High Performance Liquid

Chromatography

ALAKESH PRADHAN
COCHIN UNIVERSITY OF
SCIENCE AND TECHNOLOGY
School of Industrial
Fisheries
M.Sc IInd Sem.
 Overview:

 Chromatography and its principle

 Liquid chromatography

 High Performance Liquid Chromatography ( HPLC )

 The components of the high performance liquid chromatograph (HPLC).

 The separation process.

 The chromatogram.
Latest instrument
 Background
 Chromatography and its Principle
 Chromatography is a separation technique which is used to
separate a mixture of compounds into its individual components
based on certain physical and chemical properties.
Some important terms:
 Mobile phase: The solvent system which carries the mixture to be
separated.
 Stationary phase: Immobile surface which is particulate in nature. This is the
region over which the compound gets separated.
Principle:
 The process involves the interaction of the compounds in the analyte (which
travels along with a mobile phase) across an immobile surface (stationary
phase).

 The compounds bind at specific regions of stationary phase based on

certain physical and chemical properties. These bound molecules are then
eluted with a suitable buffer and the same are collected with time.
These are –
 Polarity
 Charge
 Molecular weight
 Present of functional group
 Introduction
 HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
 HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column, and a
detector.
 Compounds are separated by injecting a sample mixture
onto the column.
 The different component in the mixture pass through the
column at differentiates due to differences in their
partition behavior between the mobile phase and the
stationary phase.
 The mobile phase must be degassed to eliminate the
formation of air bubbles.
 Continued…
What is Liquid Chromatography?
 Liquid chromatography is a separation technique that involves:

• the placement (injection) of a small volume of liquid sample

• into a tube packed with porous particles (stationary phase)

• where individual components of the sample are transported along the

packed tube (column) by a liquid moved by gravity.
 The components of the sample are separated from one another by the
column packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.
 The separated components are collected at the exit of this column and
identified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.

 Note: The modern form of liquid chromatography is now referred to as

“flash chromatography
Principles of Liquid
Chromatography
 Terminologies for HPLC
 HPLC : High Performance Liquid Chromatography : High Pressure LC

 Now, before we go in depth of principle, lets have a basic look at few

terms as follows:

 Resolving Power: The extent of separation of the compounds present

in the mixture across the column.

 Theoretical plates : An imaginary division of the column into

equilength plates.
 Principles of HPLC
Principle:

 The table shows relation between various

parameters of HPLC.
 Trendline:

Column length No. of theoretical plates

per unit area
Resolving power Column length
Particle size Surface area

 Stationary phase have small particulate size and

high surface areas.
 Columns: 20 cm or less
 Mobile phase pumped at high pressures of
200Bar, 3000 psi.
 Flow rates: 1-3 cm3 per min
 What is HPLC?
 HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample
•into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter
called the stationary phase)
•where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.
 These components are separated from one another by the column
packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.
 These separated components are detected at the exit of this tube (column)
by a flow-through device (detector) that measures their amount. An
output from this detector is called a “liquid chromatogram”.

 In principle, LC and HPLC work the same way except the speed ,
 efficiency, sensitivity and ease of operation of HPLC is vastly
 superior.
HPLC system

Flow chart of HPLC mechanism

 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector
Varian 9010 Solvent Delivery to injector
System
through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Picture of HPLC instrument
COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM

Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors
Waste Collector
Recorder (Data Collection)
 Instrumentation of HPLC
( Describing the 5 major components and their
functions….)
Solvent
reservoirs
and degassing
1

Not shown 2
here 5
3

1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
1. Pump:

•The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed
in milliliters per min (mL /min).

•Normal flow rates in HPLC are in the 1-to 2-mL/min range.

•Typical pumps can reach pressures in the range of 6000-

9000
psi (400-to 600-bar).

•During the chromatographic experiment, a pump can deliver a

constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
Pump Module–types:
 Isocratic pump - Delivers constant mobile phase composition;

•solvent must be pre-mixed;

•lowest cost pump
 Gradient pump - Delivers variable mobile phase composition;

•can be used to mix and deliver an isocratic mobile phase or a

gradient mobile phase
–Binary gradient pump –delivers two solvents

–Quaternary gradient pump –four solvents

2. Injector:

•The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.

•Typical sample volumes are 5-to 20-microliters (μL).

•The injector must also be able to withstand the high pressures of

the liquid system.

•An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
 Sample Injection
……how is a sample actually put into an LC system
Manual Injector:
1.User manually loads sample into the injector using a syringe
2.and then turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of the
column, which is at high pressure

Auto sampler:
1.User loads vials filled with sample solution into the auto sampler tray
(100 samples)
2.and the auto sampler automatically
a. measures the appropriate sample volume,
b. injects the sample,
c. then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed ……
…….for unattended automatic operation
 Manual Injectors Sample Loop

Load - Inject

Front View Rear View

Inject

23
 Automatic Injectors

Step 1 Step 2

Step 3
24
3. Column:

• Considered the “heart of the chromatograph” the column’s stationary

phase separates the sample components of interest using various physical
and chemical parameters.

