which produces a shallow first of a long line of high performance Gel Filtration
calibration curve in the molecular weight region of a columns that have become synonymous with
typical monoclonal antibody. The calibration curve for analyzing protein molecular weights in the emerging
the TSKgel® SuperSW mAb HR column is similar to that field of biotechnology. TSKgel® SW-type columns
of the TSKgel® G3000SWxl column and has a shallower are based on highly porous silica particles with a
slope than the TSKgel® UltraSW Aggregate column surface functionalized with diol groups to prevent any
around the molecular weight range of gamma-globulin. interaction with proteins. TSKgel® SW-type columns
This shallow calibration curve produces high resolution distinguish themselves from other silica- or polymer-
separations. The TSKgel® UltraSW Aggregrate based high performance size exclusion columns by
calibration curve shows a separation range up to virtue of their large pore volumes.
around 2 million Da, which implies better resolution of
aggregate/multimer of a monoclonal antibody. TSKgel® SW (10.0 µm)
Length I.D.
(mm) (mm) G2000 G3000
300 x 7.5 805788 805789
600 x 7.5 805102 805103
300 x 8.0 808800
Guard 75 x 7.5 805371
Guard 40 x 8.0 808805

TSKgel® SW (13.0 µm) TSKgel® 5PW Anion Exchange (20.0 µm)
Length I.D. Length (mm) I.D. (mm) DEAE SuperQ
(mm) (mm) G2000 G3000 G4000
Guard 25 x 6.0 807210 818388
300 x 7.5 805790
Guard 10 x 8.0 808806
600 x 7.5 805104
Guard 35 x 10.0 816092
300 x 8.0 808801
300 x 21.5 806727 806728 TSKgel® NPR
600 x 21.5 805146 805147 Length (mm) I.D. (mm) 2.5 µm 5.0 µm
Guard 75 x 21.5 805758 35 x 4.6 813075

75 x 4.6 818249
TSKgel® SW (17.0 µm)
Guard 5 x 4.6 818253 817088
Length I.D.
(mm) (mm) G4000 TSKgel® BioAssist Q
300 x 21.5 806729 Length (mm) I.D. (mm) 10 µm 13 µm
600 x 21.5 805148 50 mm x 4.6 mm 819685
100 mm x 10 mm 821410
Ion Exchange Chromatography
TSKgel® STAT
Ion exchange chromatography separates molecules
Length I.D.
based on differences in the net charge of the (mm) (mm) 5.0 µm 7.0 µm 10.0 µm
molecules. In anion exchange, the analyte is negatively
35 x 3.0 821960
charged (anion), while the chromatographic material is
100 x 4.6 821962 821961
positively charged (cation). Ion exchange is commonly
used for protein purification, but it may be used for
Silica-based TSKgel® DEAE Anion Exchange
purification of oligonucleotides, peptides, or other
I.D.
charged molecules. For interaction to occur, the protein
Length (mm) (mm) 5.0 µm 10.0 µm 20.0 µm
of interest must have a charge opposite to that of the
250 x 2.0 818761
functional group of the sorbent particle. Because the
interaction is ionic, binding must take place under 250 x 4.6 807168
low ionic strength conditions. Elution is achieved by 75 x 7.5 807163
increasing the ionic strength of the mobile phase to Guard 10 x 2.0 842154
reduce ionic attractions, or by changing the pH of the
Guard 25 x 6.0 807648
mobile phase to alter the ionization state of the protein.
TSKgel® columns are highly efficient for sample Silica-based TSKgel® QAE Anion Exchange
purification with excellent recovery. Anion- and cation- Length (mm) I.D. (mm) SKU
exchangers are available on porous polymer-based and 250 x 4.6 807166
silica-based particles. Proteins and nucleic acids can be
Guard 25 x 6.0 807646
analyzed faster on a TSKgel® non-porous resin column,
although STAT and NPR columns have lower capacity
than particles with large pore sizes. TSKgel® BioAssist
columns are constructed from PEEK (polyether
ether ketone).
Anion Exchange HPLC Columns:
TSKgel® 5PW Anion Exchange (10.0 µm)

Length (mm) I.D. (mm) DEAE SuperQ
75 x 2.0 818757
50 x 5.0 813061
75 x 7.5 807164 818257
75 x 8.0 808802 818386
TSKgel® 5PW Anion Exchange (13.0 µm)

