High Performance Liquid

Chromatography
(HPLC)

Presented by: Dr.Qomi

2010
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 Table of Contents
1. What Does a Chemist Do?
2. How Does S(He) do it?
3. What is Analytical Chemistry?
4. High Performance Liquid Chromatography
5. Aims and Objectives
6. Origins of Liquid Chromatography
7. Why Choose Liquid Chromatography?
8. Suitable Samples for HPLC
9. Comparison with Gas Chromatography
10. Typical HPLC Data
11. Chromatography Separation Mechanisms
12. The Liquid Chromatograph
13. The Liquid Chromatographic Process
14. The Chromatogram
15. Modes of Chromatography

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 What Does a Chemist Do?

➢ Studies the atomic composition and structural architecture of

substances

➢ Investigates the varied interactions among substances

➢ Utilizes natural substances and creates artificial ones

➢ Comprehends the marvelous and complex chemistry of

living organisms

➢ Provides a molecular interpretation of health and disease

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 How Does S(He) do it?
Main Divisions of Chemistry

Organic Chemistry

Inorganic Chemistry Materials Chemistry

Physical Chemistry

Analytical Chemistry Environmental Chemistry

Industrial Chemistry
(Chemical Engineering
Forensic Chemistry
and Applied Chemistry)

Biochemistry
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 What is Analytical Chemistry?

QUALITATIVE ANALYSIS
Deals with the detection of elements or compounds (analytes) in
different materials.

QUANTITATIVE ANALYSIS
Refers to the measurement of the actual amounts of the analyte present
in the material investigated.
•Gravimetry

Chemical and Biochemical Methods •Titrimetric Analysis

•Enzymic Analysis

•Inmunochemical Analysis

•Instrumental Analysis 5
Instrumental Analytical Chemistry
• Atomic and Molecular Spectroscopic Methods

•Nuclear Magnetic Resonance (NMR)

•Electron Spin Resonance (ESR)

•Mass Spectrometry (MS)

•Vibrational Spectroscopy (IR, RAMAN)

•X-Ray Fluorescence Analysis (XPS)

•Electronic Spectroscopy (UV, VIS, Luminiscence)

•Atomic Spectroscopy (AA, ICP)

•Rotational Spectroscopy (Microwave, FIR)

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Instrumental Analytical Chemistry
• Thermal Methods
•Thermogravimetry (TG)
•Differential Thermal Analysis (DTA)
•Differential Scanning Calorimetry (DSC)
•Thermomechanic Analysis (TMA)

• Electrochemical Methods
•Electrogravimetry
•Electrophoresis
•Conductimetry, Potentiometry
•Polarography
•Voltammetry 7
 Instrumental Analytical Chemistry
Chromatographic Methods (Partition equilibrium)

•Gas Chromatography (GC)

•High Performance Liquid Chromatography (HPLC)

•Gel Permeation Chromatography (GPC)

•Thin Layer Chromatography (TLC)

•Ion Chromatography

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High
Performance
L iquid
C hromatography
9
High
Pressure
L iquid
C hromatography
10
High
Priced
L iquid
C hromatography
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12
Aims and Objectives

To give a brief History of Liquid Chromatography

To compare and contrast High Performance Liquid
Chromatography (HPLC) with Gas Chromatography (GC)
To introduce the Liquid Chromatograph (LC) and explain the
function of each component
To introduce the Chromatogram and explain the information it
gives
To outline the main separation mechanisms used in HPLC
To explain the fundamental principles of separation
To highlight the different "modes" of chromatography and
explain their uses and applications

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 Origins of Liquid Chromatography

The Russian botanist Mikhail Tswett first used the term

'Chromatography' (Greek for 'coloured drawing') in 1906, to
describe the separation that occurred when solutions of plant
pigments were passed through columns of calcium carbonate or
alumina, using petroleum ether.

HPLC was first presented by Huber, J.F.K. and Hulsman, J.A.G., in

1967.

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 Why Choose Liquid Chromatography?
The two main chromatographic techniques used in modern analytical chemistry
are Gas Chromatography (GC) and High Performance Liquid Chromatography
(HPLC).

HPLC uses a liquid mobile phase to transport the analytes (sample) through the
column, which is packed with a stationary phase material.
In contrast, Gas Chromatography uses a gaseous mobile phase to transport
sample components through either packed columns or hollow capillary columns.
In most cases, GC columns have smaller internal diameter and are longer than
HPLC columns.

GC has developed into a sophisticated technique since the pioneering work of

Martin and James in 1952, and is capable of separating some very complex
mixtures.

