Principle of chromatography:

Chromatography is the chemical method for identifying and quantifying

components of complex mixture . The method is basically a separation method
for two immiscible phases (one mobile phase and the other stationary phase)
are brought into contact ; A sample introduced into mobile phase is carried along
through a column containing a distributed stationary phase . Species in the
sample undergo repeated interactions (partitions) between the mobile and
stationary phases . When both phases are properly chosen , the sample
components are gradually separated into bands in mobile phase . at the end of
process , separated components emerge in order of increasing interaction with
the stationary phase . The least retarded component emerges first , the most
strongly retained component elutes last . Partition between the phases utilizes
the differences in the physical and / or chemical properties when the components
in the sample. The separation column is the heart of the chromatographic
process .

Classification of Chromatography:

The mobile phase can be gas or a liquid , whereas the stationary phase can be
only a liquid or a solid . When the separation involves predominantly a simple
partitioning between two immiscible liquid phase , one stationary and the other
mobile . the process is called liquid –liquid chromatography (LLC) . When
physical surfaces are mainly involved in the retentive ability of stationary phase ,
the process is called as liquid –solid (or adsorption) chromatography (LSC) . In
ion exchange chromatography , ionic compounds of the sample are concerned
by selective exchange with counteractions of the stationary phase . The use of
exclusion packing as the stationary phase brings about a classification of
molecules referred to as gel-permeation chromatography by polymer chemists
and as gel filtration by biochemists . When the mobile phase is a gas , the
methods are called gas –liquid chromatography (GLC) and gas –solid
chromatography.

General Class Specific types Stationary Phase Equilibrium types

Gas(GC) a) GLC a) Liquid a) Partitioning
b) GSC absorbed between gas and
c) GBC on a solid liquid
b) Solid
c) Organic b) Adsorption
substance
s bonded c) Either partition or
to a solid adsorption on both

1
Liquid a) LLC a) Liquid a. Partitioning
b) LSC adsorbed
c) LBC on the b. Adsorption
d) Ion surface of
exchange a solid c. Either partition or
e) Gel b) Solid adsorption on both
permeatio c) Organic
n substance
f) (GPC) s bonded
to a solid
d) Ion
exchange
resin

G=GAS , L=LIQUID ,S =SOLID , B=BONDED PHASE , C=CHOMATOGRAPHY

Chromatographic behavior of solutes:

The chromatographic behavior of a solute can be described in numerous ways .

For column chromatography , the retention volume , V R (or corresponding
retention time t R ) and the partition ratio are the terms most frequently used . By
varying the stationary-mobile phase combinations and various operating
parameters , the degree of retention can be varied from nearly total retention to a
state of free migration .

a) Retention Behavior:

Retention behavior reflects the distribution of a solute between the mobile and
stationary phases . the separation characteristics of two isomeric alkenes are
shown in the following chromatogram . The volume of mobile phase
necessary to convey a solute band from the point of injection , through the
column , and to the detector from the corresponding retention time , t R , on
the chromatogram by multiplying the latter by the volumetric flow rate , Fc ,
expressed as the volume of the mobile phase per unit time : V R  t R Fc

The flow rate , in terms of column parameters is as follows :

d c2 L Vcol  t
Fc  t  
4 tM tM
, where
d c =column bore diameter
L =column length
 tot =total porosity

2
Vcol = bed volume of the column

The porosity is expressed as the ratio of the interstitial volume of the packing
to the volume of its total mass . For solid packings the total porosity is 0.35 to
0.45 , where for porous packings it is 0.7 to 0.9 . In capillary columns the
value of porosity is unity . The average linear velocity , u , of the mobile phase
L
is thus : u  t u
M

This velocity is measured by the transit time of a non-retained solute , t M .In

interactive chromatography no material can elute prior to this time . When
Converted to volume , VM , it represents what is called the dead space ,
void volume , or hold up volume of a column . It includes the effective volume
contribution of the sample injector , any connecting tubing , the column itself ,
and the detector . The adjusted retention volume , V R' or time t R' are given
by : V R'  V R  VM or t R'  t R  t M

When mobile phase is a gas , temperature and pressure must be specified ,

and retention volumes must be corrected for the compressibility of the gas ,
and since the gas moves more slowly near the inlet than at the exit of the
column. The pressure gradient correction ( or compressibility ) factor j is
P
3[( i ) 2  1]
Po
expressed by : j  , where Pi is the carrier gas pressure at
Pi 3
2[( )  1]
Po
the column inlet and Po that at the outlet.

3
b) Partition coefficient

When a solute enters a chromatographic system , it immediately distributes

between the stationary phase and mobile phases . If the mobile –phase flow
ceases at any time , the solute reaches an equilibrium state between the two
CS
phases The partition coefficient is given by: K  , where C S and C M are
CM
the concentrations of solute and mobile phase. When K=1 , the solute is
equally distributed between the two phase . the partition coefficient
determines the average velocity of each solute zone as the mobile phase
moves down the column.

When a solute enters a chromatographic system , it immediately distributes

between the stationary and mobile phases. If the mobile phase flow is
stopped at any time , the solute assumes an equilibrium distribution between
the two phases . The concentration at each phase is given by the
thermodynamic partition coefficient given as , where and are the
concentrations of solute in the stationary and mobile phases , respectively .
When K=1 , the solute is equally distributed between the two phases . The

4
partition coefficient determines the average velocity of each solute zone
.-more specifically , the zone enters as the mobile phase down the column .
For a symmetrical peak , when the peak maximum appears at the column
exit , half of the solute has eluted in the retention volume , and half remains
distributed between the volume of the mobile phase , and the volume of the
stationary phase . Thus VR C M  VM C M  VS C S Also VR  VM  KVS or
V R  VM  KV S

It relates the retention volume of a solute to the column dead volume and the
product of the partition coefficient and the volume of the stationary phase .
This equation is correct for liquid partition columns , but for adsorption
columns , V S should be replaced by AS , the surface area of the
adsorption.

c) Partition Ratio:

The partition ratio (or capacity ratio) , k ' is the most important quantity in
column chromatography . It relates the equilibrium distribution of the sample
within the column to the thermodynamic properties of the column and to the
temperature . For a given set of operating parameters , is a measure of the
time spent in the stationary phase relative to the time spent in the mobile
phase . It is defined as the ratio of the moles of the solute in the stationary
C S VS V
phase to the moles in the mobile phase : k  K S
'

C M VM VM
, where V S = volume of stationary phase
VM = volume of mobile phase

VM
The volumetric phase ratio , is often denoted by the symbol  . Thus
VS
K
k'  .

