High performance liquid chromatography

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Idea of Chromatgraphyyy

• The mixture of substances is dissolved in a liquid or a gas and flowed through a tube (the column) containing a material to which the components of the mixture will be attracted to different degrees

• The attraction may be hydrogen bonding or van der Waals forces

• In some cases the attraction may be a chemical reaction • In biochemistry, the difference often is that some

components of the mixture will fit into pores in the column material whereas others will not; this is size-exclusion chromatography.

Thus …

• Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between two different phases. • One of these phases is a mobile phase and the other is a

stationary phase.

Developments in the field of chromatography

Year Scientist(s) Comments

1834 Runge,F.F. Used unglazed paper and/or pieces of cloth for spot testing dye mixtures and plant extracts

1850 Runge,F.F. Separated salt solutions on paper

1868 Goppelsroeder. F Introduced paper strip (capillary analysis) analysis of dyes, hydrocarbons, milk, beer, colloids, drinking and mineral waters, plant and animal pigments

1878 Schonbein, C. Developed paper strip analysis of liquid solutions

1897 -1903 Day,D.T. Developed ascending flow of crude petroleum samples through column packed with finely pulverized fuller’s earth.

1906-1907 Tswett, M. Separated chloroplast pigment on CaCO3

1931 Kuhn,R. et al. Introduced liquid-solid chromatography for separating egg yolk xanthophylls

1940 Tiselius, A. Earned Nobel prize in 1948; developed adsorption analyses and electrophoresis

1940 Wilson, J.N. Wrote first theoretical paper on chromatography: assumed complete equilibrium and linear sorption isotherms; qualitatively defined diffusion, rate of adsorption, and isotherm nonlinearity

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1941 Tiselius, A. Developed liquid chromatography and pointed out frontal analysis, elution analysis, and displacement development

1941 Martin, A.J.P. and Synge, R. L. M.

Presented first model that could describe column efficiency; developed liquid-liquid chromatography; received nobel prize in 1952

1944 Consden, R., Gordon, A.H., and Martin, A..J.P

Developed paper chromatography

1946 Claesson,S. Developed liquid-solid chromatography with frontal and displacement development analysis; coworker A. Tiselius

1949 Martin A.J.P. Contributed to relationship between retention and thermodynamic equilibrium constant

1951 Cremer, E. Introduced gas-solid chromatography

1952 Phillips, C.S.G. Developed liquid-liquid chromatography by frontal technique

1955 Glueckauf, E. Derived first comprehensive equation for the relationship between HETP and particle size, particle diffusion, and film diffusion ion exchange

1956 Van Deemter, J.J. Developed rate theoryby simplifying work of Lapidus and Ammundson to Gaussian distribution function

1957 Golay, M. Reported the development of open tubular columns

1965 Giddings, J.C. Reviewed and extended early theories of chromatography6

MOBILEPHASE

STATIONARY PHASE

PRINCIPLE OF SEPARATION

CONTAINER NAME OF THE CHROMATOGRAPHIC

TECHNIQUE

LIQUID SOLID DIFFERENCES IN ADSORPTION

COLUMN LIQUID-SOLID ADSORPTION COLUMN

CHROMATOGRAPHY

LIQUID SOLID DIFFERENCES IN ADSORPTION

THIN LAYER LIQUID-SOLID ADSORPTION THIN LAYER

CHROMATOGRAPHY

LIQUID LIQUID DIFFERENCES INPARTITIONING

COLUMN LIQUID-LIQUID PARTITION COLUMN

CHROMATOGRAPHY

LIQUID LIQUID DIFFERENCES INPARTITIONING

THIN LAYER LIQUID-LIQUID PARTITION THIN LAYER

CHROMATOGRAPHY

GAS LIQUID DIFFERENCES INPARTITIONING

COLUMN GAS-LIQUID-PARTITIONCOLUMN CHROMATIGRAPHY

GAS SOLID DIFFERENCES IN ADSORPTION

COLUMN GAS-SOLID ADSORPTION COLUMN

CHROMATOGRAPHY

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GAS LIQUID/ SOLUTION

CLASSIFICATION OF DIFFERENT CHROMATOGRAPHIC TECHNIQUES

Stationary phase is solid Stationary phase is a liquid coated on the surface of an inert solid support

gas - solid - adsorption chromatography

gas - liquid - partition chromatography

Mobile phase is Gas and all the solutes being separated are in gaseous phase under experimental conditions

Mobile phase is liquid and all the solutes being separated are in liquid/solution state

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Liquid / Solution Chromatography

ADSORPTION : Separation of solutes on the basis of their degree of adsorption on the surface of an adsorbent.