However, due to limitations of volatility and thermal stability, it is only capable

of separating around 23% of known substances.

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So ? under what circumstances would we chose
HPLC to separate our sample components?

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 Suitable Samples for HPLC
GC
Samples analysed by GC must be volatile (significant vapour pressure
below 250oC).
Derivatisation to increase analyte volatility is possible but cumbersome and
introduces possible quantitative errors.
Most GC analytes are under 500 Da Molecular Weight for volatility
purposes.

HPLC
HPLC analysis has no volatility issues, however the analyte must be
soluble in the mobile phase.
HPLC can analyse samples over a wide polarity range and is able to
analyse ionic samples. Mobile phase components are selected to ensure
sample solubility.
HPLC has no real upper molecular weight
limit and large proteins of many thousands of Daltons may be analysed.
Solubility in the mobile phase may preclude the analysis of very large
molecules.
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 Comparison with Gas Chromatograpy

GC
Temperatures in GC can exceed 350oC and samples that are thermally
unstable (labile) may decompose.
Many GC detectors such as the Flame Ionisation Detector (FID) are
destructive and the analyte does not survive analysis in-tact and therefore
cannot be recovered.
GC samples are prepared in organic solvents and extraction of analytes
from aqueous samples will be necessary.
Sample size is usually between 1 and 5µl with typical detector sensitivity
between nanograms (ng) and picograms (pg) on column.
HPLC
HPLC is usually carried out at (or around) room temperature and most
HPLC detectors apart from the Mass Spectrometer are non-destructive.
HPLC samples are prepared in a solvent system that has the same or less
organic solvent than the mobile phase and volumes of 1 to 50 µl are
common (1-10µg of analyte per 1g packing material).

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 Typical HPLC Data
HPLC - Even though the Flame ionisation detector may be more
universal and sensitive, the UV detector is non-destructive, relatively
sensitive (nanograms of analyte on column), and also has the
capability of producing spectra associated with sample components.
This can aid qualitative analysis and assist with identification of
sample components.

HPLC and GC can both use Mass Spectrometers (MS) as detection

systems to assist with analyte identification - although MS detectors
are destructive. HPLC-MS is a less mature technique and there are no
spectral libraries available for compound identification as there are
with GC instruments. However, HPLC-MS (LC-MS) is a burgeoning
technique that can assist in the characterisation of sample components
in a wide variety of application types.

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 Summary
If the sample cannot be analysed by Gas Chromatography
without lengthy sample preparation (indicating issues with
volatility), then HPLC should be the technique of choice.
HPLC is the best choice for higher molecular weight analytes
and analytes which may potentially degrade when heated.

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Scope of HPLC

Sensitive
Quantitative
Nonvolatile Compounds
Thermally Fragile
Compounds
Broad Applicability
◼ Biochemical Species
◼ Pharmaceuticals
◼ Pesticides
◼ Inorganic and Organometallics
◼ Industrial Chemicals

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 Chromatography Separation Mechanisms

HPLC separations involve both the mobile phase (a liquid) and the
stationary phase (usually materials of varying hydrophobicity chemically
bonded to a solid support). In contrast, GC separations do not involve the
mobile phase, which is only used to carry the analyte through the column.

As an illustration - the amount of water in an HPLC mobile phase will

determine how strongly a hydrophobic analyte is repelled into the
stationary phase - and how well retained it is. The chemical nature of the
stationary phase will also govern how strongly the analyte is retained. For
this reason, HPLC is a technique that is driven by the 'selectivity' achieved
using two interacting phases.

In contrast, analytes in a capillary GC column will only be retained due to

their interaction with the stationary phase (usually an immobilized
polymeric liquid of varying hydrophobicity) coated onto the inner walls of
the GC column. There are less options for improving selectivity in GC,
however, as it is very highly efficient - this is often enough to achieve the
desired separation.

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 Chromatography Separation Mechanisms
(Partitioning)

Separation is based on the analyte’s relative solubility

between two liquid phases

Mobile Phase Stationary Phase

Solvent Bonded Phase

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Mobile Phase: The compound of interest to be analysed by injection into and
elution from an HPLC or GC column.

Stationary Phase: The stationary phase is one of the two phases forming a
chromatographic system. It may be a solid a gel or an immobilised polymeric
liquid. if a liquid, it may be distributed on a solid. This solid may or may not
contribute to the separation process. The liquid may also be chemically bonded
to the solid ("Bonded Phase"). The expressions "Chromatographic Bed" or
"Sorbent" may be used as general terms to denote any of the different forms in
which the stationary phase is used. A stationary phase which is covalently
bonded to the support particles or into the inside wall of the column tubing is
known as a "Bonded Phase".