Length (mm) I.D. (mm) DEAE SuperQ
150 x 20 814016
150 x 21.5 807574 818387
Guard 20 x 20 814466
109
Cation Exchange HPLC Columns:
40
Column: TSKgel® CM-STAT, 10 cm x 4.6 mm I.D., 7 µm

30 Column temp.: 25 °C
Mobile phase: [A] 20 mM MES buffer, pH 6.0; [B] 0.5 M sodium
chloride in buffer A, pH 6.0
mV
20 E
Gradient: 0% B (0 min), 30% B (15 min), 100% B (15 min),
D 0% B (17 min), 10% B (17 min), 10% B (21 min)
10 C Flow rate: 1 mL/min
B Sample: monoclonal antibodies (mAb A through E)
0 A Injection: 20 µL
0 10 20 30 40
Min
TSKgel® 5PW Cation Exchange (10.0 µm) TSKgel® STAT Carboxymethyl

Length (mm) I.D. (mm) CM SP Length (mm) I.D. (mm) 7.0 µm 10.0 µm
75 x 2.0 818758 35 x 3.0 821965
50 x 5.0 813062 100 x 4.6 821966
75 x 7.5 813068 807161
TSKgel® STAT Sulfonic Acid
75 x 8.0 808803
Length (mm) I.D. (mm) 7.0 µm 10.0 µm
Guard 25 x 6.0 813069
35 x 3.0 821963
TSKgel® 5PW Cation Exchange (13.0 µm) 100 x 4.6 821964
Length (mm) I.D. (mm) SP
Silica-based TSKgel® Carboxymethyl Cation Exchange
150 x 20 814017
150 x 21.5 807575
250 x 4.6 807167
TSKgel® 5PW Cation Exchange (20.0 µm) 75 x 7.5 807162
Length (mm) I.D. (mm) SP Guard 25 x 6.0 807650
Guard 25 x 6.0 807211
Silica-based TSKgel® Sulfonic Acid Cation Exchange
Guard 10 x 8.0 808807
Length (mm) I.D. (mm) 5.0 µm
Guard 35 x 10.0 816093
250 x 4.6 807165
TSKgel BioAssist S
®
Guard 25 x 6.0 807644
50 x 4.6 819686
100 x 10 821411
TSKgel® NPR Cation Exchange (2.5 µm)

35 x 4.6 813076

Hydrophobic Interaction Chromatography (HIC)
Hydrophobic Interaction Chromatography (HIC) is a and are eluted by a decreasing salt gradient. As
gentle technique compared to reversed-phase LC for a result, hydrophobic interaction chromatography
the binding and desorption of hydrophobic proteins. combines the gentleness of salt precipitation with the
The use of aqueous mobile phases in HIC is less precision of chromatography for excellent recovery of
likely to disturb protein conformation and results in protein activity.
better activity recovery. Biomolecules adsorb to a
TSKgel® BioAssist Phenyl columns are constructed from
weak hydrophobic surface at high salt concentrations
PEEK (polyether ether ketone).
Fab Peaks Fc Peaks
100
Incubated with 0.1% tBHP
1 0
50 2
0
Detector Response (mAU)
10 12 14 16 Column: TSKgel® Butyl-NPR, 3.5 cm x 4.6 mm I.D.,

2.5 µm (814947)
100
Incubated with 0.01% tBHP Mobile phase: [A] 2 M ammonium sulfate, 20 mM Tris, pH 7;
50 [B] 20 mM Tris, pH 7
Gradient: 10 to 100% B in 34 min
0
10 12 14 16 Flow rate: 1 mL/min
100
Incubated with 0% tBHP (control)
50
0
10 12 14 16
Min
TSKgel® Butyl NPR TSKgel® Ether-5PW

