ANALYTICAL CHEMISTRY (Chem 276)

Chromatographic Techniques

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 Introduction

 Chromatography is one of the primary analytical

methods for the identification and quantification of
compounds in the gaseous or liquid state.
 Tswett was the first to use the term "chromatography"
derived from two Greek words "Chroma" meaning color
and "graphein" meaning to write.

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TSWETT
TSWETT EXPERIMENT
EXPERIMENT

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 …con’t
 The basic principle is based on the concentration equilibrium of
the components of interest, between two immiscible phases.
 One is called the stationary phase, because it is immobilized
within a column or fixed upon a support, while the second, called
the mobile phase, is forced through the first.
 The phases are chosen such that components of the sample have
differing affinities in each phase.
 The differential migration of compounds leads to their separation.

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 Principles of Chromatography
 Chromatography is a physical process.

 Any Chromatography system is composed of three Components :

 Stationary phase- coated or bonded on a plate or column

 Mobile phase

 Mixture to be separated

 We can only control stationary and mobile phase as mixtures are the

problem we have to deal with.

 Chromatography is a dynamic process in which the mobile phase
moves in definite direction.
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 The chromatographic process

Two different substances are partitioned between two phases.

Depending on their affinity (toward the stationary phase) will
spent different times adsorbed by the stationary phase.
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 Elution Chromatography on Columns
 Elution involves washing a species through a column by
continuous addition of fresh solvent.
 The sample is introduced at the head of a column, whereupon the
components of the sample distribute themselves between the two
phases.
 Introduction of additional mobile phase (the eluent) forces the
solvent containing a part of the sample down the column, where
further partition between the mobile phase and fresh portions of
the stationary phase occurs.
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Elution Chromatography on Columns

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 Terms in Chromatography
• Chromatograph - equipment that enables a sophisticated

separation
EX. Gas chromatography or Liquid chromatography
• Eluent - Fluid entering column/ solvent that carries the analyte.
• Eluate - Mobile phase leaving the column.

• Stationary phase - Immobilized phase

 Immobilized on the support particles or on the inner wall of the
column tubing.
 Examples : Silica layer - Thin Layer Chromatography
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 …con’t
• Mobile phase: Moves in a definite direction. The mobile phase
moves through the chromatography column (the stationary phase)
where the sample interacts with the stationary phase and is
separated.
• Retention time : Time takes for a particular analyte to pass
through the system (from the column inlet to the detector) under
set conditions.
• Sample (Anylate) :Substance analyzed in chromatography.
• Solvent : Any substance capable of solubilizing another

substance. 10
 …con’t
 Chromatogram
 Visual output of the chromatograph.

 Separation - Different peaks or patterns on the chromatogram

correspond to different components of the separated mixture.
Detector Signal

1 2

time or volume
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 Types of chromatography on the methods of
holding the stationary phase):
1- Planar or Plane Chromatography:
 The stationary phase is supported on a flat plate or in the
fibers of a paper. Here the mobile phase moves through
the stationary phase by capillary action or by gravity.
 In this type of chromatography the stationary phase is used in the
form of layer. Plane chromatography is further classified into:
a- Thin Layer Chromatography (TLC):
 The stationary phase in the form of fine powder is spread on glass
or plastic or aluminum sheets.
b- Paper Chromatography (PC):
 A specific type of papers is used as stationary phase in the form
of sheet

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 Paper Chromatography
 The papers are manufactured from highly purified cellulose, with
close control over porosity and thickness.
 Such papers contain sufficient adsorbed waters to make the
stationary phase aqueous.
 In PC the components of mixtures are separated due to the
difference in their migration rates through the stationary phase.
 The spots of the components are located by spraying dying
agents.
 Paper chromatography is a commonly used method for separation
and identification of colored compounds. 13
 Paper Chromatography
 performing a chromatographic experiment is basically a three-
step process that involves spotting, developing, and visualizing

Fig: Paper chromatography diagram 14

 …con’t
• The degree of retention of a component is called the retardation
factor (Rf) and corresponds to the distance migrated by an analyte
over the distance migrated by the solvent (also called solvent
front). In order to obtain a measure of the extent of movement
of a component in a paper chromatography experiment, we can
calculate retardation factors (so called Rf factors) for each
separated component in the developed chromatogram and
also used for qualitative analysis.

Retardation factor (Rf)=

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 Thin Layer Chromatography
 Typical thin-layer separations are performed on a glass plate that
is coated with a thin and adherent layer of finely divided
particles, This layer constitutes the stationary phase.
 A thin-layer plate is prepared by spreading an aqueous slurry of
the finely ground solid onto the clean surface of a glass or plastic
plate or a microscope slide.
 Often, a blinder is incorporated into the slurry to enhance
adhesion of the solid particles to the glass and to one another. The
plate is then allowed to stand until the layer has set and adheres
tightly to the surface. 16
 Column chromatography
 In column chromatography, the stationary phase is held
in a narrow tube, and the mobile phase is forced through
the tube under pressure or by gravity.
Theory of column chromatography
 There are 3 theories:
1) The Plate theory.

2) The Rate theory.

3) The Random walk, non-equilibrium

theory.
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 The Plate theory
The essential concept is that as the solute traverses the

column, it undergoes repeated equilibration with the

stationary phase, so that each equilibration defines one stage,
or plate.
The column length over which equilibration occurs is known

as the height of an equivalent theoretical plate (HETP), or

simply plate height.