Hydrophobic: Meaning "water-fearing", hydrophobic compounds do not

dissolve easily in water and are ususally non-polar. Oils and other long
hydrocarbons are hydrophobic.

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Selectivity: "Selectivity" is also callned the "Separation Factor" (alpha),
which describes the separating "power" of the system.
The relative retention value calculated for 2 adjacent peaks (VR2' > VR1'):

alpha = tR2'/tR1'

By definition, the value of the separation factor is always greater than 1.

Efficient: "Efficiency" is also called the plate Number (N), which describes
the broadening of the chromatographic band by using the chromatographic
peak width. The Plate Number is indicative of column performance,
calculated from the following equations, which depend on the selection of
the peak width expression (see 'Peak Width').

N = 16(tR/wb)2 H=L/N

TR = peak apex retention time, wb = width of the peak at the base,

measured using tangents to the peak sides
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 The Liquid Chromatograph

In HPLC, several instrument and column chemistry parameters

need to be optimised in order to generate a satisfactory
separation.
Each of the following items needs to be optimised in order to
generate a chromatogram that is suitable for qualitative or
quantitative purposes.

✓ Mobile Phase Composition

✓ Bonded Phase Chemistry
✓ Injection Volume
✓ Sample Pre-treatment and concentration
✓ Mobile phase Flow Rate
✓ Column Temperature
✓ Detector Parameters
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 The Liquid Chromatographic Process

The mobile phase is continuously pumped at a fixed flow rate

through the system and mixed (if required) by the pump. The
injector is used to introduce a plug of sample into the mobile
phase without having to stop the mobile phase flow, and
without introducing air into the system.

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The Chromatogram

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 TECHNIQUES
1. Solvents
2. Pumps
3. Sample Injection
4. Column
5. Detectors

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 Solvents
➢Low viscosity
➢Be free of particles and dissolved air

❖Degassing
✓Helium purge
✓Vaccum
✓Ultrasonic bath

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 Modes of Chromatography
Normal Phase.
- Polar stationary phase and non-polar solvent.

Reverse Phase.
- Non-polar stationary phase and a polar solvent.

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Common Reverse Phase Solvents
Methanol
CH3OH
• Acetonitrile CH3CN
O
• Tetrahydrofuran

• Water H2O

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 Pumps
Pressure drop:
P= Lµ/Өd2
L = column length, =solvent viscosity, µ=flow rate, Ө= constant, d=particle diameter

➢Reciprocating pumps
➢Positive Displacement pumps (Syringe pumps)

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 Sample Injection
Sample volume: 5-50 µL
Never use a gas chromatographic syringe in
HPLC injection block

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Injection Systems

Syringe Injection
◼ Possible
◼ Rarely Used
Standard Method - Loop
Injector
◼ Diagrams:
Figure 27-4 (better)
Figure 28-7
◼ Interchangeable Loops
Standard 5 to 500 mL
Micro 0.5 to 5 mL
◼ Operating Pressures to 7000
psi

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 LC Columns

Construction
◼ Mainly Smooth Bore Stainless Steel (Pressures to 10,000 psi)
◼ Occasionally Heavy Wall Glass (Pressures to 600 psi)
Costs:
◼ $200 to $500
◼ More Expensive Available
Analytical Columns
◼ 10 to 30 cm
◼ Extend Length by Coupling Additional Columns
◼ Inside Diameters: 4 to 10 mm
◼ Packing Diameters: 5 or 10 mm
◼ Common: 25 cm long; 4.6 mm inside diameter; 5 mm particles; N = 40,000 to
60,000

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Column Efficiency in HPLC
Mobile Phase Mass
Transfer
◼ CM  dp2
CM - MP mass transfer
coeff.
dp - particle diameter
◼ Effect shown in Fig 28-2

