HPLC

CHROMATOGRAPHIC MECHANISMS

• The process whereby a solute is transferred from a mobile phase to a stationary phase.

• Four sorption mechanisms– Adsorption– Partition– Ion-Exchange– Size-Exclusion

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SURFACE ADSORPTION

• Based on the relative polarities of solute, stationary phase and mobile phase.

• Components get distributed according to their relative affinity.

• Components having high affinity towards the stationary phase- Travel slower

• Components having less affinity towards the stationary phase- Travel faster

• No two components have the same affinity for a combination of SP, MP, and other conditions.

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PARTITION

• Based on the relative solubility of the solute in the two phases. (Volatility if the mobile phase is gas).

• Solutes will be distributed according to their partition coefficients.

• Components which are more soluble in the stationary phase- Travel slower

• Components which are less soluble in the stationary phase- Travel faster

• No two components have the same partition coefficient for a particular combination of Stationary phase, Mobile phase, and other conditions.

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ION EXCHANGE

• Based on the relative ion exchange capacity of the solute in the two phases.

• Stationary Phase contain fixed charged groups and mobile counter ions.

• Counter ions exchange with ions of the solute.• Reversible exchange of ions takes place between

similar charged ions of solute in the mobile phase and that of an ion exchange resin.

• Cation exchange resin used for separation of Cations.• Anion exchange resin for anion separation.

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Choice of methods• The choice of a particular mechanism depends on

following factors– The complexity of sample– Solubility and volatility of sample– Chemical and physical properties of the sample– Resolution required– Required separation efficiency, concentration of

analyte– Detection limit– Number of samples under analysis

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Technique of Column liquid chromatography

• Selection of a column• Selection of the adsorbent and activation• Selection of the mobile phase• Sample introduction technique• Development techniques• Detection and analysis

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Classification based on the purpose of Use

• Analytical Chromatography– Qualitative – Quantitative

• Preparative Chromatography

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The Chromatogram

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tR = retention time: is the time required for the analyte to reach the detector, after the sample injection.

tM = void time: is the time required for a non retained species to reach the detector. It is also known as “Dead time”

Wb = baseline width of the peak in time unitsWh = half-height width of the peak in time units

Wb

Wh

Information obtained form a chromatogram

1. Retention time of a solute – This is characteristic of a compound.

2. Area of the peak- Relates the concentration of the solute.

3. Width of the peak ( at base and at half height)- Shows efficiency of separation.

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Peak shapes

Asymmetrical peaks( Tailing or Fronting)• The extend of asymmetry is defined by the tailing

factor(TF). TF= b/a. Both a and b are measured at 10% of the peak height as shown.

b/a = 1 (Symmetric)b/a > 1 (Tailing)b/a< 1 (Fronting)

• A tailing peak will have a TF greater than one. The opposite symmetry, fronting will yield a TF less than one.

• When the asymmetry factor lies outside the range of 0.95-1.15, it reduces the column efficiency and resolution. For example, an AF of 1.3 reduces efficiency by 69% and resolution by 30%.

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• Reason for asymmetry:– Heterogeneous retention sites within a given

packing (ie., varying retention affinity). – Sample overloading– Over activation of stationary phase.– It can also occur due to sample injection problem

or because of poorly packed columns.

• Precautions to prevent asymmetry:– Take care not to over load the column. (Usually

1mg of sample/gm of stationary phase). – Selectively remove the stronger sites, ie. by

deactivating the stationary phase. 15

Theory

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Plate Theory- Defenition

• Developed by Martin and Synge.• “A chromatographic column consists of a

series of discrete, continuous horizontal layers within each of which an equilibrium of the solute exists between the stationary and the mobile phases.”

• Each of these layer is called a “plate”, and the thickness of the plate is called “Height Equivalent to a Theoretical Plate” or HETP.

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Terminologies used in plate theory

• Three terms are used as a quantitative measure of column efficiency.– Number of Theoretical Plates (N)– Plate Height (H) – Length of the column packing (L)

• The relationship governing the column efficiency is N=L/H .

• Efficiency of a column can be explained with the help of plate theory.

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Efficiency explained by plate theory

• Efficiency of a column is high – When the number of theoretical plates is more.– When the HETP is less.

• Efficiency (plate height) varies as a result of differences in the following factors– column dimensions,– stationary phase used, – mobile phase used.

• Plate numbers can be from a few hundreds to several hundred thousands.

• Plate heights can be from a few tenths to 1/1000th of a cm or even lesser.