Stated another way , the partition ratio ids the additional time a solute band
takes to elute , as compared with an non retained solute ( for which k '  0 ) ,
t R  t M VR  VM
divided by the elution time of non retained band : k  
'

tM VM

The relation states explains how many dead volumes ( or t M ) are required
to attain V R ( or t R ). Thus retention time is related to k ' by the equation :
L
t R  t M (1  k ' )  (1  k ' )
u

5
The value of k ' higher than 10 waste valuable analytical time . Values less
than unity do not provide adequate resolution among early eluting solutes .
The fraction of time that a solute spends in a particular phase is very close to
the fraction of all those particular solute molecules that are instantaneously in
the same phase . Thus , the average fraction of time spent by a solute in the
mobile phase is :

C M VM 1

C M VM  C S VS 1  k '

C S VS k'
Similarly , for stationary phase : 
C M VM  C S VS 1  k '

Sometime a factor called relative retention may be of use in case two

solutes , when solute 1 elutes before solute 2 is given by :
' '
k 2' K 2 VR , 2 t R , 2
   
k1' K1 V R' ,1 t R' ,1

The relative retention is dependent on (1) the nature of the stationary and
mobile phases and (2) the column operating temperature . One should always
be as selective as possible in choosing a pair of phases for the adjacent
solutes most difficult to separate.

Example:

On a 1000 cm wall coated open tubular column of 0.25 mm bore , the He

carrier gas velocity is 37 cm/sec . The retention time for decane is 1.27
pp , peak width at half height is 0.88 sec. The retention time for a non
L 1000
retained compound is t M    27 sec . or 0.45 min.
u 37
t R' 1.27  0.45
The partition ratio k    1.82
tM 0.45

Plate height and plate number

An important characteristic of a chromatographic system is its effectively

expressed as dimensionless quantity called “effective plate number” N eff . It
reflects the no. of times the solute partitions between the two phases during
its passage through column . The effective plate no. can be defined from the

6
 L t R' 2
chromatogram of a single band N eff   ( ) , where L is column length
H 
'
, H is the plate height , t R is the corrected retention time for elution of the
band centre , and  2 is the band variance in time unit . The width at the
base of the peak , Wb is equal to 4 times standard deviation assuming an
Wb t R' 2
ideal Gaussian distribution . Thus   and N eff  16 ( )
4 Wb
The upper portion of the peak dictates the tangent line , which minimizes any
contribution from a tailing segment of the peak . Often it is easier to measure
the width at the half the peak height . Since   W1 / 2 / 8 ln 2 ,
L t'
N eff   5.54( R ) 2
H W1 / 2
Measurement of peak width at half the peaks height is less sensitive to peak
asymmetry , since tailing often shows up below the measurement location .
Although this is not recommended , column efficiency is sometimes stated as
the number of theoretical plates . In this context no correction is made for the
transit time of a non retained solute .
Plate number is an indication only of how well a column has been packed , it
can not adequately predict column performance under all conditions . It is
designed to be primarily a measure of the kinetic contributions to band
broadening . Other contributions to peak width , such as extra column effects
and thermodynamic factors (such as peak tailing) , may play a significant role
. Plate height H , is the distance a solute moves while undergoing one
L
partition : H  N .
eff

Plate height is a good way to express column efficiency in units of length

without specifying the length of the column .

Example:

In previous example , the effective plate no . :

t' 49.2 2
N eff  5.545( R ) 2  5.545( )  17,300
W1 / 2 0.88
L 1000
The plate height : H  N  17300  0.058 cm
eff

7
d) Band Asymmetry:

Asymmetric bands are a common problem in chromatography . Symmetrical

bands are normally observed only for samples that do not exceed some
maximum size , usually 1 mg of sample per gram of stationary phase . If is
higher at lower concentrations of solute , then the low concentration wing of
the eluent peak moves more slowly than the high concentration wing . as an
initially symmetric band moves down the column , it becomes skewed and
eventually develops a sharp front and a long tail . the reverse type of
asymmetry is known as fronting . the solution in this case is to decrease the
sample size to the point where retention times and band shape for all bands
become symmetrical .
The peak asymmetry factor is defined as the ratio of the peak half –widths at
a given peak height . The lower down the peak the asymmetry is measured ,
the larger AF is . Because of detector noise , among other factors , an
acceptable compromise is to measure the AF at 10% of peak height . When
the asymmetry factor lies outside the range 0.95-1.15 for a peak of k=2 , the
apparent plate number for a column is too high . An AF of 1.3 reduces the
efficiency by 69% and the resolution by 30% . Foley and Dorsey developed
an expression for the column efficiency in terms of graphically measurable
41.7(t R' / W0.1 )
N
parameters when the elution curve is asymmetric : eff  )
(a / b)  1.25

Many band tailing problems can be attributed to the wrong combination of

sample and column packing . When system mismatch is suspected , a
different type of column should be tried .
Heterogeneous retention sites are a problem encountered most commonly
with liquid-solid or ion exchange chromatographic systems . Usually retention
sites in liquid-solid or ion exchange chromatography are not exactly
equivalent within a given packing which results in sites of varying retention
affinity . Initially retention occurs on the more active sites . When these sites
are not overloaded , there is normal elution o the sample bands . Another
approach is to selectively remove the stronger sites –that is , to deactivate the
stationary phase as is often done in liquid-solid chromatography .
Asymmetrical peaks may result from actions that occur outside the column,
particularly injection problems . They may also arise from a poorly packed
column .

e) Resolution:

The degree of separation or resolution of two adjacent bands is defined as

the distance between band peaks (or center) divided by the average

8
bandwidth . If retention and bandwidth are measured in units of time , the
t R 2  t R1
resolution is given by: RS 
0.5(W2  W1 )
Solute bands broaden gradually as they migrate through a chromatographic
column . Resolution of individual solutes into discrete bands occurs only if the
bands bandwidths of adjacent bands are almost constant , i.e. W1  W2 .
Since the baseline bandwidth is equal to four standard deviations for a given
t R , 2  t R ,1
band , resolution can also be expressed as : Rs 
4

If inadequate , the resolution of adjacent peaks can be improved either by

increasing the separation between peaks or by decreasing individual peaks
widths . This involves column selectivity when moving peaks farther apart ,
and column efficiency when attempting to narrow the peak width . Improving
the selectivity involves altering the thermodynamic of the chromatographic
system . Improving the kinetics of the system increases the efficiency of the
separation . As shown in fig ,

9
Any criterion for resolution will be somewhat arbitrary . For reasonable
quantitative accuracy , peak maxima must be at least 4 (i.e. ) apart . If so
chosen , then Rs  1.0 , which corresponds approximately to a 3% overlap
of peak areas . A value of Rs  1.5 ( for 6 )represents essentially
complete resolution with only 0.2% overlap of peak areas . Increased
resolution may be needed when a band from a major component is adjacent
to a band of a minor constituent . In real life , there are many instances where
baseline resolution for all components may be unachievable . The separation
is satisfactory when the least resolve pair of components can be quantitatively
determined to an acceptable degree.