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Picture of an HPLC column

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LC Columns
High Speed, High
Performance
◼ Smaller than Standard Columns
◼ Lengths: 3 to 7.5 cm
◼ Inside Diameters: 1 to 4.6 mm
◼ Particle Diameters: 3 or 5 mm
◼ Number of Plates: Up to 100,000
◼ Advantages:
Speed - Much Faster
Elutions
Minimum Solvent Usage
◼ Figure 28-8
8 Components in 15 s
4 cm long
4 mm inside diameter
3 mm particle diameter
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 LC Columns
Guard Columns Column Thermostats
◼ Extend Analytical Column Life ◼ Temperature Control
◼ Removes: Unimportant for Most
Particulate Matter Applications
Solvent Contaminents Constant Temperature
Sample Components Improves Chromatograms
Which Bind Irreversibly to ◼ Modern HPLC's Have Column
SP Heaters
◼ Liquid-Liquid Chromatography Range: Ambient to 100° to
Saturates MP with SP 150° C
Minimizes Analytical SP Control: a Few 0.1° C
Loss ◼ Column Water Jackets
◼ Composition Matched to Provides Heating or Cooling
Analytical Uses Standard Constant
◼ Pressure Drop Minimized with Temperature Baths
Larger Particle Size
◼ Repacked or Discarded When
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Contaminated
 HPLC Column Packings
Silica and bonded-phase silica
- Classic, Type II, and hybrid silica
Polymer reverse phase
Zirconium and MS bonded-phase
Ion exchange: polymer and silica
Size separation: polymer and silica

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 Type of Particles
Fully porous particles
Pellicular particles
Microporous particles

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 LC Columns
– Porous Particles
Column Packings " Microparticles
◼ Pellicular Beads " Diameter: 3 to 10 mm
Spherical
" Diameter Ranges
Nonporous Minimized
Glass or Polymer " Compostion:
Diameters: 30 to 40 mm – Silica
Thin Porous Layers Deposited – Alumina
◼ Silica – PS-DVB Resin
◼ Alumina – Ion-Exchange Resin
◼ PS-DVB Resin " Silica Most Common
◼ Ion-Exchange Resin " Particles Coated with
Sometimes Additional Liquid Organic Films
SP Applied to Porous Layer – By Physical
Surface Chemically Modified Adsorption

◼ Yeilds Organic Surface

– By Chemical Bonding

Mostly for Guard Columns

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 Columns
Solid Support - Backbone for bonded phases.
◼ Usually 10µ, 5µ or 3µ silica or polymeric

particles.
Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
◼ Extremely stable

-Reproducible

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 Bonded Phases

• C-2 Ethyl Silyl -Si-CH2-CH3

• C-8 Octyl Silyl -Si-(CH2)7-CH3

• C-18 Octadecyl Silyl -Si-(CH2)17-CH3

• CN Cyanopropyl Silyl -Si-(CH2)3-CN

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Synthesis of RP Packings

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Reverse Phase

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RP Mechanism (Simple)

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Common NP Packings

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Classical Bonded Silica Bonded Hybrid Silica

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 HPLC Pre-run Preparation
Volatile Buffer: mobile phase pH control
Sample and mobile phase filtration
Sample solubilizing & deproteination
SFE cartridge sample cleanup
SFE cartridge windowing
Dry System startup

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 Detectors
Ultraviolet absorption (UV)
◼ Single wavelength (filter)

◼ Variable wavelength (monochromator)

Fluorescence
Refractive Index (RI)
Electrochemical (EC)
Mass Spectrometric

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HPLC Detectors
Absorbance Detectors
◼ Eluent Measurement Cell
Minimum Volume
◼ Reduces Extra Column
Broadening
◼ Volume : 1 to 10 mL
◼ Path Length (b): 2 to 10 mm
Pressure Limited
◼ Maximum Typically 600 psi
◼ Usually Requires Pressure
Reducer
◼ Instrument Configurations
Double Beam
◼ Self Correction for Random
Intensity Variations
Single Beam
◼ Simpler, Less Expensive
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 Refractive Index (RI)
The speed of light in a medium of refractive index n
is C/n, where C is the speed of light in vaccum.

For vaccum n=1

The refractive index of a material is a function of the

wavelength of light and temperature

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 Making Sensitive Derivatives
Pre column reaction
Post column reaction