•The small particles inside the column are what cause the high
back pressure at normal flow rates.

•The pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within the
chromatograph.
 Several Column Types
( can be classified as)

 Normal phase

 Reverse phase

 Size exclusion

 Ion exchange
Normal phase
 In this column type, the retention is governed by
the interaction of the polar parts of the stationary
phase and solute.
 For retention to occur in normal phase, the
packing must be more polar than the mobile
phase with respect to the sample
 STATIONARY PHASES
(NORMAL POLARITY)

Silica or alumina possess polar sites that

interact with polar molecules.
silica
O
Polar Group HO Si
O

Components
Componentselute
eluteininincreasing
increasing
order
orderof
ofpolarity.
polarity.

Most polar…….Least polar

28
 Reverse phase
 In this column the packing material is relatively nonpolar and the solvent is
polar with respect to the sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the nonpolar stationary phase.
 Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-
organic mixtures such as methanol-water or acetonitrile-water.

Common Reverse Phase Solvents –

Methanol CH3OH

Acetonitrile CH3CN

Tetrahydrofuran

Water H2O
 STATIONARY PHASES
(REVERSE POLARITY)

If the polar sites on silica or alumina are capped with non-polar

groups, they interact strongly with non-polar molecules.
silica
C18 phase Me O
Si O Si
Me O

Components
Componentselute
eluteinindecreasing
decreasing
order
orderof
ofpolarity.
polarity.

Most non-polar…….Least non-polar

30
 Size exclusion
 In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and
is able to be filtered in an ordinarily phase
according to its molecular size.
 Small molecules penetrate into the pores within
the packing while larger molecules only partially
penetrate the pores. The large molecules elute
before the smaller molecules.
 STATIONARY PHASES
(SIZE EXCLUSION)

Size exclusion gels separate on the basis of molecular size.

Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.

Large
Largemolecules
moleculeselute
elutefast
fast(restricted
(restrictedpath),
path),
while
whilesmall
smallmolecules
moleculeselute
eluteslowly
slowly(long
(longpath
pathlength)
length)

Larger molecules…….Smaller molecules

32
 Ion exchange
 In this column type the sample components
are separated based upon attractive ionic
forces between molecules carrying charged
groups of opposite charge to those charges
on the stationary phase.
 Separations are made between a polar mobile
liquid, usually water containing salts or small
amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.
 STATIONARY PHASES
(CATION EXCHANGE)

Silica is substituted with anionic residues that interact

strongly with cationic species (+ve charged)
Cations exchange Na+ silica
O
Na O S
O

+ve
+vecharged
chargedspecies
speciesadhere
adhereto
tothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most +ve…….Least +ve

34
 STATIONARY PHASES
(ANION EXCHANGE)

Silica is substituted with cationic residues that interact

strongly with anionic species (-ve charged)
Anions exchange Cl- silica
Me
Cl Me N CH2
Me

-ve
-vecharged
chargedspecies
speciesadhere
adheretotothe
thesupport
support
and
andare
arelater
latereluted
elutedwith
withacid
acid(H
(H+))
+

Most -ve…….Least -ve

35
 HPLC Columns
Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLC

Materials of construction for the tubing

 Stainless steel (the most popular; gives high pressure capabilities)
 Glass (mostly for biomolecules)
 PEEK polymer (biocompatible and chemically inert to most solvents

Packing material:
The packing material is prepared from SILICA particle, ALUMINA particle
and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns
to retain the packing material.
According to the mode of HPLC , they are available in different size ,
diameters, pore size or they can have special materials attached ( such as
antigen or antibody ) for immuno affinity chromatography.
 Modes of High Performance Liquid
Chromatography

Types of Compounds Mode Stationary Mobile Phase

Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases

Ionics, Bases, Acids Ion C-18, C-8 Water/Organic

Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin
High Molecular Weight Size Polystyrene Gel Filtration-
Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic

37
Types of columns in HPLC:
 Guard Column
 Fast Column
 Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)
 Capillary(i.d. 0.1 -1.0 mm; various lengths)
 Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)
 Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
 Guard Column
 These are placed anterior to the separating column. This serves as
protective factor.
 They are dependable columns designed to filter or remove :
Particles that clog the separation column

Compounds and ions that could ultimately cause “ Baseline drift ”,

decreased resolution, decreased sensitivity and create false peaks.

These columns must be changed on a regular basis in order to optimize

their protective function.
 Fast Column
 One of the primary reasons for using these column is to obtain improved
sample output ( amount of compound per unit time).

