Chromatography…column type

cont…

M.phil BCH-706
Dr Sumaira Mehboob
 Liquid chromatography

• An analytical technique to separate ions or molecules that are

dissolved in a solvent.
• As it requires column so it is also known as Column Chromatography
• It require the glass column with a stopcock attached at the bottom, inserted a
cotton plug at the bottom of the column and packed the column with a slurry
of silica gel
• Once the column was packed poured the reaction mixture over the bed of
silica from the top of the column, with the aid of a glass pipette.
• The different components in the sample mixture pass through the column at
different rates due to their affinity towards mobile phase and stationary phase
• Affinity, in turn, is dictated by two properties of the molecule: ‘
• Adsorption’ and ‘Solubility’.
i. Adsorption: the property of how well a component of the mixture
sticks to the stationary phase,
ii. Solubility: the property of how well a component of the mixture
dissolves in the mobile phase.
• Higher the adsorption to the stationary phase, the slower the molecule will
move through the column.
• While Polarity” of the compounds dictates their affinities towards the
stationary and mobile phases.
• Suppose we have a mixture of two molecules A and B, where ‘A’ is a
protein and ‘B’ is a lipid.
• Our column is packed with silica, which is polar in nature;
• our mobile phase is hexane, which is non-polar in nature.
• OBSERVATIONS
i. A’, being polar in nature, will adsorb on to the polar stationary phase
(silica).
ii. ‘B’ being non-polar in nature, will readily dissolve in the non-polar
mobile phase (hexane) without adhering to silica, and will thus elute out
of the column with hexane.
Basic column chromatographic components

• The two forms of column chromatography to be discussed are:

1. liquid chromatography (LC), mainly high-performance liquid
chromatography (HPLC)
2. gas chromatography (GC)

• A typical column chromatographic system using a gas or liquid mobile

phase consists of the following components:
Analyte development and elution
• Analyte development and elution relates to
• “the separation of the mixture of analytes applied to the stationary
phase by the mobile phase and their elution from the column”

• Elution modes can be of following types:

A. Zonal development
B. Affinity development or displacement
In zonal development,
• the analytes in the sample are separated on the basis of their distribution
coefficients between the stationary and mobile phases.
• The sample is dissolved in a suitable solvent and applied to the stationary
phase as a narrow, discrete band.
• The mobile phase is then allowed to flow continuously over the stationary
phase, resulting in the progressive separation and elution of the sample
analytes.
• Types of zonal development
i. If the composition of the mobile phase is constant as in GC and some forms of
HPLC, the process is said to be isocratic elution.
ii. To facilitate separation however, the composition of the mobile phase may be
gradually changed, for example with respect to pH, salt concentration or polarity.
This is referred to as gradient elution.
• The composition of the mobile phase may be changed continuously or in a
stepwise manner.
In displacement or affinity development
• that is confined to some forms of HPLC the analytes in the sample are separated on
the basis of their affinity for the stationary phase.
• The sample of analytes dissolved in a suitable solvent is applied to the stationary
phase as a discrete band.
• The analytes bind to the stationary phase with a strength determined by their affinity
constant for the phase.
• The analytes are then selectively eluted by using a mobile phase containing a
specific solute that has a higher affinity for the stationary phase than have the
analytes in the sample.
• Thus, as the mobile phase is added, this agent displaces the analytes from the
stationary phase in a competitive fashion,
Result:
• repetitive binding and displacement of analyte along the stationary phase and
eventual elution from the column in the order of their affinity for the stationary
phase,
• the analyte with the lowest affinity being eluted first.
 CHROMATOGRAPHIC PERFORMANCE PARAMETERS

• The successful chromatographic separation of analytes in a mixture

depends upon the
1. selection of the most appropriate process of chromatography
• This involves the basic mechanisms defining the chromatographic process
such as
• adsorption,
• partition,
• ion exchange,
• ion pairing and
• molecular exclusion
2. optimisation of the experimental conditions associated with the
separation.
• optimisation requires
• an understanding of the processes that are occurring during the development
• elution,
• calculation of a number of experimental parameters
• characterising the behaviour of each analyte in the mixture

The factors involves in optimization are

diffusion, which tend to oppose the separation and which result in non-ideal
behaviour of each analyte

These processes are manifest as a broadening and tailing of each analyte band.
3. Retention time
• A chromatogram is a pictorial record of the detector response as a function
of elution volume or retention time.
• It consists of a series of peaks or bands, ideally symmetrical in shape,
representing the elution of individual analytes

• The retention time tR for each analyte has two components

i. The first is the dead time, tM time
it takes the analyte molecules to pass through the free spaces between the
particles of the matrix coated with the stationary phase.
• the volume of the free space is referred to as the column void volume, V0
• the value of tM will be the same for all analytes
• it is the time where analyte does not interact with the stationary phase but simply
spends all of the elution time in the mobile phase travelling through the void volume.
ii. adjusted retention time, t ︡ R
• in which the stationary phase retains the analyte.
• this time is characteristic of the analyte and is the difference between the
observed retention time and the dead time.
4. Retention factor
• retention factor, k
• It is simply the additional time that the analyte takes to elute from the
column relative to an unretained or excluded analyte that does not
interact with the stationary phase and which, by definition, has a k
value of 0.
5. Resolution (RS)
• It is the ratio of the difference in retention time (tR) between the two
peaks (tRA and tRB) to the mean (wav) of their base widths (wA and wB):
6. Qualitative and quantitative analysis

• Chromatographic analysis can be carried out on either a qualitative or

quantitative basis.
Quantification of a given analyte is based on
• the construction of a calibration curve obtained using a pure, authentic sample of
the analyte.

• Most commonly the calibration curve is based on the use of relative peak areas
obtained using an internal standard
• the standard must be carefully chosen it should have
a. similar physical and structural characteristics to those of the test analyte,
b. and it should be an isomer or structural analogue of the analyte.
c. it should have a retention time close to that of the analyte
d. its resolution should be greater than 99.5%.