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Determination of number of theoretical plates (N) from a chromatogram

For a Gaussian shaped peakN = 16 (t’R/Wb)2

N = 5.54 (t’R/Wh)2

Wh

Wb

Band broadening

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Peak Broadening

Rate Theory- Why

• As the solutes migrating down the column, separation occurs. At the same time broadening of the bands also takes place.

• Band broadening is inevitable, conditions are to be identified where it occurs more slowly than the separation process.

• Plate theory could not explain the band broadening, which reflects the loss of column efficiency. Rate theory explains the band broadening

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Rate theory: Definition

“The magnitude of kinetic effects on column efficiency depends upon the length of time the mobile phase is in contact with the stationary phase, which in turn depends upon the flow rate of the mobile phase ”.

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Efficiency & Band broadening explained by rate theory

• The efficiency studies generally been carried out by determining (H) as a function of mobile phase velocity (u).

• Plate height expresses the extent of band broadening, and efficiency.

• As seen in the graph, a minimum in (H) –(maximum efficiency) – occurs at low linear flow rates (optimum flow rates). 25

Van Deemter equation

• Van Deemter equation describes the quantitative relationship between the experimental variables and the plate height.

H = A+B/u +CSu +CMu• “A” is the eddy diffusion term. The coefficient that

describes the multiple path effects

• “B” is the Longitudinal diffusion coefficient.

• “CS and CM “ are the mass transfer coefficients for the stationary and mobile phases respectively.

• “u” is the mobile phase flow velocity.26

Eddy Diffusion (A)

“A” term: Eddy diffusion

“A” is defined as A= λdp’

– dp’ is the particle diameter– λ- a function of packing uniformity and column geometry

• Thus “A” depends on size of the particles, shape and manner of packing and the column diameter.

• To minimise “A” term- The mean diameter of the particle- as small as possible and packed uniformly

• There has to be a trade off between the particle size, column length, and the pressure required. 100-200mesh.

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Longitudinal diffusion (B)

“B”- term: Longitudinal diffusion

“B” is defined as B=2γDM

• γ- is an obstruction factor• DM- Solute diffusion coefficient in the mobile phase

• The magnitude of B term determined by diffusion coefficient (DM)of the analyte in the mobile phase and is directly proportional

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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

Resistance to mass transfer at Stationary phase (Cs)

“C” term: Resistance to mass transfer (Cs) existing at stationary phase

Cs=f(k)df2/DS

• Df – the effective thickness of the stationary phase

• Ds – the diffusion coefficient of the solute in the stationary phase.

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Resistance to mass transfer at the mobile phase: “C” term

Occurs due to the presence of different flow profile within channels or between particles of the support in the column.

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 mobile phase mass transfer depends mainly on:

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

“C” term: Resistance to mass transfer “CM” existing in the mobile phase

CM=f(k)dp2/DM

• dp particle diameter

• DM diffusion coeficient of solute in the mobile phase

• It is proportional to the square of the particle diameter dp

2 and inversely proportional to the diffusion coefficient DM of the solute in the mobile phase.

• Decreasing the size of the stationary phase particles is always helpful in decreasing the plate height.

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H = A+B/u +CSu +CMu

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How can band broadening be reduced? (and thus column efficiency be enhanced)

Minimum value for “H” is achieved by:• Decreasing the particle diameter• Decreasing the column width• Minimizing thickness of liquid stationary phase• Lowering the temperature (reduces diffusion

coefficient)• Using mobile phases having low viscosity and high

diffusion coefficient• Using stationary phase having low viscosity and

high diffusion coefficient in GC and partition.

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Factors affecting efficiency of a column1. Dimensions of the column.

– Increase with length. Optimum length to breadth ratio (20:1 to 30:1)- ideal.

2. Particle size of the adsorbent.– Lesser the size higher efficiency. Compromise

need to be made since the “flow rate” affected. 100-200 mesh ideal.

3. Temperature.– High temperature enhances rate of elution with

less resolution.– Low temperature affords higher resolution– Separations carried out at 20±2°C

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4. Pore diameter.– Pore diameter of ≤ 20°A affords effective polar

adsorption.

5. Nature of the solvents (Eluent).– Flow rate is inversely proportional to viscosity.

Solvents of low viscosity are selected.

6. Packing of the column.– Uniformity in packing– Neither too compact nor too much loose.– Avoid presence of air bubbles and cracks in the

column- leads to channelizing effect.

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