PARTITION : Separation of solutes on the basis of their differences in partition coefficient of solutes in two mutually immiscible solvents. ION EXCHANGE : Separation of similarly charged ions based on the differences in the degree of their affinity towards the ion exchange resin. SIZE EXCLUSION : Separation of solutes based on the differences in their molecular weights or sizes. ELECTROPHORETIC : Separation based on the movement of charged solutes to opposite poles under the applied electrical field.

AFFINITY : Separation of solutes based on specific affinity of solutes on the specific sites of the stationary phase.

Types of Chromatography

1. Liquid Column Chromatography2. Gas Liquid / Solid Chromatography3. Thin-layer Chromatography

Michael Tswett discovered liquid chromatography in

1906 and till late 1950,liquid chromatography lacked

the attributes of gas chromatography and was being

done on columns ,papers and thin layer plates.

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Modern Liquid Chromatography got a place in the

early1960’s by a combination of the experiences

gained with Gas Chromatography and Ordinary

Column Chromatography.

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Researchers working with liquid chromatography

vigorously started making attempts to achieve all

the attributes of gas chromatography to liquid

chromatography.

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IDEAL REQUIREMENTS OF HPLC

Fast analysis : in minutesSensitive :- easily detecting solutes at ppm or ppb levels Efficient :- providing high resolution Non destructive:Highly accurate :- qualitative and quantitative analysisRequires small samples :- micro litres Reliable and relatively simple Versatile:-can separate gases ,liquids and solids

( up to Molecular weight 450 )

1.Short time for analysis

2.Efficient

3.Sensitive

4.Non-destructive.

5.Accurate.

6.Small sample size.

6.Reliable

7.Relatively simple.

8.Relatively inexpensive.

9.Versatile.

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Sequence of efforts taken place during the development of modern liquid chromatography

To reduce separation time to achieve fast analysis. ( From hours to minutes)

HOW?

The solutes must move faster through stationary phase MEANS By increasing the mobility of the liquid mobile phase.

The MIGRATION VELOCITY of the liquid mobile phase should be

HIGH.

We can achieve faster migration velocities of liquid

mobile phase by-

1.Applying vacuum at the other end of the chromatographic

column

2.Applying high pressure on the liquid mobile phase.

Application of higher pressure on liquid mobile phase

seems to be an easier and more practical way for

achieving fast analysis.

CHROMATOGRAPHIC COLUMN

• Glass is normally the choice for chromatographic column material due to its chemical inertness towards stationary phase, mobile phase and solutes being separated.

• We could not increase the pressure of a glass column?

• Hence glass will not be able to withstand high pressures of liquid mobile phase because glass has relatively small mechanical strength.

• Which IDEAL MATERIAL should be selected in place of glass?

• STAINLESS STEEL due to its chemical inertness & mechanical strength.

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Previous condition :- Liquid mobile phase moving across the stationary phase in a glass column under atmospheric pressure.Present condition :- Liquid mobile phase with high migration velocity moving across the stationary phase in a stainless steel column under high pressure..

Will the column separation efficiency of the latter be good?

NO

With the increase in the liquid mobile phase

velocity under high pressures, solute molecules

will not get enough time to interact with the

stationary phase and separation efficiency will

be reduced and we will not be able to achieve

good separation of solutes.

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How can we achieve good column separation efficiency?

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1. By reducing the Particle size of the stationary phase

support.

2. By reducing the width of chromatographic column.

We use Narrow bore (3mm, id) SS columns filled

with small particles of stationary phase support (5µ)

will give us high separation efficiency with low

analytical time when liquid mobile phase moves

through the column under high pressures of the

order of 500 –5000 psi.

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Can the physically coated stationary phases used

in GC be used in narrow bore SS columns through

which liquid mobile phase moves under high

pressures?

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Obviously… NO

Because under high pressures of liquid mobile phase,

physically coated liquid stationary phases will be

removed physically or by dissolution in liquid mobile

phase.

We need physically&chemically stable stationary phase.

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Chemically treated inert (not strictly) particles have

free silinol group at their surface.

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Silica particle

CCCCCC

Si - OHSi - OHSi - OH

Free silinol groups

Early attempts were then made to chemically bonded long chain

aliphatic alcohols with the free silinol groups present on the

surface of silica particles.