The above two formulas of resolution do not relate to the conditions of

separations nor they suggest how to improve resolution . For these purposes
a resolution can be derived in a form that explicitly incorporates the terms
involving the thermodynamics and kinetics of the chromatographic systems .
To accomplish this idea , the equations : and are combined , suing as
the average baseline width , which results :

t R , 2  t R ,1 N 1  t R ,1
Rs  N  ( )
4t R , 2 4 t R,2

10
 N 1  k1' N k1'  k 2'
Substituting both t R ,1 , t R , 2 , one can get : RS  (1  )  ( )
4 1  k 2' 4 1  k 2'

k 2'
Since the relative retention is :   ' , then fundamental resolution
k1
1
1  1 k ' L
equation is : RS  ( )( )( ) 2
4  1 k H
'

From the above formula , it is seen that the resolution to be function of three
separate factors : (1) a column selectivity factor that varies with  , (2) a
rate of migration or capacity factor that varies with k ' and (3) an efficiency
factor that depends on L / H (or the theoretical plate number) . Each factor
can be calculated directly from the recorded chromatogram and can be
adjusted more or less independently . The 1 st two factors are essentially
thermodynamic , whereas the L/H term is mainly associated with the kinetic
features of chromatography .

Changes in and are achieved by selecting different stationary and mobile

phases , or by varying temperature and (less often) pressure . In addition ,
can be varies by changing the relative amounts of mobile and stationary
phases within the column . When optimizing a particular separation , the term
should be considered 1st . Maximum resolution in unit time is obtained when
k=2 . The optimum range of k values extends from 1 to 10 . Furthermore , the
1st eluted component of a given pair should have a retention time that is twice
the passage time of a non retained solute , that is . Unfortunately , in a
complex mixture of many components , it is often possible to optimize
separation conditions for only one pair of components . the only effective
solution to this problem when working with complex real samples is “ k
programming “ . In gas chromatography an optimum value of k can be
achieved by varying temperature . In liquid chromatography , it is usually
more profitable to vary systemically the composition of the mobile phase . If
resolution is still a problem , an increase in either or should be explored .

The L/H term is adjusted to provide maximum efficiency compatible with a

reasonably short analysis time . Higher L/H value always provide improved
ere solution , other factors being equal . Thus resolution may be improved by
increasing the column length . The plate height may be decreased through
improvement in kinetic features of column operation ,. Perhaps by lowering
the flow rate of the mobile phase ( but not lower than the minimum in the )
Any action that increases the efficiency of the mass transfer of solutes
between the stationary and mobile phase will decrease the plate height and
thus improve resolution.

11
 Example:

On a column 122 cm long , operated at 160 C , the retention times were

obtained : air peak , 0.9 ; heptane , 1.22 ; and octane , 1.43 . the base width
of the bands were 0.14 for heptane and 0.20 for octane . what are the relative
retention and the resolution for these bands ?
t R' , 2 t R , 2  t M 1.43  0.9
 '    1.66
t R ,1 t R ,1  t M 1.22  0.9
1.43  1.22
The resolution of the heptane/octane bands is : RS   1.24
0.5(0.2  0.14)

Column processing:

Various processes take place on a column during a chromatographic separation

that contributes to the peak variance or band broadening. Theories of band
spreading in liquid and gas chromatography are nearly identical. Plate height
expresses in simple terms the extent of band broadening and the factors that
affect the broadening. It is a function of thermodynamic and kinetic processes
within the column. These are 1) flow irregularities that lead to convective mixing
2) transverse and longitudinal diffusion in the mobile phase, and 3) a finite rate of
equilibrium of solute between the stationary and mobile phases (mass transfer) .
The effective plate height is expressed by “Van Deemter” equation famous for
B
gas-liquid chromatography as H  A   C stationary u  C mobile u
u

12
Eddy diffusion:

The elution of solutes depends on the diffusion and mass transfer phenomenon .
The eddy diffusion occurs due to the unequal length of the flow path and non
uniform particles sizes . Some solute molecules of a single species may find
themselves swept through the column close to the column wall where the density
of packing is comparatively ;low., especially in small diameter columns . Other
solute molecules pass through the more tightly packed centre of the column at a
correspondingly lower velocity . molecules that follow a shorter path elute before
those that flow a series of erratic paths . This leads to a broadening of the
column band for each solute . To minimize the eddy diffusion effect , the mean
diameter of the particles in a column packing should be as small as possible , the
higher is the inlet pressure needed to drive the mobile phase through the column
and the more difficult it is to pack the column in a uniform manner . However ,
because of the higher efficiency achieved with smaller diameter particles , the
column length can be shortened somewhat . There has to be a trade off between
particles size , column length , and the pressure required . In gas –liquid
chromatography , when films are used on the interior of capillary columns , the
eddy diffusion effect is nil . The eddy diffusion can be expressed by : A  d p ,
where  is a function of the packing uniformity and column geometry and d p
is the particle diameter .

13
Longitudinal diffusion:

In longitudinal diffusion , there is random motion within the mobile phase and is
expressed as B  2DM ,
where  is an obstruction factor that recognizes that Longitudinal diffusion is
hindered by the packing or bed structure , and DM is the solute diffusion
coefficient in the mobile phase . In coated capillary column is unity , in packing
column it has a value of about 0.6 . The foregoing B term pertains to gas –
DM
liquid chromatography . In liquid column chromatography , the ratio should
TM
be used , where TM , the interparticle tortuosity factor , corrects the diffusion
coefficient for the varying size and direction of the interstitial pores in a packing .
The contribution of longitudinal diffusion to plate height becomes significant anly
at low mobile phase velocities . Then the high diffusion rate s of a solute in the
mobile phase can cause the solute molecules to disperse axially while slowly
migrating through the column . If this happens , peak broadening occurs.

14
Mass transfer:

The plate height ratio also depends on mass transfer among stationary and
mobile phases . The term results from resistance to mass transfer at the solute
df
to stationary phase interface . It is proportional to , where d f is the effective
DS
thickness of the stationary phase and DS is the diffusion coefficient of solute in
the stationary phase . Slow molecular movement within the stationary phase
means a longer forward with the mobile phase , the faster the mobile phase
moves through the column and the slower the rate of mass transfer , the broader
is the solute band that eventually elutes from the column. Non viscous liquids
should be chosen for the stationary phase so that the diffusion coefficient is not
unduly small . Reducing the thickness of the stationary phase is beneficial ,
although the capacity of the column is lowered .
The C stationary term represents radial mass transfer resistance between
adjacent stream limits of mobile phase . It is proportional to the square of the
particle diameter of the packing material and inversely proportional to the
diffusion coefficient , of the solute in the mobile phase . Decreasing the size of
the stationary phase particles is always helpful in decreasing the plate height .