 Fast column are designed to decrease the time of chromatographic analysis

 Here internal diameter is same but length is short and packed with smaller
particles , that are 3 μm diameter.
 Advantages-
Increased sensitivity
Decreased analysis time
Decreased mobile phase usage
Increase reproducibility
 Capillary Column
 It is also known as micro columns

 It has a diameter much less than a millimeter and there 3 types:

Open tubular
Partially packed
Tightly packed

They allow the user to work with nanoliter sample volume , decreased
flow rate and decreased solvent usage volume , led to cost effectiveness
 Preparatory Column

 Used when objective is to prepare bulk ( milligrams) of sample for

laboratory preparatory application.

 It has usually a large column diameter , which is designed to facilitate

large volume injections into the HPLC system
4. Detector:

• The detector can see (detect) the individual molecules that come
out (elute) from the column.
•A detector serves to measure the amount of those molecules
so that the chemist can quantitatively analyze the sample
components.
•The detector provides an output to a recorder or computer
that results in the liquid chromatogram(i.e., the graph of the
detector response).
HPLC Detectors
 Common HPLC Detectors
•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength

•Mass Spectrometers
•Refractive Index
•Fluorescence

•Light Scattering
•Electrochemical

•Radioactivity

•Conductivity

45
UV-Vis Detectors

Principles: The fraction of light transmitted through the detector cell is

related to the solute concentration according to Beer’s Law.

Detector Flow Cell

I0 c I

Log I0 = A = abc
I

Characteristics: Specific, Concentration Sensitive, good stability,

gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).

46
Fluorescence Detection

Trigger pack Emission

Monochromator
signal &
Lens spectra mode
(condensor EX)
Xenon Slit EM Slit PMT
flash Lamp, Slit EX
PMT detector
15 W

Lens (condensor EM)

Exitation
Mirror Diffuser
Monochromator,
signal & Reference Diode
spectra mode

8 µl Flow Cell, auto-recognition

47
 Electrochemical Detectors

• Gold for carbohydrates.

• Platinum for chlorite, sulfate, hydrazine, etc.
• Carbon for phenols, amines.
• Silver for chloride, bromide, cyanide.

48
Variable UV/Vis Detector
ABS AUFS λ RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min

Ready
5. Computer:

• Frequently called the data system,

The computer not only controls all the modules of the

HPLC instrument but it takes the signal from the
detector and uses it to:

1. determine the time of elution (retention time) of the

sample components (qualitative analysis) and
2. the amount of sample ( quantitative analysis) .
 Ready
UV Spectrum
UV Spectrum {shows full UV abs
UVmax
UVmax

Chromatogram

ABS.
Reset
Wavelength

Rt Rt

ABS.
Time

Chromatogram
Varian 9060 {shows peaks, Rt}

Polychrom
Detector
 What is HPLC used for ?
Separation and analysis of non-volatile or
thermally unstable compounds

HPLC is optimum for the separation of chemical and biological

compounds that are non-volatile .

NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in

gasoline, etc.), gas chromatography is a better separation technique .

Typical non-volatile compounds are:

 Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
 Salts like sodium chloride and potassium phosphate
 Proteins like egg white or blood protein
 Organic chemicals like polymers (e.g. polystyrene, polyethylene)
 Heavy hydrocarbons like asphalt or motor oil
 Many natural products such as ginseng, herbal medicines, plant extracts
 Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
Separation Technique
 How can We Analyze the Sample?
For example:
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose 5
6. isomaltose

2
3
mAU
4
1
6

time

54
 Separations
Injector
Separation in based upon differential
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Pumps
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

55
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

56
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

57
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

58
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

59
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

60
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

61
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

62
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

63
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

64
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

65
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

66
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

67
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

68
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

69
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

70
 Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

71
 The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
72
HPLC used for Qualitative
Analysis
HPLC used for Quantitative
Analysis
 HPLC uses
This technique is used for -

chemistry and biochemistry research analyzing

complex mixtures

purifying chemical compounds

developing processes for synthesizing chemical

compounds

isolating natural products, or predicting physical

properties.

It is also used in quality control to ensure the purity of

raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product
stability and monitor degradation.
In addition,

it is used for analyzing air and water pollutants, for

monitoring materials that may jeopardize occupational
safety or health, and for monitoring pesticide levels in
the environment.
HPLC Applications

Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine

77
 References
 HPLC instrumentation – Agilent Technologies
 Introduction to HPLC – Agilent Technologies
 Principles and Technique of Biochemistry and Moleculer Biology –
Wilson.Keith and Walker. John
 HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE
6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical
Chemistry (Separation Science) at KCL

WEB REFERENCES
 http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
 Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed.
Orlando: Harcourt Brace & Co., 1998.
 http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
 http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
 http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLC
HP1090/HPLCINJ.HTM
 http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analyti
cal_Instruments/Chromatographs/HPLC_Columns
• THANK YOU…