Length (mm) I.D. (mm) SKU Length (mm) I.D. (mm) 10.0 µm 20.0 µm
35 x 4.6 814947 10 x 2.0 842156
100 x 4.6 842168 75 x 2.0 818760
50 x 5.0 814013
TSKgel® Phenyl-5PW
75 x 7.5 808641
Length (mm) I.D. (mm) 10 µm 13 µm 20 µm
10 x 8.0 814025
75 x 2.0 818759
75 x 8.0 814014
50 x 5.0 813063
Guard 25 x 6.0 808643
75 x 7.5 807573
50 x 7.8 820023
75 x 8.0 808804
150 x 21.5 807656
Guard 25 x 6.0 807652
Guard 35 x 10.0 816095
111
Affinity Chromatography
Affinity chromatography allows purification of biomolecules on the basis of biological function or

three-dimensional structure. The molecule to be purified is specifically and reversibly adsorbed by
a complementary binding ligand immobilized on a matrix. The natural specificities of the interacting
molecules offer high selectivities that can greatly reduce the time needed to purify the molecule. High-
efficiency resin-based TSKgel® affinity columns separate or purify many enzymes and other proteins.
Mid activity
ABS 280nm
Low activity
High activity
0 5 10 15 20
Retention time (minutes)
Chromatographic Conditions
Column: TSKgel® FcR-IIIA-NPR, 7.5 cm x 4.6 mm I.D., 5 μm, PEEK (823513)
Mobile phase: [A] 50 mM Citrate, pH 6.5 ; [B] 50 mM Citrate, pH 4.5
Flow rate: 1 mL/min
Injection: 30 µL
Sample: Rituximab, 1 µg/µL, Rituximab kindly provided by Rentschler Biopharma
TSKgel® Iminodiacetic Acid TSKgel® Tresyl (10 µm)

Length (mm) I.D. (mm) 10 µm 13 µm 20 µm Length (mm) I.D. (mm) SKU
50 x 5.0 814440 40 x 6.0 814455
75 x 7.5 808645 75 x 7.5 814456
50 x 7.8 820022
TSKgel® FcR (5.0 µm)
150 x 21.5 808646
Guard 25 x 6.0 808647
75 x 4.6 823513
TSKgel Boronate
®
Length (mm) I.D. (mm) 10 µm 20 µm

50 x 5.0 814449
75 x 7.5 813066
Guard 25 x 6.0 813125
Guard 10 x 8.0 814451
Chromolith® Protein A monolithic columns for Affinity Chromatography,

please see page 55

Protein Purification Chromatography
You have a diverse set of molecules that need to be

purified. We have a full suite of industry-trusted and
proven chromatography resins and membranes to help
you to tackle them all from lab to process scale.
• Membrane Chromatography • Process Chemicals

• Affinity Chromatography • Multi-use Systems
• Ion Exchange Chromatography • Single-use Systems
• Prepacked Columns
Chromatography Resins
Our portfolio of reliable, trusted chromatography solutions has been optimized for
your molecule across its entire life cycle, from early-phase development through
commercial manufacturing.
Our proven tentacle technology offers a number of advantages compared to
conventional resins due to increased accessibility of functional groups and binding
of target molecules. This unique feature allows reliable purification and efficiency
for our process with high selectivity, excellent yield and purity. We optimized the
tentacular surface chemistry of our resins for different applications.
Eshmuno® CP-FT resin Eshmuno® CPX resin Eshmuno® CPS resin
For more information on protein purification products,