In liquid column , as contrasted with gas –liquid chromatography , major

differences arises from the 10,000 fold decrease in the value of DM when a
liquid constitutes the mobile phase as opposed to a gas , (2) the presence of
stagnant pockets of mobile phase trapped within pores and channels of the
stationary phase . Solute molecules move into and out of these pores by diffusion
. These molecules are retard in their forward motion relative to the main band of
a given solute and again there is an increase in the molecular spreading . Also
the velocity of the mobile phase differs from point to point owing to the
perturbation caused by the support particles . Liquid stream lines of the mobile
phase near the particle boundaries move slowly . Solute molecules transfer
constantly by lateral diffusion to a different stream line . Hence , the obstructive
path of a solute molecules is due to both to stationary phase particles . Molecular
diffusion , coupled with unequal stream lines ( multipath effect) gives rise to a
A
C
convective mixing , or coupled , term : 1  mobile
u

Plate height in liquid column chromatography can be expressed by :

B A
H   C mobile u  C stat u
u C
1  mobile
u
st
, where the 1 term=longitudinal or axial diffusion ,
2nd term= convective mixing ,

15
3rd term= resistance to mass transfer in mobile phase and
4th term= resistance to mass transfer in stationary phase

The individual contributions of the four terms in plate height is shown in the
following plots . In both gas –liquid column chromatography , longitudinal
diffusion is a significant factor only at mobile phase velocities less than the
H
minimum in the composite curves . Ideally an operator might wants to
u
untilize a velocity corresponding to the minimum plate height . Actually the plate
height in the region to the right of the minimum usually rises only gradually for
most gas-liquid or liquid column chromatography columns. Consequently , a
slight loss in column efficiency may be traded for shortened analysis time when
velocities somewhat higher than u th are used .

Time of Analysis & resolution:

It is needed much to determine the time required to get the solute band through
one plate , t p , multiplied by the number of plates required , N req , for the
desired resolution . Thus t R  t p N req
Since t p is given by the plate height divided by the band velocity , , where is
the fraction of time spent by the solute in the mobile phase .then
H  2 (1  k ' ) 3 H
t R  N req (1  k ' )( ) Also , t R  16 R 2 ( ) [ ]( )
u  1 (k ' ) 2 u
Differentiating , w.r.t. k and placing all other variables into one constant C :
'

dt R ( k ' ) 3  3k '  2
 C[ ]
dt (k ' ) 3

16
Now t R is a minimum when k '  2 which is when t R  3t M . There is little
increase in analysis time when k ' lies between 1 and 10 , provided k ' has no
effect on the other variables .

Quantitative Analysis:

Detectors in chromatography respond either to the concentration of solute or to

the mass flow rate . Those that respond to the concentration yield signal that is
proportional to the solute concentration around the detector. An elution peak
results when the signal is plotted against time . For such detectors the area
under the peak is proportional to the mass of a component and inversely
proportional to the flow rate of the mobile phase . It is crucially important that the
flow of the mobile phase be kept constant for such detectors if quantitative
analysis is to be carried out . In differential detectors that respond to mass flow
rate , the peak area is directly proportional to the total mass and there is no
dependency on the flow rate of the mobile phase . In column chromatography the
analog signal generated by the detector is graphically recorded in the form of the
familiar chromatographic peaks . the area under these peaks can be then
integrated in a variety of ways and the resulting data related to the composition of
the unknown samples.

a) Peak Area integration:

Half Times width at half -height :

In this method a baseline of the peak is fixed first . Peak height is measured
from this baseline .Peak width is measured at the position half of this height .
The zero base line is not used because large deviations may be caused by
tailing.

Peak height:

Measurement of peak height is inherently simple . the only operations

involved are drawing the baseline and measuring the height . The precision is
better than measuring the peak area , particularly of narrow peaks . However
, peak heights are sensitive to small changes in the technique of sample
injection and in operating conditions . Peak height does not always remain
directly proportional to sample size . As later increases , there is a point at
which the peak begins to broaden and no longer increases in height at the
same rate.

b) Computing integrator:

17
On line computer based data system provide complete automation . This
includes automatic acquisition and reduction of data , storage of calculation
methods and a printout of analylitical results . Initially the analog
chromatographic signal is digitized by an analog to digital converter . The
software then can detect the presence of peaks , correct for baseline drift ,
calculate areas and retention times , determine concentrations of components
using stored calibration factors , and generate a complete report of type
analysis .
Peaks area counts are accumulated when the signal leaves the baseline .
Thus departure from the baseline is usually determined by monitoring the
slope of the signal . The retention time and signal heights of each peak
maximum detected by the programme are stored in memory . The termination
of a component peak is established when the signal returns to the baseline .
During isothermal runs the software can automatically increase the slope
sensitivity with time , thus ensuring the programmer’s ability to detect both
initial sharp peaks and later low flat peaks with equal precision . In the case of
fused peaks , areas can be allocated to each component by dropping
perpendiculars from valley points to the corrected baseline . In the case of
overlapping peaks , special algorithms allot areas to each component .

c) Evaluation method:

The three principals evaluation methods are I) calibration by standards , 2)

area normalization , and 3) internal standards .

1) Calibration by standards:

18
When the sample volume is known , calibration by standard is often
used . It has the advantage that only the areas of the peak of interest
need to be measured . It does not require that the same amount of
sample be injected each time . the necessary calibration should be run
under the same operating conditions of the sample . The %
concentrations are obtained by calculating the ratio of the volume of
each component of interest to the sample size . In practice , standard
solutions of the components of interest are prepared and injected into
the chromatograph . then for an unknown , X  (area ) x K , where K is
the constant proportionality
Relative response factors must be considered when converting area to
volume and when the response of a given detector differs for each
molecular type or class of compounds . Response factors are best
obtained by analyzing standard samples.

2) Area normalization:

19
 When it is known that the chromatogram represents the entire sample ,
that all components have been separated , and that each peak has
been completely resolved , area normalization may be used for
evaluation . To use this method , the area under each individual peak is
measured and then divided by its response factor to give the peak’s
calculated area . adding together all the peaks areas gives the total
calculated area . the percent by volume for individual components is
obtained by multiplying the individual calculated area by 100 and then
dividing by the total calculated area.

3) Internal standard:

The internal standard method permits the operating conditions to vary

from sample to sample and does not require repeatable sample
injection . The internal standard has to be a component that can be
completely resolved from adjacent peaks , is not present in the
unknown mixture , and does not have any interference effects . A
known quantity of this standard is chromatographed , and area vs.
concentration is plotted . any variation in sample size will be
immediately apparent by comparing the peak area of the internal
standard in different runs. A correction factor can then be used when
determining the exact concentration of the other components.

Components of Gas chromatography:

A gas chromatograph consists of several basic modules joined together to (1)

provide a constant flow of carrier gas (mobile phase) , (2) permit the introduction
of sample gas into flowing gas stream , (3) contain the appropriate length of
stationary phase , (4) maintain the column at appropriate temperature ,(5) detect
the sample components as they elute from the column , and (6) provide a
readable signal proportional in magnitude to the amount of each component .

