Chromatographic Techniques

CHE 361
(A Course of Department of Chemistry, BSMRSTU)

Dr. Bijan Mohon Chaki

Associate Professor
Department of Chemistry
Begum Rokeya University, Rangpur-5400
 Syllabus Content
Introduction to chromatography
History
Principles
Importance
Chromatographic terms
Classification of chromatography
Adsorption chromatography
Partition chromatography
 Syllabus Content of Chapter 1

Introduction to Chromatography: General Description,

classification of chromatographic methods, chromatograms,
migration rates of solutes, retention time, selectivity factor,
retention factor, rate theory, kinetic variables affecting
column efficiency, theory of band broadening, optimization
of column performance.

Book Reference:
1) Chromatographic Methods by A. Braithwaite and F. J Smith, 5th
Ed.
2) Principles of Instrumental Analysis by D. A. Skoog, F. J Holler and
S. R. Crouch.
 Chromatographic Techniques
Purpose of Chromatography
Analytical:
 Identifying unknown compounds/determine chemical composition of a compound;
 Monitoring product formation in chemical reactions, pharmaceutical and
biotechnology industries;
 Forensic teams use it to analyse blood and urine samples for drugs, for paint
analysis, testing for the presence of explosives or any other forbidden materials to
carry.
Preparative:
 Separating mixtures of compounds;
 Establishing the purity or concentration of compounds;
 Chromatogram

Chromatogram: Visual output of the chromatograph. It is

represented as Gaussian plot.
Separation - Different peaks or patterns on the chromatogram
correspond to different components of the separated mixture.
 Chromatographic Techniques
 X- axis - Retention time
 Y-axis – Detector Signal
 Signal is proportional to the concentration of the specific analyte separated.

 The retention time tR solute A can be defined as the time from the injection
of the sample to the time of compound elution, taken at the maximum
(apex) of the peak that belongs to the specific molecular species A (known
or unknown). i.e. the time at which a component elutes from a column
 The retention time indicates how long it takes for a compound A to elute
from the column (from the injector to the detector).
 Chromatographic Techniques

 Retention time is usually characteristic for a specific compound in a given

separation. For this reason, the retention time is critical in identifying
analytes once their retention time is known (e.g., by using standards).
 Retention time (tR) depends:
(i) structure of the specific molecule
(ii) the nature of the mobile and stationary phases,
(iii) the flow rate of the mobile phase, and
(iv) dimensions of the chromatographic column.
 Chromatographic Techniques

 The separation uses a Column (stationary phase) and Solvent (mobile

phase).
 The components are separated from each other based on differences in
affinity for the mobile or based on differences in affinity for the mobile or
stationary phase.
 The column is filled with the carrier (liquid or gas) before the sample is
injected. Thus if there is no interaction between the sample and the
column, then the fastest that the sample can get to the detector is the dead
time denoted by tM in the diagram.
 If the sample does interact with the column, then it is retarded by a time tS
shown in the diagram. Thus the total retention time from injection to
detection is tR = tM+ tS

 The goal of the separation is to have the best RESOLUTION possible

between components. RESOLUTION possible between components.
 The mobile phase (MP) moves over a
stationary phase (SP)
 The SP is present as a film on the surface of
small particles or the walls of capillary
columns therefore presents a large surface
area to the MP
 The sample mixture (analyte) is introduced
into the MP and undergoes a series of
partition or adsorption interactions at the MP-
SP boundary as it moves through the
chromatographic system.
 The differences in the physical and chemical
properties of the individual components
determine their relative affinity for the SP &
MP
 Therefore components will migrate through
the system at differing rates depending on
their retardation resulting from attraction onto
the stationary phase.
 Chromatographic Resolution
 The least retarded component, having an equilibrium ratio which least
favours the stationary phase, will be eluted first, i.e. moves fastest through
the system.
 The most retarded component moves the slowest and is eluted last.
 The fig 1.2 show the separation of three components A, B & C being the last
retained on the SP (A<B<C). A therefore moves fastest down the column
and furthest up the TLC
 The components therefore move along at a slower rate than the eluent, the
rate being determined by the relative affinity of each component for the SP
to the MP, that is the distribution ratio:
 

=  or, 
 
 Where CSP= conc. of the component in stationary phase
CMP= conc. of the component in mobile phase

 A component in a sample mixture will be distributed the MP and SP

according to its distribution ratio, K. The larger the value of K, the greater
the affinity that component has for the stationary phase, that is CSP is larger
than CMP
 Chromatographic Resolution