please visit
SigmaAldrich.com/safc/bioprocess/purification.html
113
Synthetic Carbons for
Chromatographic Applications
Our portfolio includes many different carbon These particles are used for an analyte size relative to
adsorbents, tailored to meet customer needs. These C3-C20+ n-alkanes. These also have lower adsorbent
particles can be designed with: strength than the CMS products.
• The desired shape – either spherical or granular Supel™ Carbon LC – These innovative, porous
• No pores, or more or less of the desired pores sub-3 µm particles are used in HPLC applications, and
(micro-, meso-, macro-) to meet the application exhibit high pressure stability, elevated temperature
of interest stability, unique retention mechanisms, and are
compatible with any solvent system. The figure on
• Tapered pores, which increase thermodynamic and
the next page highlights the repeatability and narrow
kinetic efficiency
particle size distribution of this material.
• A through-pore or a closed-pore structure,
which influences microporous strength and
kinetic effectiveness
About Supel™ Carbon LC HPLC Particles
• Surface pH adjustments, from 2.5 to 10.5 Supel™ Carbon LC particles are a fully porous graphitic
carbon-based material designed specifically for HPLC
These carbons can be used in gas chromatography and UHPLC applications. Supel™ Carbon LC is the first
(GC), liquid chromatography (LC), solid phase commercially available sub-3 µm particle. This new,
extraction (SPE), and bulk scale applications. A patented, porous graphitic carbon (PGC) exhibits much
sampling of these carbon particles includes: narrower particle size distributions compared to pre-
Carbosieve™ – These are spherical, non-friable, highly existing technology, as the plots on the next page
porous particles and used for an analyte size relative indicate. PGC materials are very similar to graphitized
to the C2-C5 n-alkanes. These particles have non- carbon blacks (GCBs), but PGC has a spongy structure
tapered pores and very high adsorptive strength. These which is able to withstand the shearing forces occuring
particles are effective for small, volatile analytes that in HPLC. As a packing for HPLC, PGC can act as a
most adsorbents have trouble retaining. strong hydrophobic adsorbent behaving similar to
reversed-phase (RP) chromatography. However,
Carboxen® – These particles are carbon molecular where PGC differs to RP chromatography is in its
sieves (CMS), much like the carbosieve materials, ability to retain more polar analytes that tend to
but include an expanded selection of physical elute too early by RP. In addition, planar (especially
characteristics. Many of the carboxen materials include aromatic) compounds due to their shape have intense
a combination of micro-, meso-, and macropores to suit interactions with the surface resulting in strong
customer needs. retention as consequence of the graphitic nature of the
Graphsphere™ – Graphsphere™ particles are particles. There are no alkyl ligands off the support,
spherical, graphitized, polymer carbon (SGPC) with but rather the top layer has flat, hexagonally arranged
a porous or non-porous core, and a graphitized shell carbon atoms that are all covalently bonded to three
of controlled thickness. This shell imparts a variable carbon atoms. The remaining electron on each carbon,
amount of surface area and capacity. These carbons needed for bonding, is transposed perpendicular to
are used for an analyte size relative to the C5-C12 the plane in p-orbitals which subsequently hybridize
n-alkanes. Graphsphere™ particles generally have a together to form a continuous pi orbital allowing the
weaker adsorbent strength as compared to the carbon delocalized electrons to freely roam across the plane.
molecular sieves discussed above. For chromatography, this “sea” of electrons is believed
to allow for electrostatic interactions with the pi-cloud
Carbotrap® and Carbopack™ – These materials are of graphite.
graphitized carbon blacks (GCB) which can be porous
or non-porous and are generally granular and friable.

Particle size distribution of Supel™ Carbon LC HPLC lots vs. Competitor
12
Incremental Number Percent
10 Competitor
Lot 1
Lot 2
8
Lot 3
Lot 4
6 Lot 5
Lot 6
4 Lot 7
0
1 1.5 2 2.5 3 3.5 4 4.5 5
Particle Diameter (µm)
(Right) Simplified view of graphite (Left) P-orbitals on a section of graphite. This quality gives the
surface not only conductive properties, but also the ability for electronic interactions at the surface
An example of compound alignment with the graphitic surface. Left: Naphthalene is planar and can
align well against the graphitic plane. Right: Triptycene is rigid and not able to align completely flat
against the surface . This better alignment of naphthalene results in a stronger interaction with the
surface and increased retention.
115
Supel™ Carbon LC HPLC Columns
Porous graphitic carbon (PGC) particles, designed by a Unique Retention Mechanism: The Polar Retention
unique and patented synthetic process, constitute the Effect on Graphite (PREG) mechanism allows for the
packing of Supel™ Carbon LC U/HPLC columns. The key retention of polar or charged compounds without
advantages of using PGC columns over silica particle- hydrophilic interaction liquid chromatography (HILIC)
packed columns include: conditions. The mechanism also allows the resolution of
geometric isomers.
Elevated Temperature Stability: Columns can be
readily operated at temperatures up to 250 °C, thereby Compatibility with Any Solvent: Any polar or non-
allowing faster and more efficient separations. polar solvent can be used for the resolution of an
analyte of interest.
pH Stability: Compatible with mobile phases in the pH
range of 1 – 14 at any temperature without causing a Pressure Stability up to 700 bar.
decline in the column lifespan.
Supel™ Carbon LC Specifications

Bonding Particle Size Surface Area Max Shipping
Phase Bonding Chemistry (µm) Pore Size (Å) (m2/g) pH Stability Temperature Endcapped Solvent
N/A (porous N/A 2.7 200 155 1 to 14 250 °C No Acetonitrile/
graphitic carbon) Water
Peaks Compound Ret. Time (min)