1. Sample injection system

The sample is injected by a hypodermic syringe through a self sealing silicone

rubber septum onto glass liner within a metal block , where it is vaporized and
swept onto the column . The block is heated at a fixed temperature sufficiently
high to convert virtually instantly the liquid sample into a “lug” of vapor .
Samples , both liquid and gas , can be measured with a calibrated loop, then
introduced into the flowing gas stream , by means of valve . reduction in sample
volume is necessary when working with capillary columns . This is accomplished
by an injector-splitter , where typically a 1-ul sample is injected but only 0.001 ul
enters the capillary , the remainder is vented . This technique prevents column
overload but wastes a significant portion of the sample. When analyses are

20
performed on small amounts with some component concentrations in the parts –
per-billion range , too little material is placed on the column if splitting is used for
these samples . Split less injection is required for these samples . the entire
sample , including any solvent is injected onto the open tubular column through a
modified heated flush vaporizer . A large solvent tail is avoided by venting the
injection port to the atmosphere at that time of about 30 sec. , when most of the
solvent and essentially all the sample have entered the column . The proper time
until venting is critical ; too short a time causes loss of sample components ,
whereas too long a time causes a solvent peak larger than necessary which
might bury some of the peaks of interest .

a) Automatic sampler

An automatic sampler duplicates manual sample measurements

and injection . Sample valves are a glass , throw –away type with
vapor tight septum caps. The samples flushes the syringe with a new
sample to remove traces of the previous sample, pumps new sample
to wet the syringe completely and eliminate any bubbles, takes in a
precisely measured amount of sample , and injects the sample into the
gas chromatograph .The automatic samplers are machine reproducible
and consistently more precise than a skilled chromatographer ; also
unattended operation releases the operator for other works.

21
2. Chromatographic column

a) Packed column:

Packed column are constructed from tubing stainless steel , nickel , or

glass with inner diameter ranging from 1.6 to 9.5 mm and length about
3 m . These columns are packed with an inert support , usually
diatomaceous earth whose internal diameter range from 2um for
material derived from firebricks to 9 um for materials derived from filter
aids . The internal column diameter should be at eight times the
diameter of the support particles.

b) Capillary column:

Capillary column have an internal diameter of 1 mm or less . These are

usually constructed of fused silica ( a very high –purity glass) , which
has a much higher degree of cross linking within the silicon-oxygen
matrix than does ordinary glass. The high tensile strength of the silica
tubing permits the construction of thin walled , flexible columns. To
protect the thin wall against scratches , a protective coating of
polymide is applied to the outer wall. There are two main types of

22
capillary columns. (1) packed column with solid particles over the
whole dia of the column and (2) open tubular column with an open and
unrestricted flow path through the middle of column . the latter are
divided into wall –coated open tubular columns., support-coated open
tubular columns and porous-layer open tubular columns. The inner
surface of the wide-bore (WCOT) capillary column (0.53 mm ID) is
coated with the stationary liquid phase . Uniform coating of the surface
was difficult to obtain until bonded –phase columns were introduced .
The polymer wets the surface of a fused silica column well , with the
result that a very uniform film covers the column walls . A variety of
functional groups can be blended into the polysiloxane chain to provide
stationary phase of different polarity or selectivity .

The major advantage of wide –bore capillaries regardless of

separations is an increase in the speed of analysis . Using packed
column flowrates , the total run time roughly three times faster .
However , when the flowrates is optimized for the tubing diameter , the
wide –bore column produces far superior efficiency and therefore
superior separations , with analysis times approximately equal to those
for packed columns to wide –bore capillaries shorter retention times ,
greater inertness , longer life , lower bleed , higher efficiency , and
greater reproducibility .

With He as the carrier gas , the optimum flowrate for a 0.53 ID column
is approximately 2.5 mL/min . This provides 2100 plates per metre ,
giving 21,000 theoretical plates with a 10-m wide-bore capillary column
. This is far superior to the 3000-4000 theoretical plate total column
efficiency obtained with a typical 20-m packed column . Nothing is
superior to a capillary column for resolving mixtures with many
components.

The chromatographic columns are coiled and held in a basket that is

mounted inside an oven . The column oven must be able to be rapidly
heated and cooled . This requires a well designed and adequate
system of air flow . In most design the air is blown past the heating
coils then through baffles that make up the inner wall of the oven , past
the column , and back to the blower to be reheated and recirculated .
Ovens are usually constructed of low –mass stainless steel . For
temperature programming it is desirable to have a range of
temperature program rates from 0.1 to 50C/min . Hold times anywhere
within the program should be available . Temperature should be
maintained within  1 C
For isothermal runs and within  2  C of the desired temperature
during programming .

23
3. Detector:

a) Thermal conductivity detector:

The thermal conductivity detector (TCD) uses a heated filament placed

in the emerging gas stream . the amount of heat loss from the filament
by conduction to the detector wall depends on the thermal conductivity
of the gas phase . In construction , a tightly coiled filament made of
tungsten material , tungsten rhenium alloy , or tungsten sheathed with
gold is contained in metal block cavity . the filament is heated to a
constant temperature but less than a dull red condition by a regulated
DC current supply . Heat loss from the filament to the metal block is
constant when only carrier gas is flowing through the detector. The
thermal conductivities of hydrogen and helium are roughly six to tem
times greater than those of most organic compounds . Thus the
presence of even small amounts of organic materials causes a
relatively large decrease in the thermal conductivity of the column
effluent . The filament retains more heat , its temperature rises , and its
electrical resistance goes up. The standard detector consists of 4 nos
identical filaments mounted within one brass block . the filaments make
up the arms of a whetstone bridge . In analysis , the column effluent is
passed through one pair of filaments and the second pair is placed in
the gas stream ahead of the sample injection point. Any unbalance
between the pairs of filaments is recorded . In this way the thermal
conductivity of the carrier gas is cancelled and the effects of variation
in flow rate and pressure are minimized . an initial baseline reading is
established by passing only carrier gas through both pairs of filaments
and then adjusting the setting of the pot. D. A TCD can also be
constructed with thermistors . The thermistor is made of metal oxide
bead with two leads . The thermistor has negative temperature
coefficient of resistance . One head is mounted in the pure carrier gas
stream , the other in the column effluent . The Whetstone bridge circuit
is completed by a matched pair of 500 ohm , 3W resistors .
Thermsitors are not exchangeable either mechanically or electrically
with filaments .

24
b) Flame ionization detector:

25
The flame ionization detector (FID) adds hydrogen to the column
effluent . Subsequently , the mixture is passed through a jet where it is
mixed with external air and burned . In construction , the flame jet and
a cylinder positioned 0.5 to 1.0 cm above the flame tip constitutes the
twin electrodes . In an alternative arrangement , two parallel plates are
mounted above the flame tip. A potential of about 400V is applied
across the two electrodes , which lowers the resistance between the
electrodes and causes a current to flow. The current arises from the
ions and free electrons generated in a pure hydrogen/air flame. When
ionizable material from the column effluent enters the flame and is
burned , the current markedly increases . The current flows through an
external resistor , is sensed as a voltage drop , amplified , and then
finally sent to an output device . An opposing voltage equal to this
signal from the hydrogen/air flame when only pure carrier gas is
passing permits the baseline to be adjusted . The FID is enclosed
within a chimney so that it is unaffected by drafts and can be heated
sufficiently to avoid condensation of water droplets from the
combustion process . An ignitor coil and flame out sensor are placed
above the jet to reignite the flame if it becomes extinguished . The FID
responds proportionately to the number of ---CH2--- groups introduced
into the flame .

