Chromatography

Introduction
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase
(which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced
through an immobile, immiscible stationary phase. The phases are chosen such that
components of the sample have differing solubilities in each phase. A component which
is quite soluble in the stationary phase will tae longer to travel through it than a
component which is not very soluble in the stationary phase but very soluble in the
mobile phase. As a result of these differences in mobilities, sample components will
become separated from each other as they travel through the stationary phase.
Thus, chromatography is an analytical technique used for the separation of complex
chemical mixtures into individual components.
Chromatography is a separations method that relies on differences in partitioning
behavior between a flowing mobile phase and a stationary phase to separate the
components in a mixture. Chromatographic separation process based on the difference in
the surface interactions of the analyte and eluent (mobile phase) molecules.
A column (or other support for T!C) holds the stationary phase and the mobile phase
carries the sample through it. "ample components that partition strongly into the
stationary phase spend a greater amount of time in the column and are separated from
components that stay predominantly in the mobile phase and pass through the column
faster. As the components elute from the column they can be quantified by a detector
and#or collected for further analysis.
Gas Chromatography and Liquid Chromatography
At its simplest, gas and liquid chromatography are methods to separate sample
components from each other and from matrix components and the solvent. This is
accomplished by partitioning the compounds between a stationary phase (the column
pacing or coating) and a moving phase (a gas in $C and a liquid in %&!C). Compounds
with little partitioning into the stationary phase pass through the column more swiftly and
elute first. Those that spend a lot of time in the stationary phase are retarded and elute
later. 'n gas chromatography, boiling point or vapor pressure is the ma(or factor in elution
order, although polarity has a small but important effect. 'n %&!C, polarity is the primary
factor in elution order.
"ince elution is based on physical properties, retention time or elution pattern is an
attribute of a molecule and can be used to determine identity. A particular compound will
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have a characteristic retention time (time from in(ection to detection) when run on similar
columns under similar conditions. Two different compounds may have very close or
identical retention times on one column type, but when analy*ed on a column of differing
polarity usually have different retention times. This is the basis for identification based on
chromatographic retention time.
$C and %&!C are also used in quantitation. The signal from the detector is in the shape
of a pea, and the height or area of the pea is proportional to the amount in(ected.
+uantitation utili*es standard curves, where nown amounts from standards of nown
concentration are in(ected into the instruments, and the response compared to a sample of
unnown concentration.
CHROMATOGRAPHY THEORY
Chromatography theory consists of empirical relationships to describe chromatographic
coumns and the separation o! pea"s in chromatograms. The underlying principle that
controls chromatographic separations is a dynamic behavior that depends upon
partitioning and mass transport.
#escription o! Chromatograms
"eparation of solutes depends on the rates at which the species are eluted. These
migration rates are determined by the partition coefficient, $ or the equilibrium
constant of the distribution of the solutes between the mobile phase (elution solvent) and
stationary phase (column pacing).
#istri%ution o! anaytes %et&een phases
The distribution of analytes between phases can often be described quite simply. An
analyte is in equilibrium between the two phases,
A
mobile
A
stationary
The equilibrium constant, $, is termed the partition coefficient, defined as the molar
concentration of analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase.
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[A] stationary
[A] mobile
$ '
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detector
signal
time
t
0
t
1
t
2
t
3
t
4
packed
column
sample
A + B
mobile phase
A
B
B
B
A
A
B
.
B
A
Retention factor capacity factor!, "#, is often used to describe the migration rate of an
analyte on a column. The retention factor for analyte A is defined as,
k'A ' t R ( tM ) tM
Retention time,( t
R
)
Retention time, t
R
is the time for the analyte to pass through the column or the time
between sample in(ection and an analyte pea reaching a detector at the end of the
column. /ach analyte in a sample will have a different retention time.
Dead Time (t
M
)
The dead time is the time a non0retained compound spends in the mobile phase which is
also the amount of time the non0retained compound spends in the column. The time taen
for the mobile phase to pass through the column is therefore called tM.
Adjusted Retention Time (t
R
'
)
The ad(usted retention time is the time a compound spends in the stationary phase. The
ad(usted retention time is the difference between the dead time and the retention time for
a compound.
t R and tM are easily obtained from a chromatogram. 1hen an analytes retention factor is
less than one, elution is so fast that accurate determination of the retention time is very
difficult. %igh retention factors (greater than -2) mean that elution taes a very long time.
'deally, the retention factor for an analyte is between one and five.
Selectivity Factor (alpa)
The selectivity is a ratio of the capacity factors of two peas which describes the
separation of two species (A and 3) on the column. The selectivity is always greater than
one. 'f the selectivity equals one, the two compounds cannot be separated. The higher the
selectivity, the more separation between two compounds or peas.
' k '* ) k 'A
1hen calculating the selectivity factor, species A eutes !aster than species *+
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#escription o! chromatographic coumns
The resolution of chromatographic columns is described by the teoretical plate ei!t,
", or the number o# teoretical plates, $. % and 5 provide useful measures to compare
the performance of different columns for a given analyte.
The efficiency is related to the number of compounds that can separated by the column.
The efficiency is also expressed as the number of theoretical plates (5, unitless) or as the
height equivalent to a theoretical plate (%/T&, generally in millimeters). The efficiency
increases as the height equivalent to a theoretical plate decreases, thus more compounds
can be separated by the column. The efficiency increases as the number of theoretical
plates increases, thus the column6s ability to separate two closely eluting peas increases.
The Theoretica Pate Mode o! Chromatography
The plate model supposes that the chromatographic column is contains a large number of
separate layers, called theoretical plates. "eparate equilibrations of the sample between
the stationary and mobile phase occur in these 7plates7. The analyte moves down the
column by transfer of equilibrated mobile phase from one plate to the next.
It is important to remem%er that the pates do not reay e,ist, they are a figment of
the imagination that helps us understand the processes at wor in the column.They also
serve as a way of measuring column efficiency, either by stating the number of
theoretical plates in a column, $ (the more plates the better), or by stating the plate
height, the %eight &quivalent to a Theoretical 'late (the smaller the better).
'f the length of the column is (, then the %/T& or % is
H 8 ( ) $
The number of theoretical plates that a real column possesses can be found by examining
a chromatographic pea after elution, - ' ./ 0t
R
) 12
3