1 3-epi-25-hydroxyvitamin D3 (50 µg/mL) 8.294
2 25-hydroxyvitamin D3 (25 µg/mL) 9.125
3 3-epi-25-hydroxyvitamin D2 (50 µg/mL) 11.126
4 25-hydroxyvitamin D2 (100 µg/mL) 12.052
200
8.294
12.052
9.125
11.126
mAU
100
0.818
0
0 10 20 30
Time (min)
Chromatographic separation of Vitamin D2 and D3 metabolites on

Supel™ Carbon LC
Chromatographic Conditions
Column: Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 μm
Mobile phase: [A] 2-Propanol; [B] Tetrahydrofuran
Gradient 0% B to 70% B in 15 min; hold at 70% B for 5 min
Injection: 2.0 μL
Sample: Vitamin D2 and D3 metabolites mix, varied concentration
in ethanol

Supel™ Carbon LC column - 20 Underivatized Amino Acids
Ser
120200 300 Ala

Arg
250 Glycine
Asn
100200
200 Asp
Height (cps)
Cys
80200 150 Gln
Glu
100
60200 His
50 Ile
Leu
40200 0
1 3 Lys
Met
20200 Phe
Pro
200 Thr
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Trp
Time (minutes)
Separation of 20 underivatized amino acids by LC-MS/MS. Conditions: Column: Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 µm; Mobile Phase:
[A] Water (0.1% (v/v) DFA); [B] Acetonitrile (0.1% (v/v) DFA); Gradient: Hold at 0% B for 7 min; 0% B to 5% B in 5 min; 5% B to 100% B in 10
min; Flow Rate: 0.2 mL/min; Column temp.: 12 °C; Detector: MSD; Injection: 1.0 µL; Sample: Amino Acid Mix, varied concentration, water (0.1%
(v/v) DFA)
Elution Order Compound Retention Time (min) Elution Order Compound Retention Time (min)
1 Glycine 1.27 11 Glutamic Acid 5.13
2 Serine 1.43 12 Leucine 5.18
3 Alanine 1.45 13 Cystine 7.34
4 Lysine 1.54 14 Isoleucine 8.93
5 Threonine 1.75 15 Methionine 12.61
6 Proline 2.03 16 Histidine 12.81
7 Asparagine 2.29 17 Arginine 13.30
8 Aspartic Acid 2.65 18 Phenylalanine 16.03
9 Valine 2.85 19 Tyrosine 16.29
10 Glutamine 3.69 20 Tryptophan 18.42
Supel™ Carbon LC
50 x 2.1 59984-U
100 x 2.1 59986-U
150 x 2.1 59987-U
50 x 3.0 59991-U
100 x 3.0 59993-U
150 x 3.0 59994-U
50 x 4.6 59997-U
100 x 4.6 59998-U
Guard 3pk x 2.1 59981-U
Guard 3pk x 3.0 59988-U
Guard 3pk x 4.0 59995-U
Guard Kit x 2.1 59982-U
Guard Holder x N/A 59999-U
117
Special HPLC phases
Polymeric particles, Zirconia and Alumina
apHera™ HPLC Columns amino group. Since apHera™ uses a strong alkaline
compatible polymer, these problems are eliminated.
apHera™ reversed phase columns were developed Stable retention time and long column lifetime are also
specifically to provide the superior advantages of characteristics of the column.
both silica and polystyrene columns, without the
disadvantages of either. This trait was accomplished 5
using a vinyl alcohol copolymer base that keeps the 1. D-(-)-arabinose
surface wetted even with high carbon loads. The 2. D-(+)-xylose
columns are packed with butyl (C4), octyl (C8) or 3. D-(+)-mannose 2
4. D-(+)-galactose
octadecyl (C18) packings obtained by the introduction 5. D-(+)-glucose
of the alkyl function on the hydroxyl groups of vinyl
alcohol copolymers. The porous structure has an
average pore diameter large enough to produce ideal
results for small analytes, peptides and small proteins.
These columns equal silica-based columns in separation
efficiency with organic solvents but provide efficiency 3
with buffered and alkaline solutions not possible
on silica. Shrinkage and swelling are minimal in a
1 4
broad range of solvents, and the high NTP values of
these columns are practically unaffected by differing
solvent polarities, unlike polystyrene based polymer
columns. One of the most significant features is the
logical elution order of alkylated bases where retention
increases proportionately with increasing chain length. 0 10 20
Min
apHera™ amino columns are based on covalently
Column: apHera™ NH2, 15 cm x 4.6 mm I.D., 5 µm
bonded polyamine specifically optimized for the
separation of mono- and oligosaccharides. The elution Column temp.: 25 °C
order mono-, di-, tri-saccharide shows increased Mobile phase: [A] water: [B] acetonitrile; (20:80, A:B)
elution volume with increased acetonitrile concentration Flow rate: 1.0 mL/min
and complete stability for both acidic and alkaline
Sample: 500 µg/mL in 30:70, water: acetonitrile
eluates. The small, robust PVA copolymer bead
provides mechanical and chemical strength as well as Injection: 10 µL
high column efficiency. Conventional amino columns Detector: ELSD, 45 °C, 3.5 psi nitrogen
based on silica do not show long column life, perhaps
due to hydrolysis of the silica particle by the basic
apHera™ (5.0 µm)