26
c) Thermionic emission detector:

The thermo ionic emission detector (TED) uses a fuel –poor hydrogen
plasma , a low temperature flame that supposes the normal flame
ionization response of compounds that do not contain nitrogen or
phosphorus . A non volatile bead of rubidium silicate is centered 1.25
cm above the flame tip. In all other respect The physical arrangement
resembles a FID . the bead is electrically heated and can be adjusted
to between 600 and 800C . this option permits adjustment of the
bead’s temperature independent of the flame as a source of thermal
energy . With a very small hydrogen flow , the detector responds to
both nitrogen and phosphorus compounds . enlarging the plasma size
and changing the polarity between the plasma tip and collector causes
the TED to respond to only phosphorus compounds.

d) Electron capture detector:

In electron capture detector (ECD) the column effluent passes between

two electrodes . One of the electrodes has on its surface a
radioisotope that emits high-energy electrons (  particles) as it
decays . these electrons bombard the carrier gas (nitrogen) , resulting
in the formation of a plasma of positive ions , radicals , and thermal

27
electrons by a series of elastic and inelastic collisions . this process is
very rapid (< 0.1 usec) . the applications of a potential difference to the
electron –capture cell allows the collection of the thermal electrons that
constitute the detector standing current , or baseline signal , when only
carrier gas is passing through the detector standing current , or
baseline signal , when only carrier gas is passing through the detector.
Electron –absorbing compounds in the carrier gas stream react with
the thermal electrons to produce negative ions of larger mass. The rate
of recombination between –ve and +ve ions is many times faster than
between thermal electrons and +ve ions . the decrease in detector
current due to removal of thermal electrons by recombination in the
presence of electron capturing compounds forms the quantitative basis
of the detector operation.

Effluent electron collection with nitrogen gas as the carrier gas is

achieved with a displaced coaxial cylindrical cell configuration . A 0.2
ml volume is formed by two closely spaced cylinder in which the carrier
gas flow is opposite to the electron flow . Radioactive sources include
tritium adsorbed in titanium or scandium , and higher energy source
than tritium and can be used up to 400C . although the sensitivity of
the nickel detector is about five times less than that of a tritium unit ,
the standing current remains constant and the detector is much less
susceptible to contamination. To prevent the formation of contact
potentials and space charge effects , the ECD voltage is applied as a

28
sequence of narrow pulses with a duration and amplitude (0.6 usec
and 50V) sufficient to collect the very mobile electrons but not the
heavier , slower negative ions. During the interval between pulses
(100-150 u sec ) the electron concentration builds up inside the cell .
When the pulse is applied , the concentration of electrons is essentially
reduced to zero . This mode of operation has several advantages .
During most of the time no field is applied ; this enables the free
electrons to reach thermal equilibrium with the gas molecules . The
opportunity for electron capture is maximized , and stable response
factors are thereby attained . Negative ions formation occurs in a
region where positive ions are also present and recombination can
efficiently take place . There is no collection of negative ions . The
formation of a space charge is prevented by the brevity of pulse
interval . The linearity of ECD is function of the cell design , the
composition of the carrier gas , and the method of applying the
potential difference to collect the thermal electrons . For the direct
current and pulsed ECD , the linear range is about 100 , and for pulse-
modulated , constant –current ECD about four decades . Argon mixed
with 5% -10% methane is the makeup gas of choice for pulsed ECDs .
It is added to the column effluent . At the voltages used , the electron
velocity is ten times greater in this mixture than in nitrogen . Through
inelastic collisions with methane , the metastable ions formed from
argon are eliminated before they can cause undesirable sample
ionization.

29
The chromatographic scheme:

The basic scheme comprises of six parts :- These are (i) Carrier gas supply with
regulator and indicator (ii) sample injection system (iii) Chromatographic column
(iv) Detector (v) The thermal compartments housing the different part , and (vi)
Recorder to produce the chromatogram .

Carrier gas from the cylinder passes through a pressure regulator and a direct
indicating flow meter to enter the column on the one side before that a sample
injector injects a sample into the stream and the reference cell of the detector on
another side . This bifurcation is accomplished by a pressure –T . Flow rate
depends on the sample to be analyzed but once the flow rate is selected it must
be maintained constant . The usual flow rate limits are from 0 to 250 cc/min at a
line pressure varying from 0 to 4kg/cm2 . The flow rate may be adjusted by
controlling a needle valve . Commonly used carrier gas is nitrogen and the gas
must be perfectly pure , sometimes called white nitrogen . The purity is required
for proper detection whereas efficiency of operation depends on constancy in
carrier supply condition and the volume of the injected sample . Other gases
used as carrier are argon , helium , hydrogen etc. Choice of the gas depends on
types of the detector .

30
Injection system:
It introduces same quantity of the sample in the carrier stream in the vapour
state as many times as is necessary . For liquid samples , a heater to vaporize
the sample when injected , is required . Such instantaneous vaporization
sometimes may be associated with decomposition . Care must be taken to avoid
it . Gas sample injection is comparatively simply done by injecting through a gas
tight syringe or through a three-cock stream splitter . In the latter , a measured
volume is trapped in a loop between two stopcocks . On simultaneous rotation by
90 and 180 of these valves by a sliding mechanism , the trapped volume is
flushed into the main carrier stream . The liquid sample is introduced by a micro-
syringe through a self –sealing silicone rubber septum . the portion of the line
where the sample is injected is heated by electrical means . The injection
operation should b quick enough so that the pressure of the sample in carrier can
be considered a ‘pulse’ . the sample is between 0.1 to 10 u sec depending on the
carrier flow rate .

31
The column:

The chromatographic column is made from a tube of copper , stainless steel ,

alloys of copper nickel or even glass . It may be shaped in the form of a U or may
be coiled to increase the length . the latter shape is preferred for capillaries
whereas the packed types generally are of U –shape . The length of a column
varies from 100 to 150 mm for the packed type with a diameter of the order of
0.5 to 1 mm . Golay or capillary columns have lengths ranging from 30 to 300 m
with a diameter between 0.02 and 0.05 cm . The packing may be in the solid
phase for adsorption or liquid phase for partitioning as . The packing material is
chosen from a wide variety depending on the specific use . A few common
materials include silica gel , alumina , charcoal activated with proper chemical
and other silica products . Good resolution characteristics with retentive property
are necessary for the materials . The Golay capillary columns or the open tubular
columns (OTC) have their stationary phase – a liquid coated on the inner wall of
the columns . Pressure drop across it is quite small and the tube/capillary is
made of glass or fused silica to avoid any catalyzing action within the column
due to the presence of metal ( column material) at the operating temperatures .