where 1 is the width of the pea at its base or
where %.)3 is the pea width at half0height.
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As can be seen from this equation, columns behave as if they have different numbers of
plates for different solutes in a mixture.
Chromatographic Resoution
Although the seecti4ity !actor5 5 describes the separation of band centres, it does not
tae into account pea widths. Another measure of how well species have been separated
is provided by measurement of the resolution.
The resolution (:
s
) between two peas in a chromatogram is given by;
where delta < is the separation between peas A and 3, and 1
a
and 1
b
are the widths at
the base of peas A and 3, respectively.
Acceptable resolution is on the order of :
s
8 ).2, and baseline resolution between two
peas (as shown in the figure) requires an :
s
= ).9.
't is useful to relate the resolution to the number of plates in the column, the selectivity
factor and the retention factors of the two solutes,
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>
To obtain high resolution, the three terms must be maximised. An increase in $, the
number of theoretical plates, by lengthening the column leads to an increase in retention
time and increased band broadening 0 which may not be desirable. 'nstead, to increase the
number of plates, the height equivalent to a theoretical plate can be reduced by reducing
the si*e of the stationary phase particles.
't is often found that by controlling the capacity factor, "#, separations can be greatly
improved. This can be achieved by changing the temperature (in $as Chromatography)
or the composition of the mobile phase (in !iquid Chromatography).
The selectivity factor, , can also be manipulated to improve separations. 1hen is
close to unity, optimising "# and increasing $ is not sufficient to give good separation in a
reasonable time. 'n these cases, "# is optimised first, and then is increased by one of the
following procedures;
). Changing mobile phase composition
-. Changing column temperature
.. Changing composition of stationary phase
4. ?sing special chemical effects (such as incorporating a species which complexes
with one of the solutes into the stationary phase)
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@

The Rate Theory o! Chromatography
A more realistic description of the processes at wor inside a column taes account of the
time taen for the solute to equilibrate between the stationary and mobile phase (unlie
the plate model, which assumes that equilibration is infinitely fast). The resulting band
shape of a chromatographic pea is therefore affected by the rate of elution. 't is also
affected by the different paths available to solute molecules as they travel between
particles of stationary phase. 'f we consider the various mechanisms which contribute to
%and %roadening, we arrive at the Aan Beemter equation for plate height,
HETP ' A & ' ( u & ) u
where u is the flow rate (cm#s) or the average velocity of the mobile phase, A is /ddy
diffusion term, 3 is a longitudinal diffusion term, and C is a mass transfer term. A, *, and
+ are factors which contribute to band or *one broadening
A ( Eddy di!!usion 6 &ach molecule ta"es a different path through column
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Optimi7ation o! Coumn Per!ormance
Can be achieved by;
increasing the difference in migration rates (retention time) of components
reducing pea width (*one broadening)
d
e
t
e
c
t
o
r