 A solute is introduced onto the SP as a narrow band (Fig 2.1)

 As this band moves through the chromatographic system, dispersion of the
molecules occurs due to lateral diffusion and hence the band becomes
broader
 Too much dispersion can result in overlapping bands or peaks and
incomplete separation
 Therefore, in order to obtain efficient separations, the MP, SP and
equilibrium conditions need to be carefully selected to achieve the desired
separation as rapidly as feasible but with minimum dispersion or band
broadening.
 A is least retained component whereas C has greatest affinity for the SP, in
the fig: KA<KB<KC
 The component can be considered to move through the column or plate in a series of
theoretical separation steps with distribution between the two phases as defined by K
 Chromatographic Techniques

 In planar chromatography the separation would be viewed as a series of

spots, while in instrumental method such as GC or HPLC the composition is
monitored continuously by detector: the signal or peak produced being
proportional to the amount of solute present in the MP, the eluant, and is
plotted against time to form the chromatogram.
 The longer a component takes to move through the system the greater the
band broadening.
 The symbol and notation used to describe features of the peaks and
chromatogram are shown in fig 2.2
 The volume of mobile phase required to elute or transport a component
through the system is the actual parameter to measure.
 The detector signal is processed by an electronic integrator which plots the
chromatogram and calculates retention time, peak area, peak height and
peak width.
Chromatographic Techniques
 Chromatographic Techniques
 Peak width: Ideally any chromatographic peak should be symmetrical and
Gaussian shaped, though this may not be the case always. Depending upon
sample characteristics, many times we get tailing peaks. The common
measures of peak width are: width w1/2 , measured at ½ height of the peak
and width w measured at baseline between tangents drawn to the inflection
points (steepest part of the peak on either side).

 Resolution: Rs: Resolution is the most important parameter in

chromatography. Resolution is that provides complete information about
the sample composition by way of the total number of components. The
resolution depends on two factors:
1. Width of the solute peak (it should be narrow, related to column
efficiency)
2. Distance between the peaks (related to selectivity).
Rs can be calculated as : 2 ∆ T / (WA + WB), where, ∆T is the distance
between peak A & B (tRB-tRA) (measured between the peak maxima) and WA &
WB are the widths of the peaks at baseline.
(

 )
So, Resolution (RS ): RS =
  
Resolution (Rs) : Solute moving through a column spreads into a
Gaussian shape with standard deviation σ. Common measures of
breadth are:
 The width w½measured at half-height
 The width w at the baseline between tangents drawn to the steepest
parts of the peak (inflection points).

It can be shown that: w½= 2.35σ and w = 4σ

 Chromatographic Resolution

In the left figure, the minimum time that a

non-retained chemical species will remain in
the system is tM.
A Curve Width (W): The width of the
concentration profile curve of the
different samples in the chromatogram in
units of time

(

 )
RESOLUTION (RS ) : RS =   

Retention volume, VR, is the volume of mobile

phase required to elute a particular solute from the
column:

VR= tR×μ
where μ is the mobile phase flow rate
•The dead time, tM, is the time of travel of
unretained mobile phase through the column
In chromatography, the resolution of two peaks from each other is
defined as
∆ ∆ 0.589 ∆
 = = =
   ! 


VR= tR×μ
where ∆ or ∆ is the separation between peaks and  is the
average width of the two peaks

What is the resolution

of two Gaussian
peaks of identical width
(3.27 s) and height
eluting at 67.3 s and
74.9 s, respectively?
•ANS: Resolution = 2.32
 Chromatographic Techniques
 Retention Factor (Capacity factor): k': It is an important parameter which
shows how long a compound can be retained by the stationary phase.
Ideally, there should be sufficient difference between k’ values of different
sample components so as to get a desirable separation.
(

 )
Retention factor can be calculated as k’ =


A range of k’ values from 2 to 10 will be most desirable.
"#
Or, k’ = #  , Where K= distribution ratio,

VS= is the volume of the SP and VM= is the volume of the MP

'() *+ ,*-.
) ,/)01, '0 ,

'*023 /4,)
k’=
'() *+ ,*-.
) ,/)01, '0 (*5'-) /4,)

 Relative Retention: α: This factor gives an idea as to how far two

compounds can be separated by a given stationary phase. For any two
compounds A and B, the relative retention, α is the ratio of their adjusted
retention times.