Liquid packing materials should have a good differential partitioning property ,

low vapour pressure and high thermal stability , i.e. should not vaporize or
deteriorate in the operating temperature range . The common materials are solid
petroleum products dissolved in silicone coils or other organic solvents . Capillary
column has a higher efficiency and is useful in a greater number of components
in a gas mixture but is more expensive as well . For a temperature Tc in K of
vg
the column , the normalized retention figure is given by : FR   273 / wTc
vR

32
, where w is the weight of the liquid phase , v g is the specific retention volume
and v R is the total retention volume corrected for pressure gradient in the tube .
The efficiency of substances as column material is judged by what is known as
v gt
the relative retention ratio as given by :  
v gs
, where v gt is the specific retention volume of the test substance and v gs is
that of standard substance .
In LSC and LLC the stationary phases are solids and liquids . The solids in the
stationary phase may consists of .Al 2O3 , MgO , charcoal , silica gel , CaO ,
MgCO3 , K2CO3 , Na2CO3 , CaCO3 , starch , Cellulose etc. and for LLC water on
silica gel or some other absorbent , benzene or carbon tetrachloride on rubber
powder may be sued . For mobile phase , solvents used for both LLC and LSC
are varied in nature such as petroleum ether , CCl4 , cyclohexanne , benzene ,
toluene , chloroform , ethyl ether , dichloroethelhane , alcohols water many
others including organic acids which are high polarity solvents an have relatively
low retention time . High polarity increases adsorption . Retention time can be
controlled by polarity control , i.e. choice of stationary as well as mobile phases .

High performance liquid chromatography (HPLC) is a type of liquid

chromatography where the column is specially packed to provide greater surface
area . For a useful flow rate through this rightly packed column , a greater
pressure is to be maintained across the column which is usually done by usng a
pump . A typical scheme is below:

33
The working theory of chromatography:

The chromatographic operation basically differentiate the components in a

mixture by separating the components by elution process as the mixture of two
phases ( mobile and stationary phases) passes through chromatographic column
. If the partition coefficient of individual components is large , it would lag during
the process migration through column , and considering two components in the
mixture , with movement , the distance between the two components increases
and simultaneously band –broadening also takes place , which decreases the
column efficiency . The theory that accounts for the relative rates at which the
solutes migrate and also the bands broaden during migration , is the trade theory
also called the kinetic theory . During the interaction between stationary and
mobile phases , the particles is eluted . If now a no. of particles are considered ,
their time of stay in the two phases , no of times they change their stay in each
phase – all become quite random but because the no. of transfer is very large the
random individual processes generate most common or average particle . If more
time is required for migration , bandspread increases , the bandwidth is thus
directly proportional to resistance time in the column and inversely proportional to
the velocity of the mobile phase . For giving a quantitative form , the plate
theory , in line with the distillation column , was first proposed by Martin and
Synge , where it is assumed that the column consists of N no . of discrete but
contiguous narrow horizontal layers called “ theoretical plates “ of height
equivalent H i.e. of thickness H so that the column length L is given by : L  NH

It is argued that the efficiency of a column increases with increasing N or

decreasing of H . The term “ theoretical column” is used to indicate that in reality
such a thing does not exists . As the breadth of Gaussian curve is related to
variance , efficiency of a column can be defined in terms of as well . Thus either
H   2 / L or N  L2 /  would mean efficiency , because as  decreases
H decreases and N increases . In fact N and H can be evaluated from the
chromatogram of a component . In a typical chromatogram peak  2 is in cm
corresponding to length L , this would changed to  2 corresponding to retention
time t R so that one can write :  / t R   / L

It can be shown that approximately 96% of the area under the peak of the
chromatogram is the same as the area of the triangle drawn with W as the base
and with the analogy that approximately 96% of the area under the curve of
Gaussian type is within  2 of the variable value at the peak , then one can
write : W  4 so that H   2 / L  L 2 / t R2  LW 2 / 16t R2 and N  L2 /  2  16(t R / W ) 2
Thus H and N are the performance parameters of the column chromatography .

L
Also  u s , the average velocity of the solute . The peak at time t M is for a
tR
constituent that is not eluted by the column packing and hence is the velocity of

34
the mobile phase . Now and are related by the equation : u s  u M ( No. of
moles of the solute in the mobile phase / Total No. of moles of the solute )

This is because a solute migrates only when it is in the mobile phase . If V s

and VM are the total volumes of the stationary and mobile phases , then :
 M VM uM u
us  uM ( )  M
 M VM   S V S V 1  K c , where K c is known as the capacity
1 K S
VM
factor and is obviously a measure of the migration of the solute . K c can be
found out from the chromatogram .
Substituting the values of u M and u S , one can get : K c  (t R  t M ) / t M

Both and are obtained from the chromatogram . The ratio of the partition
coefficient of two components A and B is called the selectivity factor as given by :
K
FS  B
KA
Component B is considered to be more strongly retained here , so that t RB  t RA
K cB
In terms of K c s thus : FS 
K cA
t RB  t M
So that from the chromatogram , FS 
t RA  t m
Resolution o a column is another important factor to be considered . Considering
two component chromatogram , the resolution R S is defined as
P 2 P 2(t RB  t RA )
RS    , where P is the difference
(W A / 2  WB / 2) (W A  WB ) (W A  WB )
between the peak of A and B compound.
If is 1.5 , there is about 0.3% overlapping and for being 1.0 , there is about 4%
overlapping . By increasing can be increased . Resolution can be expressed in
terms of and for a paired components . Thus
N K cB  K cd N FS  1 K cB
RS  ( )( ) ( )( ) , where
4 1  K cB 4 FS 1  K cB
16 RS2 H FS 2 (1  K cA ) 3
The elution time : t RA  ( ) [ ]
uM FS  1 K 2 cA

35
Signal detection and data processing of chromatography:

36
The input voltage to the A/D converter will be proportional to the detector output
signal . For a flame ionization detector , the detector output current is
proportional to the number of molecules of the i-th component , eluting from the
column per sec. . In this case the converter input voltage is :
.
, Where is a constant , ideally the same is : Vi IN  G n i

1 1
G
On integration , ni  Vi IN dt  Ai ,
G
This shows that the total no. of molecules of the i-th component in the sample
is proportional to the integral of converter input voltage , i.e. to the area under
the i-th peak . Thus the total number M of molecules present in a sample of m
m
1 m
components is given by : M  
i 1
n i   Ai
G i 1
i.e. is proportional to the sum of the areas under all peaks . The % molar
concentration of the component in the sample is therefore given by :
n A
ci  i  100%  m i  100
M
 Ai i 1

Thus , in ideal case , the composition of the sample can be found by evaluating
the area under each component peak . The output voltage of the detector
however , is different components , and is approximately proportional to the
.
concentration x i of the component in the component/ carrier mixture . If n c is
the number of molecules of carrier leaving the column per sec. , then :
. .
nc ni
xi  . .
 . for small x i
n c  ni nc

.
Thus for dilute mixture xi , is proportional to n c , provided that is a constant ,
so that there is tight control of carrier gas flow rate . The A/D converter input
.
voltage for a detector is therefore given approximately by : Vi IN  Gi n i

, where G I is the sensitivity for the i -th component , which depends on

thermal conductivity k i . In this case the corresponding expression for the
concentration of the component in the original sample is :

(1 / Gi ) Ai Ai*
ci  m
 100%  m
 100%
1

i 1 G
Ai 
i 1
Ai*

37
, where Ai* the are the corrected peak areas Ai*  (1 / Gi ) Ai

The input to the computer is a sampled , coded version of the A/D converter
input signal . The converter has , typically a conversion time o a few milli-sec ,
so that samples can be read by the computer at intervals of a few milli-sec .
These sampled values may be affected by high -frequency interference or
noise and should be filtered . One sample filtering method is to calculate the
average value of a group of several samples . Thus if the A/D conversion time is
5 ms an average of 40 samples is available every 0.2 sec . The computer uses
these filtered values to estimate the area of the peak by dividing it into a series of
rectangular elements .

