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CHROMATOGRAPHY

BY-RICHA CHAUHAN

AKSHATA ADHYAPAK

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Chromatography is an analytical method that is widely used for separation, identification and determination of the chemical components in complex mixtures.

It consist of a mobile phase and stationary phase. Components of the mixture are carried through the stationary phase by the flow of gaseous or liquid mobile phase, separations being based on differences in migration rates among the sample components.

Chromatographic methods: Based on technique:Column Chromatography (HPLC, GC)Planar Chromatography (PAPER, TLC) Based on affinity:Adsorption chromatography ( TLC, HPLC, GC)Partition chromatography ( GLC, PAPER) Based on non-affinity:Gel permeation chromatography

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TERMINOLOGIES A theoretical plate in many separation processes is a

hypothetical zone or stage in which two phases, such as the liquid and vapour phases of a substance, establish an equilibrium with each other.

It is expressed as N, N= L / H where N= No. of plates L= length of column H= Height equivalent to theoretical plates (HETP)

The plate height H is known as height equivalent oftheoretical plate. H= ϭ2 / L where ϭ2 = Variance , L= length of column (cms)

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Plate Theory / Separation Efficiency / Efficiency factor It is described as plate height (H) and plate no. (N). This is an important characteristic in chromatography as it

reflects no. of times the solute partitions between two phases while travelling through the column.

Generally N is expressed as Neffective and given by Neffective = L / H N can also be calculated using following equation N = (tr’ / wb)2 x 16 where tr’= adjusted retention time wb = width of peaks

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ISOCRATIC ELUTION: A separation that employs a single solvent orsolvent mixture of constant composition.

GRADIENT ELUTION: Here two or more solvent systems that differsignificantly in polarity are employed. After elution is begun; the ratio ofthe solvents is varied in a programmed way, sometimes continuously andsometimes in a series of steps. Separation efficiency is greatly enhancedby gradient elution.

To decide whether to go for isocratic or gradient elution, we need to note the elution of solute with general gradient of mobile phase.

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tg= total time of the gradient i.e., 18 minstg = difference in the retention time of last peak and first peak.In the adjacent diagram, tg = 13- 3 = 10mins tg/ tg = 10/18 = 0.8If this ratio 0.25, go for gradient elutionIf this ratio 0.25, go for isocratic elution

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CAPACITY FACTORIt is used to describe the migration rates of solutes on columns. For asolute A, the capacity factor k’A is defined as:

k’A = KAVS/VM where, KA is partition ratio for the species A

VS is the volume of the stationary phase

VM is the volume of the mobile phaseIt is a measure of the retention of a peak that is independent of column geometry or mobile phase flow rate.

k’ = (tr-t0) / t0

tr = retention time of peakt0= dead time of column

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k’ can be altered by varying stationary phase.When k’=0 indicates no retention in the stationary phase or no affinity

to the stationary phaseThis will lead to no resolution and separationIn such cases alteration in the capacity factor becomes essential to

increase or to get better resolution or separation.

SELECTIVITY FACTORIt is denoted as αThe selectivity factor of a column for the two species A and B is defined

as α= KB/KA KB is the partition ratio for more strongly retained species B and KA is the constant for less strongly held or more rapidly eluted

species A. when α = 1 resolution =0 irrespective of large no of N larger α, better and easier separation and resolution small changes in α can bring large changes in resolution

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In case of GC, changes in the stationary phase can bring large changes in α factor.

While in case of HPLC α can be changed by changing both stationary phase and mobile phase.

RESOLUTIONThe resolution Rs of a column provides quantitativemeasures of its ability to separate two eluted peaks.It is defined as the difference in the retention timesbetween two peaks, divided by the combined widths ofelution peaks.

Rs = tr2-tr1

½ (ω1+ω2)

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TAILING FACTORThe tailing factor is a measure of peak tailing. It is defined

as the distance from the front slope of the peak to the back slope divided by twice the distance from the centre line of the peak to the front slope, with all measurements made at 5% of the maximum peak height. The tailing factor of a peak will typically be similar to the asymmetry factor for the same peak, but the two values cannot be directly converted. 