%& "
Hence, α =  =  = 

 %& "
Relative retention has to be always more than 1.
 Chromatographic Techniques
 Retention Factor (Capacity factor):

789 :; <:=>9 <?9@A< 7@ <B7:@BCD ?ℎB<9

k& =
789 :; <:=>9 <?9@A< 7@ 8:F7=9 ?ℎB<9

8:=9< :; <:=>9 7@ <B7:@BCD ?ℎB<9

=
8:=9< :; <:=>9 7@ 8:F7=9 ?ℎB<9

&
G 
k =
GH H

but, K =

#


&
So, k & =
#  =

=



t’R= is the adjusted retention time of the solute. The adjusted retention time, t’R, for a solute is
the additional time required for solute to travel the length of the column beyond the time
required by unretained solvent: t’R = tR–tM

In GC, tM is usually taken as the time needed for CH4 to travel through the column
 Chromatographic Techniques
 Gaussian curve and chromatographic peak:
 Relationship between Gaussian curve and chromatographic peak: Gaussian
Curve is also known as normal distribution curve or normal error curve, it
resembles with the valley shape, it looks like a valley. When replicate
values of a measurement are plotted as a function of frequency of their
occurrence then Gaussian curve is obtained. A chromatographic peak is
resembles with the Gaussian curve, now how chromatographic peak is
obtained
 When solute molecules eluted from column they are eluted at a time.
It looks like
 Chromatographic Techniques
 But this is not the case, when sample molecules are injected, to the column
they are travelling with different velocities due to column. Some solute
molecules travel fast and come out of the column first, and detected which
are belonged in the first portion of the peak (up to the red line).
 Most of the molecules travel with average velocity and get eluted from
column, they will form central portion or middle portion of the peak(up to
the green line), and remaining molecules lag behind and come out of
column last and form late portion of the peak.

 The ideal shape of a chromatographic peak is

as look

 As the solute molecules travel through the column the band (peak)
broadening take place
 Chromatographic Techniques
 As the solute molecules travel through the column the band (peak)
broadening takes place. Peak width increases. This increase in peak width
causes poor resolution (separation)

 Why band broadening-

In order to understand the separation process in chromatography, the
chromatographer should know 2 fundamental theories,
1. Plate Theory (old theory developed by Martin and Synge in 1941)
2. Rate Theory (current theory developed by van Deemter in 1956)

 Plate Theory (old theory developed by Martin and Synge in 1941):

According to this theory, a chromatographic column consist of a series of
discrete set of continuous horizontal layers which are termed as Theoretical
plates
 Chromatographic Techniques
 Solute molecules get equilibrated between stationary and mobile phase at
each of these plates. Now consider this is the column
 After equilibrium, the solute is carried by the mobile phase from one plate
to another and the process is continued till the solute elutes out of the
column with it’s characteristic retention time and peak width.

 As the number of theoretical plates is

increased in a column, the solute peak
becomes narrower and this results in a much
better resolution between different sample
components in a run. The number of
theoretical plates is denoted by N.
 A large N value indicates greater resolving or
separation power of the column. This is what
is referred to as the Efficiency of the column.
 Theory of Chromatographic Techniques
 A large N value indicates greater resolving or separation power of the
column. This is what is referred to as the Efficiency of the column.
 Another term which describes the efficiency of the column is HETP
(Height Equivalent to a Theoretical Plate)
which can be calculated as H = L/N,
where, L is the column length in mm and
N is the number of theoretical plates.
Hence a large value for N or a very low value for HETP indicates high
efficiency of the chromatographic column.
 The chromatographic resolution is related to the number of theoretical
plates of the column, the selectivity factor and the retention factors of two
solutes by the following equation:

 RS=1/4(√N ) (α-1/α) (k’/k’+1)

 If all the variables in the resolution equation are kept constant, except the
number of theoretical plates, then the resolution is proportional to the
square root of N. Therefore, increasing the number of theoretical plate by 4
will increase the resolution by a factor of 2.
 Chromatographic Techniques
 Due to the several limitations of the plate theory, rate theory has been
widely used.

