Presentation of chromatography by Shaiq Ali

ReviewReviewof of

ChromatographyChromatographyPresenting By:Presenting By:

Mr. Muhammad Kamran

&&

Mr. Shaiq Ali

Muhammad KamranMuhammad Kamran

22

33

HistoryHistory

Mikhail Tswett, Russian, 1872-Mikhail Tswett, Russian, 1872-19191919

Botanist Botanist In 1906 Tswett used to chromatography to In 1906 Tswett used to chromatography to

separate plant pigmentsseparate plant pigments

He called the new technique chromatography He called the new technique chromatography because the result of the analysis was because the result of the analysis was 'written in color' along the length of the 'written in color' along the length of the adsorbent columnadsorbent column

Chroma means Chroma means ““colorcolor”” and graphein means to and graphein means to ““writewrite””

4444

1903 Tswett - plant pigments separated on chalk columns1931 Lederer & Kuhn - LC of carotenoids1938 TLC and ion exchange1950 reverse phase LC1954 Martin & Synge (Nobel Prize)1959 Gel permeation1965 instrumental LC (Waters)

Milestones in ChromatographyMilestones in Chromatography

55

Introduction to Chromatography Definition

Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. The chromatographic process occurs due to differences in the distribution constant of the individual sample components.

Components: mobile phase: a solvent that flows through the supporting medium

stationary phase: a layer or coating on the supporting medium that interacts with the analytes

supporting medium: a solid surface on which the stationary phase is bound or coated

ChromatographyChromatographyIs a technique used to separate and identify the components of a mixture.

Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium.

Molecules that spend most of their time in the mobile phase are carried along faster.

77

Chromatography has application in every branch of the Chromatography has application in every branch of the physical and biological sciencesphysical and biological sciences

1212 Nobel prizes were awarded between 1937 and 1972 alone Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital rolefor work in which chromatography played a vital role

Applications of Chromatography

88

Applications of Chromatography

Forensics

Research Pharmaceutical industry

separating mixtures of compounds

identifying unknown compounds

establishing the purity or concentration of compoundsmonitoring product formation in the pharmaceutical and biotechnology industries

99

Types of Chromatography…

Paper

HPLC Gas

Thin layer

Column

Chromatographic Chromatographic methods classificationmethods classification

1111

((11 ) )Classification of chromatography according to Classification of chromatography according to Phase involvedPhase involved: :

1.) 1.) The primary division of chromatographic techniques is based The primary division of chromatographic techniques is based on the type of mobile phase used in the system:on the type of mobile phase used in the system:

Type of ChromatographyType of Chromatography Type of Mobile PhaseType of Mobile Phase

Gas chromatography (GC)Gas chromatography (GC) gasgas Liquid chromatograph (LC)Liquid chromatograph (LC) liquidliquid

12121212

2.) Further divisions can be made based on the type of 2.) Further divisions can be made based on the type of stationary phase used in the system:stationary phase used in the system:

Gas ChromatographyGas Chromatography

Name of GC MethodName of GC Method Type of Stationary PhaseType of Stationary PhaseGas-solid chromatographyGas-solid chromatography solid, underivatized support solid, underivatized supportGas-liquid chromatographyGas-liquid chromatography liquid-coated supportliquid-coated supportBonded-phase gas chromatographyBonded-phase gas chromatography chemically-derivatized chemically-derivatized

supportsupport

13131313

Liquid ChromatographyLiquid ChromatographyName of LC Method Type of Stationary PhaseAdsorption chromatography solid, underivatized supportPartition chromatography liquid-coated or derivatized supportIon-exchange chromatography support containing fixed chargesSize exclusion chromatography porous supportAffinity chromatography support with immobilized ligand

1414

((22 ) )Classification according to the packing of the Classification according to the packing of the stationary phasestationary phase::

In column chromatography the stationary phase is contained in a tube called the column.

Planar chromatography. In this geometry the stationary phase is configured as a thin two-dimensional sheet.

(i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase.

(ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet.

1515

(3) Classification according to the force of separation:

Mode or type Stationary phase Mobile phase

Mechanism

Adsorption Chromatography

Solid that attracts the solutes

Liquid or gas

Solutes move at different rates according to the forces of attraction to the stationary phase.

Partition Chromatography

Thin film of liquid formed on the surface of a solid inert support

Liquid or gas

Solutes equilibrate between the 2 phases according to their partition coefficients

Ion Exchange Chromatography

Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds

Liquid containing electrolytes

Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions.

Molecular Exclusion Chromatography

Porous gel with no attractive action on solute molecules

Liquid Molecules separate according to their size:1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate.

Affinity Chromatography

Solid on which specific molecules are immobilized

Liquid or gas

Special kind of solute molecules interact with those immobilized on the stationary phase

1616

Term Definition

SolventMobile liquid phase with no affinity to the stationary phase

(i.e. inert towards it) & no effect on solutes.

DeveloperAny liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move

them through the column.

Effluent Any liquid that passes out of the column.

EluentAny liquid that has lesser affinity to the stationary phase

than solutes but is capable to move them out of the column.

Eluate Fraction of eluent containing a required specific substance.