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 The height equivalent to a theoretical plate (HETP or H)
will be given by equation (1):

H = L/N Plate
HETP

The analyte moves down the column by transfer of equilibrated

mobile phase from one plate to the next plate.

Hypothetical zone in which two phases establish an equilibrium

with each other.
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column in which the solute is complete equilibrium
with the mobile and the stationary phases.
This equilibrium is defined by the distribution
coefficient, KD (partition coefficient).

i.e. The distribution of analytes between phases can often be

described quite simply. An analyte is in equilibrium
between the two phases;
[ A] stationary
Amobile ↔ Astationary K 
[ A] mobile

Where: – CS: concentration of analyte on the stationary phase

– CM: concentration of analyte on the mobile phase
20 –K = is termed the partition coefficient
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• The retention time (tr) is the time elapsed between the injection
point and the peak maximum. Each solute has a characteristic
retention time.

• The retention volume (Vr) is the volume of mobile phase passed

through the column between the injection point and the peak
maximum. Thus, Vr = Qtr where Q is the flow rate in ml/min.

• The peak height (h) is the distance between the peak maximum
and the base line geometrically produced beneath the peak.

• The peak width (w) at 0.606h is the distance between each side
of a peak measured at 0.6065 of the peak height .

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 Column efficiency
The column efficiency is measured in terms of the number

of theoretical plates (N) in the column and plate height

( H)
It can be measured, either by stating the number of

theoretical plates in a column, N (the more plates the

better), or by stating the plate height; the Height Equivalent
to a Theoretical Plate ( HETP) (the smaller the better).
More N, Lower HETP, Longer column are the better for
separation efficiency. N = L/H
23How column efficiency can be improved or affected?
 Theoretical efficiency (number of theoretical plates, N)

 As the analyte migrates through column, it

occupies a continually expanding zone (Figure
below).

Fig. Dispersion of a solute in a column and its

translation on a chromatogram

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 This linear dispersion 1 measured by the variance 21
increases with the distance of migration. When this
distance becomes L, the total column length, the variance
will be:

But: H = L/N

 Any chromatogram that shows an elution peak with

the temporal variance 2 permits the determination
of the theoretical efficiency N for the compound
25 under investigation
 On the chromatogram,  represents the half-width of the peak at 60.6
per cent of its height and tR the retention time of the compound.

 tR and  should be measured in the same units (time, distances or eluted

volumes if the flow is constant).
 If  is expressed in units of volume (using the flow), then 4 corresponds
to the ‘volume of the peak’, that contains around 95 per cent of the injected
compound.

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 Column efficiency parameters

Column efficiency parameters:

1. The number of theoretical plates (n).
2. The height equivalent to a theoretical plate, H (HETP).

Column performance parameters:

3. Resolution (Rs).
4. Peak asymmetry.
5. Separation factor.

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 Retention (or capacity) factor k

 is often used to describe the migration rate of an analyte on a

column. It is also called the capacity factor
 More practical than distribution ratio
• Describe the ability of s. phase to retain components
• When a compound of total mass mT is introduced onto the column,
it separates into two quantities: mM, the mass in the mobile phase
and mS, the mass in the stationary phase.
• During the solute’s migration down the column, these two
quantities remain constant. Their ratio, called the retention factor k,
is constant and independent of mT:

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  Knowing that the retention time of a compound tR
is such that tR = tM +tS, the value of k is therefore
accessible from the chromatogram (tS = tR );

This important relation can also be written:

 Bearing in mind the relations below, the retention volume VR of

a solute can be written:

Or
This final expression linking the experimental parameters to the
thermodynamic coefficient of distribution K, is valid for the
ideal chromatography. 29
Fig. Retention factors and separation factor between two compounds. Each
compound has its own retention factor. On this figure, the separation factor is
around 1.3. The separation factor is also equal to the ratio of the two
retention factors.  alone is not enough to determine whether the separation
is really possible. 30
 Separation (or selectivity) factor between two solutes

 The separation factor, , enables the comparison

of two adjacent peaks 1 and 2 present in the
same chromatogram (Fig. on slide above).
 Using Equations discussed before, it can be

concluded that the separation factor can be

expressed by Equation
a = k 'B / k ' A
t B  tm

t A  tm

 By definition , is greater than unity (species 1

elutes faster than species 2):
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 Resolution factor between two peaks

• Although the selectivity factor, , describes the

separation of band centers, it does not take into account
peak widths. Another measure of how well species have
been separated is provided by measurement of the
resolution.

• Thus, contrary to the selectivity factor which does not

take into account peak widths, the following expression
is used to calculate R between two compounds 1 and 2

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 The larger the value of Rs ,the greater the separation of the solute A and B.
 The resolution of 1.5 usually show baseline separation.
 Large difference in retention time
 Narrow peak width

WbA WbB WbC

trA trB trC time 33
Resolution
 Resolution Rs defines the degree of separation of two
adjacent bands
 The more efficient column has greater degree of
resolution, it will produce between closely eluting peaks.
 The resolution between two peaks – A and B is expressed
in the equation:
Rs = 2 (trB - trA) / (WbB + WbA)

trB and trA are the retention times of peaks A and B

WbB and WbA are the widths of peaks A and B at baseline.

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 αA/B = trA / trB

A B A B

 Two chromatograms with the same αA/B , but different

resolution factors.

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