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Instrumentation

57
 Instrumentation

Gradient
Controller
•
Pump Column
Detector
Injector
Mobile Phases

58
````````````````````````````

59
 HPLC System Maintenance
Reverse Order Diagnostics
Injector and tubing clearing
Gradient checking with acetone spike
Water Diagnostics
Column cleaning and column killers

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Column Blank for System Pacification

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63
 FOUR TYPES OF LIQUID
CHROMATOGRAPHY

Partition chromatography

Adsorption, or liquid-solid

Chromatography

Ion exchange chromatography

Size exclusion, or gel, chromatography

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Quantification in Chromatography

Four methods
◼ Normalizing peak areas
◼ Internal standard
◼ External standards
◼ Standard addition

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 Normalizing Peak Areas
Inject mixture with equal amounts of all components
The area of each peak is measured precisely
◼ Replicate (5+) injections
◼ Need good precision
◼ One component is chosen as the reference
The areas of the others are normalized with respect to
that one
◼ Detector response factors (DRF)

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 Internal Standard
Eliminate the need for accurate injections
A reference standard is included in each
sample at accurately known concentration
Eluted in a gap in the chromatogram

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 External Standard
Inject standard before and/or after analyzing
the sample
Standard will be on a different chromatogram
Depends on good injection reproducibility
HPLC multiport valves < 1% difference

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 Standard Addition
Sample is analyzed
Extra analyte is added, re-analysis
Can use to verify linearity

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 HPLC Applications
Drug and compound discovery
Proteomics and other biologicals
Metabolites, Impurities, degradations
Toxicology: Drugs of Abuse
Clinical: Therapeutic Drug monitoring
Arson residue determination
Water and pesticides analysis

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Analysis of Diet Cola Additives
Conditions:
◼ Cartridge column
20x2.1mm HAISIL HL
C18
◼ 200µL/min
◼ 15% acetonitrile/ 10mM
phosphate buffer (pH
2.2)
Detector: 210nm

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 Chromatograms

A Supelcosil LC-PAH Columns. B

Conditions: A: 150mm x 4.6mm, 5µ. Conditions: B: 50mm x 4.6mm, 3µ.
Flow Rate: 1.5 mL/min Flow Rate: 3.0 mL/min
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 HPLC Methods
Parameter Group Method Compounds

• SDW05.23000’s EPA 555 Cl-PhenoxyAcids

• WPP05.06000’s EPA 605 Benzidines

• WPP05.13000’s EPA 610 PAHs

• SHW06.26000’s SW-846 8316 Acrylics

• SHW06.28000’s SW-846 8330’s Explosives

• SHW07.06000’s SW-846 8325 Benzidines and
N- Pesticides

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Compounds

2,4,5-T

Benzidine

Fluorene

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 Compounds

TNT (2,4,6-Trinitrotoluene)

H2C=CH-CN Acrylonitrile

Carbaryl

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Determination of chlorophenoxy acid herbicides in water by liquid phase
microextraction and high performance liquid chromatography detection

Peak Chlorophenoxy Enrichment LOD Linearity range

RSD%a R2
No. acid hrebicide factor (ng/ml) (ng/ml )

1 192 0.4 3.9-6.8 0.9992 5.0-500.0

Dicamba

2 293 0.3 2.2-5.8 0.9994 5.0-500.0

2,4-D

3 390 0.4 1.1-5.1 1.0000 5.0-500.0

MCPA

4 352 0.3 2.4-6.2 0.9990 5.0-500.0

2,4-DP

5 344 0.4 3.1-6.5 1.0000 5.0-500.0

Mecoprop

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General structure of chlorophenoxy acid herbicides considered in this work

Chlorophenoxy acid IUPAC name

Dicamba (3,6-dichloro-2-methoxy) benzoic acid
2,4-D (2,4-dichlorophenoxy) acetic acid
MCPA (2-methyl-4-phenoxy) acetic acid
2,4-DP 2-(2,4-dichlorophenoxy) propanoic acid
Mecoprop 2-(2-methyl-4-chlorophenoxy) propionic acid

OH OH
H3C
O O
O OH CH2 O CH2 O
Cl
O Cl Cl CH3
Cl

Cl
OH
OH
O
O CH2 O
CH O
Cl CH3 CH
3
Cl Cl CH3

77
Injection procedures were carried out using a 25 µl Hamilton syringe, model 702 NR.
Chromatographic analysis was carried out on a HP SERIES 1100 (USA) HPLC system.
The chromatographic system consisted of injector equipped with a 20 ml sample loop,
a HP SERIES 1100 pump, and a HP SERIES 1100 UV–vis spectrophotometric
detector. A column (250mm×4.6mm I.D.) from Waters (CA, USA) packed with C18
(µBondPak™) was used. Data was collected and processed by ChemStation (Agillent
Technologies, USA) data analysis software. A flow-rate 1.0 ml/min was applied in
laboratory temperature of 20 (±2) °C. The mobile phase was methanol–25 mM
phosphate buffer (60:40, v/v; pH = 2.75) and the detection wavelength was 240 nm.

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Procedures for analysis

Step 1 – Extraction
Step 2 – Cleanup
Step 3 – Analysis

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 Extraction methods
Liquid-liquid
Soxhlet
Automated soxhlet
Microwave
Supercritical fluid
Accelerated solvent extraction
Ultrasonic
Solid phase extraction (SPE)
Blender

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