Length (mm) I.D. (mm) C18 C8 C4 NH2
150 x 2.0 56100AST 56400AST
150 x 4.6 56102AST 56202AST 56302AST 56401AST
250 x 4.6 56103AST 56303AST 56403AST
250 x 10.0 56208AST 56308AST 56408AST
Guard 10 x 2.0 56129AST 56429AST
Guard 10 x 4.6 56130AST 56230AST 56430AST
Guard 50 x 7.5 56332AST

Discovery® Zr Zirconia HPLC columns Discovery® Zr (3.0 µm)
Length I.D.
Discovery® Zr HPLC columns consists of spherical, (mm) (mm) PBD PS Carbon Carbon-C18
porous zirconia particles allowing the use of the full
50 x 2.1 65725-U
range of mobile phase pH from pH 1 to 13. Discovery®
Zr HPLC columns are available with different coatings: 75 x 2.1 65702-U
150 x 2.1 65703-U
Discovery® Zr-PBD: with a durable coating of
polybutadiene. It operates via reversed-phase and 50 x 4.6 65740-U
ion-exchange modes. 150 x 4.6 65718-U 65742-U 65730-U 65706-U
Discovery® Zr-PS: with cross-linked polystyrene. It Discovery® Zr (5.0 µm)

operates via reversed-phase and ion-exchange modes.
Length I.D.
Because it is weakly hydrophobic, it can be used with (mm) (mm) PBD PS Carbon Carbon-C18
100% aqueous mobile phases. It has unique selectivity
150 x 2.1 65720-U 65744-U 65732-U
especially for aromatic compounds.
150 x 4.6 65723-U 65735-U
Discovery® Zr-Carbon with carbon-coating. It is ideal
for the reversed-phase separation of positional isomers,
Guard Kit 4.0 65828-U
diastereomers, and many other compounds.
Discovery® Zr-CarbonC18 with carbon-clad covalently
modified with octadecyl (C18) groups. It is intended
for the reversed-phase separation of acidic and
neutral compounds.
Aluspher® RP select B Alkaline stable HPLC separations

Due to its stability, alumina, together with alkaline Aluspher® 100 RP-select B is ideal for use with basic
eluents, has enabled new applications to be found for eluents, as ionization of basic compounds is suppressed
HPLC. Advanced formulation techniques permit the and peak-tailing is avoided. Due to its stability in
production of spherical alumina particles as a base for the range of pH 2-12, Aluspher® 100 RP-select B
Aluspher® 100 RP-select B. permits the use of basic eluents such as NaOH for the
separation of neutral, basic and acidic compounds.
Aluspher®
Phase USP Particle Pore Surface Area pH Max Shipping
Bonding Designation Bonding Chemistry Size (µm) Size (Å) (m2/g) Stability Temperature Endcapped Solvent
RP-Select B L7 Alumina particles, coated 5 100 170 2-12 60 No Acetonitrile/
with polybutadiene (PBD) Water
Aluspher® LiChroCART® HPLC Cartridge

