Chapter 10

CHROMATOGRAPHY

What is Chromatography?

• Chromatography is the science of separating the components of materials from each other.

• Such separations are achieved using a wide variety of techniques

• For example, molecules can be separated by their differences in molecular charge, molecular size, molecular mass, bond polarities, redox potential, ionization constants, and arrangement of bonds such as isomer.

• Separations that use electric fields to drive charged molecules so that they separate is called electrophoresis.

• It is believed that the separation method in its modern form originated at the turn of the century from the work of Tswett to whom we attribute the terms chromatography and chromatogram

• The method was used for preparation and purification purposes until the development of sensitive detectors

• The detector signal, which is registered, leads to a chromatogram that indicates the variation of the composition of the eluting phase with time.

(a)Diagram showing

the separation of a

mixture of

components A and B

by column elution

chromatography.

(b)The output of the

signal detector at the

various stages of

elution shown in (a).

Separation Experiment

• Sample is dissolved in a mobile phase (a gas, a

liquid or a supercritical fluid)

• The mobile phase is forced through an immiscible

stationary phase which is fixed in place in a column or

on a solid surface.

• The two phases are chosen so that the components of

the sample distribute themselves between the mobile

and stationary phase to a varying degree.

The Current Equipment that Replaced Michael Tswett Experiment

Diagram of high-performance liquid chromatography

(HPLC).The pressures needed to obtain an adequate

flow of fluid through a bed of particles in the column

Nomenclature of Chromatographic Separations

• Chromatographic separations can be carried out in a liquid (liquid chromatography) or a gas (Gas chromatography) phase (mobile phase).

• In both liquid chromatography (LC) and gas chromatography (GC), the sample is introduced rapidly into a moving fluid phase - a liquid or gas, respectively-more generally called the mobile phase.

• In common usage, the term eluent refers to liquids.

• During all separations, the components of the sample are carried by the mobile phase through a column packed with particles of solid material.

• The solid materials are called solid support, packing material, and commonly known as stationary phase.

• The crucial interactions occur at the surface of the stationary phase.

• The interaction of the analytes may occur with the surface of the support itself or with liquids coated on the surface or with some molecules bonded to the surface.

• The eluent that passes through a column is often called the effluent.

• The results of chromatographic separations can be obtained by collecting the effluent in a series of fractions and carrying out further tests on them.

• On the other hand, liquids are passed through an instrumental detector.

• The detectors are placed at the end of the column. A chromatogram will be recorded

The Chromatogram

• The components entering the detector will be shown as a series of peaks that would be more or less resolved from one another as they rise from the baseline.

• If the detector signal varies linearly with the concentration of analyte, the same variation will occur for the area under the peak in the chromatogram.

• A constituent is characterized by its retention time, tR,

• Retention time is defined by the time taken between the moment of injection into the chromatograph and the peak maximum recorded on the chromatogram.

A

BC

D

E

Sample: mixture of volatile liquids (~1L)

Gas Chromatogram

Gas Chromatogram

0 5 10 15 20

Time (minutes)

Ab

un

dan

ce

A

B

C

D

E

Gas Chromatograph

• In an ideal case, the retention time tR is independent of the quantity injected.

• A compound not retained will elute out of the column at time tM, called the void time or the dead time (sometimes designated by to ).

• The separation is complete when as many peaks are seen returning to the baseline as there are components in the mixture.

• In quantitative analysis, it suffices to separate only the components that need to be measured.

• When tM = tR ; there would be no separation, why?

Parameters of Chromatography

• Chromatogram showing the parameters that are used to characterize a

chromatogram.

• Each band can be described by a peak position and a peak width.

• Pairs of bands are characterized by a separation factor or by resolution

of the corresponding peaks.

Parameters for Individual Bands (Peaks)

• Hold-up volume volume, VM • Volumes that elutes from the column

between the injection of the sample and the maximum of the first peak that elutes.

• Its value, written, corresponds to the liquid (or gas) volume surrounding the stationary phase.

• It is the minimum volume of eluent that can carry any component of the sample from the point of injection to the detector.

Retention volume, or elution volume: VR

• The volume (or time) at which the

maximum of the peak appears.

• This is called the total retention volume

(preferred), retention volume, or elution

volume or retention time

• The IUPAC recommends the

abbreviations VR or tR

• Peakwidth or Bandwidth, (fwhm) or W1/2

• Fwhm = full width at half maximum.

• For a band having height h; the fwhm is the volume eluted between the edges of the band at a position one-half of the total height.

• Peak width at the base, W

• This is found by drawing tangent lines at the inflection points on both sides of the band outline and extending them down to the baseline.

• The time between these points on the baseline is a useful measure of the width.

• W = 1.698 fwhm = 4 ; (Gaussian band shape)

Classification of chromatographic techniques

• Chromatographic techniques can be classified into

three categories depending on

– the physical nature of the phases,

– the process used,

– or the physico-chemical phenomenon, which is at

the basis of the Nernst distribution coefficient K,

also defined as:

• We will take here the classification based on the

nature of the phase present

Classification of chromatographic techniques

1. Liquid-solid chromatography

• The mobile phase is a liquid and the stationary

phase is a solid.

• This category, which is widely used, can be

subdivided depending on the retention phenomenon

into:

– Adsorption chromatography

– Ion chromatography

– Molecular exclusion chromatography

a. Adsorption chromatography

• The separation of organic compounds on a

thin layer of silica gel or alumina with solvent

as a mobile phase

• Solutes bond to the stationary phase

because of physisorption or chemisorption

interactions.

• The physico-chemical parameter involved is

the coefficient of adsorption.

b. Ion chromatography

• The mobile phase in this type of chromatograph; is a buffered solution and the stationary phase consists of spherical m diameter particles of a polymer

• The surface of the particles is modified chemically in order to generate ionic sites.