s
i
g
n
a
l
Time
Criginal Chromatogram
'ncrease difference in
migration rates
:educe *one broadening
D
The mobile phase moves through the column which is paced with stationary phase.
"olute molecules will tae different paths through the stationary phase at random. This
will cause broadening of the solute band, because different paths are of different lengths.
' ( Longitudina di!!usion
The concentration of analyte is less at the edges of the band than at the center. Analyte
diffuses out from the center to the edges. This causes band broadening. 'f the velocity of
the mobile phase is high then the analyte spends less time on the column, which
decreases the effects of longitudinal diffusion.
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A 8 -dp
A is directly proportional to the pacing particle diameter, dp
is a function of pacing uniformity and column geometry
'n unpaced columns, A is *ero
At low mobile0phase velocities, the rate at which each molecule moves down the
column tends to approach that of the average. Thus the molecules are not
significantly dispersed by the multipath nature of the pacing.
At high velocities, sufficient time is not available for diffusion averaging to occur and
band broadening due to the different path lengths is observed.
Thus, A depends on the stationary particles (,ant small) and their pacing (,ant
uniform)
E
The longitudinal diffusion is the usual diffusion due to a concentration gradient, with
analyte in a band moving through a column diffusing from the center of the band forward
and bacward.
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*) 8 ' 3#
M
) 8
B
F
is smaller in liquids than in gases, so 3# G is less pronounced in !C than in $C

The obstructive factor, shows that longitudinal diffusion is hindered by the
pacing. is lower for a paced column (ie 8 2.>) than an unpaced
(capillary) column (ie 8 ))
The longitudinal diffusion effect on % is inversely proportional to G because the
solute residence time is shorter at high G and the extent of diffusion is less
3 is directly proportional to the solute diffusion coefficient in the mobile phase, B
F
)2
-olute molecules di##use to regions of lo,er solute concentration in front of . behind
zone/ inversely proportional to mobile phase flo, rate
) ( Resistance to mass trans!er
The analyte taes a certain amount of time to equilibrate between the stationary and
mobile phase. 'f the velocity of the mobile phase is high, and the analyte has a strong
affinity for the stationary phase, then the analyte in the mobile phase will move ahead of
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Fobile phase
Analyte band
Horward and bacward
diffusion in mobile phase
))
the analyte in the stationary phase. The band of analyte is broadened. The higher the
velocity of mobile phase, the worse the broadening becomes.
C 8 ' C
s
8 9 C
m
8
C
s
is a mass transfer term for the analyte in or on the stationary phase, and C
m
is a mass
transfer term for the analyte in the mobile phase
C
s
G 8 (f
"
(I)d
f
-#B
"
)u
d
f
8 film thicness of stationary phase (most important factor)
C
F
G 8 (f
F
(I)d
p
-#B
F
)G
d
:
8 diameter of support particle
J The mass0transfer effect on % is directly proportional to 8 because the solute
residence time is longer at low 8 , the deviation from equilibrium is less, and *one
broadening or % is smaller.
J C
s
G is less if the liquid stationary phase film thicness, d
f
, is smaller , or the
solute diffusion coefficient in the stationary phase, B
"
is larger.
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"o analyte molecules at the front of a band are swept ahead without
equilibrating with the stationary phase and those at the trailing edge are left
behind for a longer time in the stationary phase.
The equilibrium between
the mobile phase and
stationary phase is
established so slowly that
a chromatographic
column always operates
under non0equilibrium
conditions.
Mobile
phase
Stationary
phase S!"
Analyte
attracted to
S!
Mass(Trans!er Coe!!icients 0C
:
; C
M
2
)-
J C
F
G is less if d
p
is smaller (hence greater surface area), or the solute diffusion
coefficient in the mobile phase, B
F
is larger
Therefore, the mass transfer terms depend on the diffusion coefficients for the analyte in
the stationary and mobile phases, and can be thought of as interactions that delay the
analyte and lead to band broadening. The stationary phase mass transfer term, C
s
, also
depends on the thicness of the stationary phase, and the mobile phase mass transfer
term, C
m
, also depends on the particle si*e of the pacing material due to its effect on
eddy diffusion.
<an #eemter pots
A plot of plate height vs. average linear velocity of mobile phase.
"uch plots are of considerable use in determining the optimum mobile phase flow rate.
*lot o# " (and components) vs+ #lo% rate
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5ote that there is an optimum flow rate to obtain the minimum theoretical plate height.
=one *roadening can be reduced by using;
K smaller particles,
K narrower columns,
K lower temperature in $C,
K thinner liquid stationary phases
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H#
cm
u# cm$s
A
B$u
%u
)4