Column dimension
Length (mm) I.D. (mm) RP-select B
LiChroCART HPLC Cartridge [1 unit]
®
125 x 4 1.51315.0001
250 x 4 1.51318.0001
The LiChroCART® columns (75, 100, 125, 150 and 250 mm length)
in the list above (2, 3, 4 and 4.6 mm I.D.) require part number
1.51486.0001 manu-CART® cartridge column holder, which can be used
to hold one cartridge column with or without a 4-4 mm guard column.
119
Customized packings
On top of the very extensive column assortment Supelco® offers, customized packed columns for highest flexibility
and professional solutions are available. The sorbents and the packed HPLC columns are tested before delivering.
Each finished column is provided with a Certificate of Analysis.
Easy Ordering: Please combine the ordering number of the column hardware
(LiChroCART®, Hibar® RT or Hibar® HR) and the sorbent number:
Example: Customized packing ordering number of Hibar® RT 250-4.6 1.00424.
Sorbent Number of Purospher™ STAR Si, 5 µm 7180
Ordering Number of Hibar® RT 250-4.6 Purospher™ STAR Si, 5 µm 1.00424.7180
I.D. I.D.
Length (mm) (mm) LiChroCART® Hibar® RT Hibar® HR Length (mm) (mm) LiChroCART® Hibar® RT Hibar® HR
30 x 2 150229 25 x 4 150172
50 x 2 151928 30 x 4 150302 151196
55 x 2 150234 50 x 4 151927
100 x 2 151939 151929 55 x 4 150228
125 x 2 150195 151930 75 x 4 150171
150 x 2 151940 151931 125 x 4 150170 150181
250 x 2 150190 151932 250 x 4 150174 150182
30 x 2.1 151934 100 x 4.6 151448 150013
50 x 2.1 151935 125 x 4.6 151442 150012
100 x 2.1 151936 150 x 4.6 151432 150009
150 x 2.1 151937 250 x 4.6 151431 100424
250 x 2.1 151938 75 x 10 151449
30 x 3 150233 100 x 10 151445
50 x 3 151923 125 x 10 151443
55 x 3 150236 150 x 10 151444
100 x 3 151941 151924 250 x 10 150179 150183
125 x 3 150175 151925 75 x 25 151449
150 x 3 151942 151926 Guard Cartridge
250 x 3 150177 100423 10 x 2 150201
300 x 3.9 151943 151933 4 x 4 150173
10 x 10 150178

Sorbent Sorbent Sorbent
Code Packing Material Code Packing Material Code Packing Material
Purospher™ STAR Superspher® 7084 LiChrospher® 100 RP-18e 10µm
7174 Purospher™ STAR SI 3µm 7137 Superspher® 100 RP-18 4 µm 7085 LiChrospher® 100 RP-18e 5µm
7175 Purospher™ STAR SI 5µm 7138 Superspher 100 RP-18e 4 µm
®
7087 LiChrospher® 100 RP-8 5µm
7177 Purospher™ STAR NH2 5µm 7139 Superspher 60 RP-8 4 µm
®
7088 LiChrospher® 100 RP-8 10µm
7184 Purospher™ STAR RP-18e 3µm 7140 Superspher® 60 RP-8e 4 µm 7091 LiChrospher® 100 RP-8e 10µm
7185 Purospher™ STAR RP-18e 5µm 7141 Superspher 60 RP select B 4 µm
®
7092 LiChrospher® 100 RP-8e 5µm
7194 Purospher™ STAR RP-8e 5µm 7142 Superspher 60 Si 4 µm
®
7093 LiChrospher® 60 RP select B 5µm
7220 Purospher™ STAR RP-8e 3µm 7143 Superspher® 100 Si 4 µm 7094 LiChrospher® 60 RP select B, 10 µm
7232 Purospher™ STAR Phenyl 2µm LiChrospher
®
7104 LiChrospher® 60 Si 10µm
7234 Purospher™ STAR Phenyl 3µm 7070 LiChrospher 100 CN 10µm
®
7109 LiChrospher® 60 Si 5µm
7235 Purospher™ STAR Phenyl 5µm 7071 LiChrospher® 100 CN 5µm ChiraDex®
7236 Purospher™ STAR RP-18e 2µm 7075 LiChrospher 100 DIOL 5µm
®
7004 ChiraDex® 5µm
7237 Purospher™ STAR RP-8e 2µm 7076 LiChrospher 100 NH2 5µm
®
7222 ChiraDex® HR 5µm
Purospher™ 7077 LiChrospher® 100 NH2 10µm Aluspher®
7127 Purospher™ RP-18 5µm 7078 LiChrospher PAH 5µm
®
7002 Aluspher® 100 RP-select B, 5µm
7130 Purospher™ RP-18e 5µm 7079 LiChrospher 100 RP-18 5µm
®
7180 Purospher™ Si 5µm 7081 LiChrospher® 100 RP-18 10µm
Custom Column requests for Ascentis® Express and BIOshell™ Fused-Core® HPLC and UHPLC Columns,
Discovery®, Discovery® BIO, Ascentis®, SUPELCOSIL™, CHIROBIOTIC®, CYCLOBOND™ Fully porous silica
particulate HPLC columns and Titan™ UHPLC columns as well as Supel™ Carbon LC Carbon U/HPLC columns, can
be submitted through our Customer Service or on our web page.
Custom HPLC Column Request
121
Merck KGaA
Frankfurter Strasse 250
64293 Darmstadt, Germany
SigmaAldrich.com/HPLC
To place an order or receive technical assistance:
Order/Customer Service: SigmaAldrich.com/order