• These phases allow the exchange of their mobile counter ion, with ions of the same charge present in the sample.

• This separation relies on the coefficient of ionic

distribution

c. Molecular exclusion chromatography

• The stationary phase is a material containing pores, the dimensions of which are chosen to separate the solutes present in the sample based on their molecular size.

• This can be considered as a molecular sieve allowing selective permeation.

• This technique is known as gel filtration or gel permeation, depending on the nature of the mobile phase, which is either aqueous or organic.

• The distribution coefficient in this technique is called the coefficient of diffusion.

2. Liquid-liquid chromatography (LLC)

• Stationary phase is a liquid immobilized in the column.

• It is important to distinguish between the inert support which only has a mechanical role and the stationary phase immobilized on the support

• The stationary phase still acts as a liquid and the separation process is based on the partition of the analyte between the two phases at their interface.

• The parameter involved in the separation mechanism is called the partition coefficient.

3. Gas-liquid chromatography (GLC)

• The mobile phase is a gas and the stationary phase

is a liquid.

• The liquid can be immobilized by impregnation or

bonded to a support,

• The partition coefficient K is also involved

4. Gas-solid chromatography (GSC)

• Stationary phase is a porous solid (such as graphite or

silica gel) and the mobile phase is a gas.

• This type demonstrates very high performance in the

analysis of gas mixtures or components that have a

very low boiling point.

The Theoretical Plate Model

• Many theories have been suggested to explain the mechanism of migration and separation of analytes in the column.

• The oldest one, called the theoretical plate model,

• In this model, each analyte is considered to be moving progressively through the column in a sequence of distinct steps (theoretical plates), although the process of chromatography is a dynamic and continuous phenomenon.

• Each step corresponds to a new equilibrium of the entire column.

• In liquid-solid chromatography, for example, the elementary process is described as a cycle of adsorption/desorption.

Column Efficiency

• Assuming L, is the column length, H is the value for the height

equivalent to one theoretical plate

• Since N = L/H

• The more appropriate equation for N is

N = 5.54 t2R/w2

1/2

Where tR is the retention time and w1/2 is the

width of the peak at half its height.

Isochronic image of

the concentration

of an eluted

compound at a

particular instant.

Dispersion of a solute in a column and

its translation into a chromatogram

Variation of the

concentration at

the outlet of the

column as a

function of time.

On the chromatogram,

Represents the peak width

At 60.6% of the height

Effective plate number

• If the performance of different columns has to be compared for a given compound, more realistic values are obtained by replacing the total retention times tR, by the adjusted retention times t’R

• t’R does not take into account the void time tM spent by the compound in the mobile phase.

• The mathematical relationships:

Separation factor between two solutes

• The separation factor, , allows the comparison of two adjacent

solutes 1 and 2 present in the same chromatogram

Thus, the separation

factor is given

by the equation:

Resolution factor between two peaks

• To quantify the separation between two peaks, the resolution

factor R is used and can be obtained from the chromatogram

Origins of band broadening

The van Deemter equation in chromatography

• The length of time it takes a compound to pass

through the column depends on its capacity factor,

K’

• Capacity factor is a measure of the degree to which

the compound partitions (adsorbs) into the

stationary phase from the mobile phase.

• K'= VR-Vm /Vm= tR-tm /tm • The more rapidly the peak broaden the less efficient

the column

• The longer the analyte takes to travel through a column, the

more the ndividual molecules of the sample spread out and the

broader the band becomes

• The causes of band broadening are expressed in Van Deemter

equation

van Deemter equation in chromatography

• When the characteristics of a separation were expressed previously, the speed of the mobile phase in the column did not appear.

• However, the speed has to affect the progression of the solutes, hence their dispersion within the column, and must have an effect on the quality of the analysis.

• These kinetic considerations are collected in a famous equation proposed by van Deemter.

• The simplified form of this equation is given below

Van Deemter Equation

• The three experimental parameters A, B and C are

related to column parameters and also to experimental

conditions. H= Height Equivalent to Theoretical Plate. It expresses the efficiency

of the column (the smeller the H the more efficient the column.

Ū = Average linear velocity (cm/s) of the mobile phase in the column

A= Eddy diffusion term. Broadening occurs because some molecules

take longer distorted paths, while some take direct paths, thus

eluting first.

B = The rate of diffusion of the molecules in the gaseous (mobile)

phase. It contributes to the band (peak) broadening through diffusion

either with or against the flow of the mobile phase. (It is very small in

liquid chromatography). Its contribution decreases as flow rate

increases and it only becomes significant at very slow flow rates.

Eddy diffusion

• Cs is the resistance to the mass transfer of a molecule

in the stationary phase and is dependent on the

diffusion coefficient in the stationary phase and upon

the thickness of the stationary phase coated onto the

solid inert support.

C = (d2 thickness)/Ds

d2 thickness = the square of the stationary film thickness

Ds = diffusion coefficient of the molecules of the

component in the stationary phase

The thinner and more uniform stationary phase coating,

the smaller the contribution to the band (peak)

broadening

• If H is expressed in cm, A will be expressed in cm, B

in cm cm2/s and C in s (where velocity is measured in

cm/s).

• The function is a hyperbolic function that goes

through a minimum (Hmin) when

Van Deemter plot for gas phase chromatography

showing domains for A, B and C.

Optimization of a chromatographic analysis

• For quantitative analysis, it is crucial to precisely measure the areas of the peaks.

• Therefore, the substances to be determined must be well separated.

• In order to achieve this, the analysis has to be optimized using all the resources of the instrumentation and, when possible, software that can simulate the results of temperature modifications, phases and other physical parameters.

• This optimization process requires that the chromatographic process is well understood.