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• ASSYMETRY FACTOR The asymmetry factor is a measure of peak tailing. It is

defined as the distance from the centre line of the peak to the back slope divided by the distance from the centre line of the peak to the front slope, with all measurements made at 10% of the maximum peak height. The asymmetry factor of a peak will typically be similar to the tailing factor for the same peak, but the two values cannot be directly converted.

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Rate Theory

In the rate theory, a number of different peak dispersion processes were proposed and expressions were developed that described

• the contribution of each of the processes to the total variance of the eluted peak

• the final equation that gave an expression for the variance per unit length of the column

The rate theory has resulted in a number of different equationsAll such equations give a type of hyperbolic function that predicts

a minimum plate height at an optimum velocity and, thus, a maximum efficiency. At normal operating velocities it has been demonstrated that the Van Deemter equation gives the best fit to experimental data

The Van Deemter Equation H = A + B/u + u [CM + CS]

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H = A + B/u + u [CM +CS]

Van Deemter model

u = L/ tM

A: random movement through stationary phase

B: diffusion in mobile phase

C: interaction with stationary phase

H: plate height

u: average linear velocity

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Term A- molecules may travel unequal distances- independent of u- depends on size of stationary particles or coating (TLC)

H = A + B/u + u [CM +CS]

Van Deemter model

time

Eddy diffusionMP moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

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Term B

H = A + B/u + u [CM +CS]

Van Deemter model

Longitudinal diffusion

B = 2γ DM

γ: Impedance factor due to packing DM: molecular diffusion coefficientB term dominates at low u, and is more important in GC than LC since DM(gas) > 104 DM(liquid)

One of the main causes of band spreading is DIFFUSIONThe diffusion coefficient measures the ratio at which a substance moves randomly from a region of higher concentration to a region of lower concentration

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Term B

H = A + B/u + u [CM +CS]

Van Deemter model

Longitudinal diffusion

B = 2γ DM

γ: Impedance factor due to packing DM: molecular diffusion coefficient

B term dominates at low u and is more important in GC than LC since DM(gas) > 104 DM(liquid)

B - Longitudinal diffusionThe concentration of analyte is less at the edges of the band than at the centre. Analyte diffuses out from the centre to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time in the column, which decreases the effects of longitudinal diffusion.

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Cs: stationary phase-mass transferCs = [(df)2]/Ds

df: stationary phase film thicknessDs: diffusion coefficient of analyte in SP

CM: mobile phase–mass transfer

CM = [(dP)2]/DM packed columnsCM = [(dC)2]/DM open columns

H = A + B/u + u [CM +CS]

Van Deemter model

Term C

dP: particle diameterdC: column diameter

BandwidthStationary

phase

Mobilephase

Elution

Broadened bandwidth

Slow equilibration

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H = A + B/u + u [CM +CS]

Van Deemter model

Term C (Resistance to mass transfer) BandwidthStationary

phase

Mobilephase

Elution

Broadened bandwidth

Slow equilibration

The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

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Van Deemter plotA plot of plate height vs average linear velocity of mobile phase

Such plot is of considerable use in determining the optimum mobile phase flow rate

A

BC

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• APPLICATIONS OF CHROMATOGRAPHYQualitative analysis: to determine presence or absence of components in mixtures that contain a limited number of species.Quantitative analysis:It is based upon a comparison of either the height or the area of an analyte peak with that of one or more standards. Both of these parameters vary linearly with concentrations.

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• REFERENCES:• D. A. Skoog and J. J. Leary, principles of instrumental

analysis, 4th edition, page no. 592-598, 1992.• Chromatography: fundamentals and applications of

chromatography and electrophoretic methods, E. Heftmann, New York, Elsevier, 1983

• Chromatographic theory and basic principles, J. A. Jonsson, New York, 1987

• www.lcresources.com

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