 Greater separation occurs with:

–greater number of theoretical plates (N)
–as plate height (H or HETP) becomes smaller
- no explanation of peak broadening

 Chromatographic rate theory: The rate theory provides a more realistic

explanation of the processes that take place inside a chromatographic
column. It takes into account the time taken by the solute to equilibrate
between the stationary and mobile phase unlike the plate model which
assumes that the equilibration is infinitely fast. When the solute elutes out
of the column, the band shape is affected by the rate of elution. There are
various mechanisms that contribute to broadening of the band and it can
be shown by van Deemter equation:
 Chromatographic Techniques
 Due to the several limitations of the plate theory, rate theory has been
widely used.
 Chromatographic rate theory: The rate theory of chromatography describes
the shapes and breadths of column bands in quantitative terms based on a
random-walk mechanism for the migration of molecules through a column.
It describes the effect of an elution band as well as its time of elution. It is
based upon the van-Deemter equation that describes the relation of a
theoretical plate H and the average linear velocity of the mobile phase.

H (HETP) = A + B/μ + (CS+CM)μ

 Where H = Height equivalent to theoretical plates
A = Eddy’s diffusion
CS and CM = mass transfer of analyte into stationary and mobile phase
 μ = flow rate of mobile phase/average velocity mobile phase velocity, μ is
the average velocity of the mobile phase and A, B & C are the factors
contributing to band broadening. A low value for HETP will increase the
efficiency of the column, To get a low value for HEPT, all the three
factors, A, B & C must be minimized.
 Chromatographic Techniques

 Factor A: Eddy Diffusion: In a packed column, the solute molecules take

different paths at random while passing through the column. This leads to
broadening of the peak because different paths in a packed column are of
different lengths. To minimize A term, eliminating column packing (open
tubular columns), reducing the particle size of the packing, packing the
column more uniformly (spherical particles of similar/uniform smaller
size)

 Factor B: Longitudinal Diffusion: As the analyte is passing through the

column, it diffuses out towards the edges of the column. Hence the
concentration of the analyte is always more at the centre as compared to
the edges. This leads to band broadening. If the velocity of the mobile
phase is high, then the analyte will spend less amount of time in the
column which will reduce the effect of longitudinal diffusion.
 Chromatographic Techniques

 Factor C: Resistance to Mass Transfer : The analyte takes a certain amount

of time to equilibrate between the stationary and mobile phases. If the
velocity of the mobile phase is high and the analyte has a strong affinity
towards the stationary phase, then the portion of analyte in the mobile
phase will move ahead of the portion of analyte in the stationary phase.
This will lead to band broadening. The higher the velocity of mobile
phase, more will be the band broadening. The effect of C term can be
reduced by decreasing the stationary phase content (film thickness in case
of capillary column), reducing the column radius and increasing the
temperature.

 The ultimate goal of the chromatographer is to achieve the highest possible

resolution in the shortest possible time by optimizing various parameters.
Chromatographic Techniques
 Chromatographic Techniques

 The term ‘A’ is independent of flow rate of the mobile phase

 The term B/μ decreases drastically in the beginning with increase in the
flow rate of mobile phase, increase in the flow rate beyond particular
value, leads to slow decrease in the value of B/μ
 The term Cμ increases with increase in the flow rate.
 Such plot is considerable use in determining the optimum mobile phase
flow rate.
 Chromatographic Techniques

 Theoretical plates: Assume a chromatographic peak that has a Gaussian

shape. H is height of peak, W1/2 is with ½ height (If true Gaussian W1/2=
2.35σ, where σ = standard deviation, W= 4 σ) which at baseline should be
4σ

 For chromatography we retain the name of theoretical plate, but calculate it

using the retention time and the width at ½ height

5.54 
I= = 5.54
K / !

N is the number of theoretical plates, Again the bigger the N the better, because
that means the width of the peak is small compared to its retention time.
 Factors affecting resolution
 A more usable expression for resolution is

 Required Plate Number: If k2’and α are known, the required number of

plates can be calculated:

The Rs value is set at the 6σ level or 1.5

 The minimum analysis time tmin

 Required Column Length

 Chromatography Optimization Techniques
 Optimization Techniques of Chromatography
Minimizing plate height:
–Reducing diffusion rate
–Reducing column diameter
–Changing column temperature
–Reducing the thickness of the liquid film
–Optimizing the flow rate of the mobile phase

 Increases in k’ enhance resolution but at the expense of elution time

– The optimum k’ values are from 1 to 5
– The easiest way to improve Rs is by optimizing k’
– In GC, k’ can be improved by temperature programming
– In LC, k’ is improved by gradient elution

 Resolution also improves with L, but it expensive in terms of time of analysis

Variation in the selectivity factor:
–Changing the composition of the mobile phase (in HPLC)
–Changing the column temperature
–Changing the stationary phase
–Using special chemical effects