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Si - OH + HO - R- H2O

Si - O - R

However these chemically bonded phases with

Si - O - C bonds are stable only in acidic media.

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We cannot restrict our separation work always in acidic media

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In basic media Si– O- C bond undergoes hydrolysis.

Si - O - R basic medium Si - OH + R - OH

We therefore would like to use chemical bonding

with ‘ Si ’ of free silinol groups which will be

hydrolytically stable over a wide acidic to basic

pH range.

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Attempts were then made to carry out chemical

reactions with substituted silane ( Si H4) having

suitable long chain of hydrocarbons attached to it.

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Si - OH1 2 18

+ Cl – Si – C – C C - H

CH3

CH3

H

H

H

H

H

H

Substituted siliane

1 2 18 – Si – C – C C - H

CH3

CH3

H

H

H

H

H

H

Si - O

-H Cl

This chemically bonded phase ( Si-O-Si bond )

is therefore mechanically strong and hydrolytically

stable over a wide pH range (2-9).

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At this stage we can say that the combination of pumping systems capable of giving liquid mobile phase flow under high pressures and chemically bonded, mechanically stable liquid stationary phases with desired polarity, pore size ,and their hydrolytic stability over acidic to basic pH range ,has allowed us to achieve faster analytical times and versatility of separating solutes with diverse nature.

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About Pumping:

Do our pumping systems allow us to have a pulseless

flow of liquid mobile phase through chromatographic

columns?

Different types of Pumps available are

1. Reciprocating Pumps

2. Motor driven Syringe type Pumps

3. Pneumatic Pumps.

• A reciprocating piston pump operation results in a pulsating flow of liquid mobile phase.

• But for chromatographic separations we need pulseless flow of liquid mobile phase.

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A reciprocating pump for HPLC

The piston expels the liquid through a one way valve (Check valve)

The pumping rate can be adjusted by controlling the distance the piston retracts or by the cam rotating speed.

This limits the amount of liquid pushed out by each stroke.

How do we achieve a pulse less flow?

If we introduce a second reciprocating piston pump and synchronize its function with the first one the pulsation is reduced considerably.

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A dual piston reciprocating pump for HPLC

Using common eccentric cam, it allows one piston to pump and other to refill.

Thus we do get pulseless flow of liquid mobile phase with two synchronized piston pumps

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When the liquid mobile phase is moving under high

pressures through chromatographic columns,

introduction of a sample on to the column will require

some specially designed injection system.

Introduction of sample?

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The most popular and commonly used injector system,

is the syringe –loop injector of the Rheodyne type.

It is a fixed volume universal injector which allows

introduction of micro litres of samples on to the

chromatographic column.

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With Rheodyne injector we have achieved

“small sample” attribute of GC with liquid

chromatography.

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Thus so far with we have achieved the following attributes with LC

Fast Analysis

Small Sample Size

Versatility

Efficiency

Non-destructive

NOW WHAT IS LEFT?

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DETECTORS:

As soon as the solutes are eluted they should be

detected and quantitated with sensitive and

specific or universal detectors.

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We have universal detectors like :

Refractive index detector is relatively less sensitive

but can detect and quantitate all types of solutes

Mass spectrometry detector is highly sensitive

detector and can also help in structure

elucidation.

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With specific and universal detectors we have achieved the following attributes:

High sensitivity

High reliability and accuracy

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Thus in place of glass column liquid

chromatography now we have

1 4 5

2

3

1- Pumping system

2- Universal injector

3- Column

4- Detector

5- Recorder

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1.Pumping system capable of giving

pulseless flow of liquid mobile phase under

high pressures of the order of 500 to

5000psi.

*This makes fast analysis.

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2.Universal injector which allows to introduce samples in microlitres.

*This reduces sample size

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3.Narrow bore SS columns with suitable chemically

bonded phases with small particle size (3-5µ), desired

polarity and pore size and hydrolytically stable over a

wide acidic to basic pH range.

*This gives high separation efficiency , high versatility in separating solutes with diverse nature.

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4.Sensitive and Specific or Universal detector

*This gives high sensitivity , reliability and accuracy of

detection and quantitation of solutes.

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5.Computing system allows to record and compute chromatographic results.

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All the above factors add

“High Performance” to normal Liquid

Chromatography.

It is therefore known as

“High Performance Liquid Chromatography”

abbreviated as HPLC.

Thank You