Retention volume (VR)

)or retardation volume :(Volume of mobile phase that passes out of the column, before elution of a specific

substance.

SHAIQ ALISHAIQ ALI

1717

1818

Theory of Chromatography1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

Wh

Wb

Where:tR = retention timetM = void timeWb = baseline width of the peak in time unitsWh = half-height width of the peak in time units

Inject

1919

Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation

system)

A similar plot can be made in terms of elution volume instead of elution time

Peak width & peak positiondetermine separation of peaks

2020

2.) Solute Retention:

A solute’s retention time or retention volume in chromatography is directly related to the strength of the solute’s interaction with the mobile and stationary phases.

Retention on a given column pertain to the particulars of that system:- size of the column- flow rate of the mobile phase

Capacity factor (k’): more universal measure of retention, determined from tR or VR.

k’ = (tR –tM)/tM

or

k’ = (VR –VM)/VM

capacity factor is useful for comparing results obtained on different systems since it is independent on column length and flow-rate.

2121

3.) Efficiency:

Efficiency is related experimentally to a solute’s peak width.- an efficient system will produce narrow peaks- narrow peaks smaller difference in interactions in order to separate two

solutes

Efficiency is related theoretically to the various kinetic processes that are involved in solute retention and transport in the column

- determine the width or standard deviation () of peaks

Wh

Estimate from peak widths, assuming Gaussian shaped peak:

Wb = 4

Wh = 2.354

Dependent on the amount of time that a solute spends in the column (k’ or tR)

2222

Number of theoretical plates )N(: compare efficiencies of a system for solutes that have different retention times

N = (tR/)2

or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2

The larger the value of N is for a column, the better the column will be able to separate two compounds.

- the better the ability to resolve solutes that have small differences in retention- N is independent of solute retention- N is dependent on the length of the column

© RGR© RGR 2323

Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of columns with different lengths:

H = L/N

where: L = column length N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical plate

H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?a. Eddy diffusionb. Mobile phase mass transferc. Stagnant mobile phase mass transfer (The fraction of the mobile phase

contained within the pores of the particle)d. Stationary phase mass transfere. Longitudinal diffusion

3/18/20093/18/2009 © RGR© RGR 2424

2525

a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column.

As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances.

Longer path arrives at end of column after (1).

2626

A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to band-broadening

The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on:

1) the size of the packing material2) the diffusion rate of the solute

b.) Mobile phase mass transfer – a process of peak broadening caused by the presence of different flow profile within channels or

between particles of the support in the column.

2727

c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the rate of diffusion of the solute molecules between the

mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase).

Since a solute does not travel down the column when it is in the stagnant mobile phase, it spends a longer time in the column than solute that remains in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass transfer depends on:

1) the size, shape and pore structure of the packing material2) the diffusion and retention of the solute3) the flow-rate of the solute through the column

2828

d.) Stationary phase mass transfer – band-broadening due to the movement of solute between the stagnant phase and the stationary phase.

Since different solute molecules spend different lengths of time in the stationary phase, they also spend different amounts of time on the column, giving rise to band-broadening.

The degree of band-broadening due to stationary phase mass transfer depends on:

1) the retention and diffusion of the solute2) the flow-rate of the solute through the column3) the kinetics of interaction between the solute and the stationary phase

2929

e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase.

The degree of band-broadening due to longitudinal diffusion depends on:

1( the diffusion of the solute2( the flow-rate of the solute through the column

3030

Van Deemter equation: relates flow-rate or linear velocity to H: H = A + B/ + C

where:

= linear velocity )flow-rate x Vm/L(H = total plate height of the columnA = constant representing eddy diffusion & mobile phase mass transferB = constant representing longitudinal

diffusionC = constant representing stagnant mobile

phase & stationary phase mass transfer

One use of plate height (H) is to relate these kinetic process to band broadening to a parameter of the chromatographic system (e.g., flow-rate).

This relationship is used to predict what the resulting effect would be of varying this parameter on the overall efficiency of the chromatographic system.

Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2peak width (Wh)

H = L/N

3131

optimum

Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of the mobile phase

Optimum linear velocity (opt) - where H has a minimum value and the point of maximum column efficiency:

3232

4.) Measures of Solute Separation:

separation factor () – parameter used to describe how well two solutes are separated by a chromatographic system:

= k’2/k’1 k’ = (tR –tM)/tM

where:k’1 = the capacity factor of the first solutek’2 = the capacity factor of the second solute,

with k’2 k’1

A value of 1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.

3/18/20093/18/2009 © RGR© RGR 3333

resolution (RS) – resolution between two peaks is a second measure of how well two peaks are separated:

RS =

where:tr1, Wb1 = retention time and baseline width for the

first eluting peaktr2, Wb2 = retention time and baseline width for the

second eluting peak

tr2 – tr1

(Wb2 + Wb1)/2

Rs is preferred over since both retention (tr) and column efficiency (Wb) are considered in defining peak separation.

Rs 1.5 represents baseline resolution, or complete separation of two neighboring solutes ideal case.

Rs 1.0 considered adequate for most separations.

3434

Importan Chromatographic formulae

3535

THANK YOUTHANK YOUTHANK YOUTHANK YOU