Technical Service: SigmaAldrich.com/techservice
Safety-related Information: SigmaAldrich.com/safetycenter
© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the vibrant M, and
Supelco, Supel, SUPELCOSIL, LiChrospher, Discovery, Ascentis, SeQuant, Purospher, BIOshell, Eshmuno, Chromolith,
Astec, CHIROBIOTIC, CYCLOBOND, ChiraDex, LiChrosorb, SUPERSPHER, apHera, SUPELCOGEL, Samplicity, Millex,
Pelliguard, Supelclean, Suplex, Supelguard, SiLu, LiChroSolv, Carbosieve, Carboxen, Graphsphere, Carbotrap,
Carbopack, Titan, manu-CART, LiChroCART, Hibar, CapRod, ZIC, and Aluspher are trademarks of Merck KGaA,
Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners.
Detailed information on trademarks is available via publicly accessible resources.
MK_BR7614EN Ver. 2.0
40190
05/2022

HPLC Columns Guide Br7614en MK

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HPLC and

The Life Science business

Supelco® HPLC and UHPLC Column selection 5

Monolithic Silica HPLC Columns 46

Fully Porous Particulate Silica (FPP) Columns 60

Chiral HPLC Columns 94

Biomacromolecule Characterization: A Multipronged Separation Challenge 100

Synthetic Carbons for Chromatographic Applications 114

Customized Packings 120

We provide a premier selection of proven

Sample Collection Sample Preparation Standardization & Chromatographic

Supel™ Carbon HPLC Columns

Type A Fully porous silica particles (FPP) Silica FPP

SiO2 (Na, K, Fe)

Other stationary phase materials

Fully porous polymeric particles Polymeric

Zirconia and Alumina Particles

Reversed Phase Ascentis® Express 28 BIOshell™ MS

Reversed Phase Chromolith® 50 Chromolith® WP 300 54

Reversed Phase Purospher™ STAR MS

SeQuant® HILIC HPLC columns and 82

Size Exclusion Sepax 102

Reversed Phase LiChrospher® 86

Reversed Phase Supel™ Carbon 114 Supel™ Carbon MS

Note: As chromatographic separation depends on many physical and

Need for Speed

8 HPLC and UHPLC Column Selection Guide

Compound ZIC®- ZIC®- ZIC®- RP- Propyl Phenyl RP-

1 Most suitable column 2 Possible alternative column

Alcohols Ethyl alcohol -0.1

Aldehydes Benzaldehyde 1.5

Alkaloids Quinine 2.9

Amino Acids Aspartic acid 2

Aromatic amines Aniline 0.9

C Carboxylic acids Glucuronic acid -2.3

Carotenoids Canthaxanthin 11.4

D Dyes Rhodamine 4.4

E Enatiomers Thalidomide 0.3

Essential oils Safrole 3

Esters Atropine 1.8

F Fat soluble vitamins Retinol 5.7

Fatty acids Stearidonic acid 5.9

Flavonoids Quercetin 1.5

10 HPLC and UHPLC Column Selection Guide

I Inorganic ions Chloride Cl- 0.8

N Nitrosamines N-Nitrosodimethylamine -0.6

P PAH Anthracene 4.4

Peptides Neurokinin B -1.6

Pesticides Glyphosate -4.6

Phenols Bisphenol A 2.2

Phospholipids Phosphatidylserine -3.5

S Steroids Progesterone 3.9

Sugars Lactose -4.7

Sugar Alcohols Maltitol -5.2

Sweeteners Aspartame -2.7

W Water soluble vitamins Folic Acid -1.1

Too much Poor Peak

RP- F5 Phenyl ES- Si

C18 C8 Diol Cyano Amino Si