If y j is the j-th filtered value and T the interval between values , then the
P P

area of the j-th reactangle is , giving : A   y j T  T  y j

j 1 j 1

, where P is the number of filtered values used in the calculation . The above
expression is accurate if T is small compared with peak standard deviation 
. Thus if   2.5 sec . , a T is 0.2 sec is suitable , giving P  75 for the
complete peak of width 6

For a simple system involving one column and on carrier stream , , the sequence
of operations is shown below:

38
There are however , many analysis which require multiple column and carrier
operation . One example is the measurement of small concentrations of benzene
, ethyl benzene and paraxylene in a mixture which is mainly toluene . In the
resulted chromatogram , it is seen that the peaks are not clearly resolved ; the
small ethyl benzene and paraxylene peaks sit high on the tail of the large toluene
peak . The problem is solved using the technique of heartcutting which involves
switching gas streams between two columns . two sliding plate valves are used ,
one for sample injection (V1) and one for column switching (V2) . the sample is
injected into column 1using V1 , in the normal way . Valve V2 is initially in
position C , which enables the benzene peak to pass from column 1 to column 2 .
as the toluene peak is about to elute from column 1 , VB2 is switched to position
D , so that the gas leaving column 1 is vented and carrier gas 2 is passed from
through coC c . Thus the remainder of the toluene peak , together with the
associated ethyl benzene and paraxylene peaks , enter column 2 . These peaks
can then be effectively separated in column 2 without having to use very long
retention times . The resulting chromatogram is shown together with operation
sequence .

Another switching technique involving two columns and two carrier supplies is
called backflushing . This technique allows components of interest to pass

39
through columns 1 and 2 to the detector . Components not required for the
analysis , or which may harm the detector , are prevented from entering column 2
and flushed to even by carrier 2 flowing through column 1 in a reverse direction .

Examples:

40
1. From a two components chromatogram following data are obtained:
Retention time of A component: t RA  19.92 sec
Retention time of B component : t RA  18.8 sec
Bandwidth of A component: WB  1.22 m
Bandwidth of B component: W A  1.02 m
Column length: L  50 cm .
Calculate the no. of plates , plate height and resolution

N B  16(19.92 / 1.22) 2  4265 and N A  16(18.8 / 1.02) 2  5435

Hence , the average of : N avg  4850 and assuming the retention ,
L
H   1.03  10 3
N avg
Resolution : RS  2(19.92  18.8) /(1.22  1.02)  1.0 . To change
R s1 N1
resolution , length needs must be changed . . Now 
RS 2 N2
And then new N can be calculated , from which the new L also.

2. On a 1000 cm wall coated open tubular column of 0.25 bore , the He

carrier gas velocity is 37 cm/sec. The retention time , for decane is 1.27
min. ; peak width at half height is 0.88 sec.

L
The retention time for a non retained compound is t M   1000 / 37  27
u
sec or 0.45 min.
t R' 1.27  0.45
The partition ratio , k    1.82
'

tM 0.45
t R' 2 49.2 2
The effective plate no. : N eff  5.545( )  5.545( )  17,300
W1 / 2 0.88
L 1000
The plate height : H  N  173000  0.058 cm
eff

3. On a column 122 cm long , operated at 160 C , these retention times (min)

were obtained : air peak , 0.9 ; heptane , 1.22 ; and octane , 1.43 . The
base width of the bands were 0.14 for heptane and 0.2 for octane . What
are the relative retention and the resolution for these bands ?

t R' , 2 t R,2  t M 1.43  0.9

Relative retention:      1.66
t '
R ,1 t R ,1  t M 1.22  0.0

41
 1.43  1.22
Resolution : RS   1.24
0.5(0.2  0.14)

4. Assuming 100 mg of internal standard is added to 1.0 mg of mixture .

Measurement of the resultant chromatogram shows four components
(including the internal standard ) with areas (in arbitrary unit) as follows :

A1  27, Astd  80, A2  70 and area sum =197 . The amount of component
A3 70
3 in the sample is W3  Wstd ( )  100( )  87.5 mg.
Astd 80
0.0875
% of component 3 :  100  8.75%
1.0

5. The data in the table were extracted from the chromatogram of a two
components mixture of x and yu . Calculate a) the capacity ratios of x and
y (b) parameters affect the capacity ratio , (c) separation factor (d)
resolution

6. Consider a 50 –cm column with a plate height of 1.5 mm that provides a

theoretical plate no. of 333 at a flow rate of 3 mL/min ; VM  1.0mL .
Calculate solute retention time and retention volume when k '  1,2,5 and
10 also peak width for each of the foregoing values of k '

7. Chromatograms with a standard test mixture were obtained using porous

alumina . Assume the total porosity is 0.75 , the inlet pressure was 22.5
atm for all columns . Operating conditions and retention times are given in
the table . Calculate k ' a) for each solute , (b) average linear velocity
and plate height for each column (c) free column volume and flow rate for
each column.

Test substance , Column 1 Column 2 Column 3

t R (sec.)
Nitrobenzene 538 182 91
Anisole 232 76 36
Biphenyl 168 56 26

42
 Toluene 124 40 19
t M (sec.) 104 34 16
L (cm) 50 13.5 9
N 3200 4450 5000
dp (um) 20 10 6.5

8. A packed column of length 25 cm when used in a chromatograph, three

species A,B and C show retention times as 5,14 and 20 min respectively
and peak base widths as 0.4 , 1.1 an 1.7 min respectively . An unretained
species passes in 3 min . Calculate the column resolution , the plate no.
in the column and the plate height .

9. A sample containing O2 and N2 is injected into a He carrier at time t=0 .

The sample is swept through a column 1.0 m long packed with molecular
sieve . The eluting components are detected by a detector which has
equal sensitivity for O2 and N2 . The chromatogram is given below.

a) Assuming that the distribution ratio K for O 2 2.0 , estimate the

mean carrier velocity u and K for N2.
b) Estimate base width W for peaks and hence find resolution
c) Estimate the number of theoretical plates N
d) Estimate the % composition of the sample ( assuming peaks are
approximately triangular)

43