WEEK 5: ANALYSIS OF LIQUID CHROMATOGRAPHY

INTRODUCTION
Liquid chromatography is a technique used to separate a sample into its individual parts. This
separation occurs based on the interactions of the sample with the mobile and stationary phases.
Because there are many stationary/mobile phase combinations that can be employed when separating
a mixture, there are several different types of chromatography that are classified based on the
physical states of those phases. Liquid-solid column chromatography, the most popular
chromatography technique and the one discussed here, features a liquid mobile phase which slowly
filters down through the solid stationary phase, bringing the separated components with it.

EXPLORE
History of Liquid Chromatography
The first known chromatography is traditionally attributed to Russian botanist Mikhail Tswett who
used columns of calcium carbonate to separate plant compounds during his research of chlorophyll.
This happened in the 20th century (1901). Further development of chromatography occurred when
the Nobel Prize was awarded to Archer John Porter Martin and Richard Laurence Millington Synge
in 1952. They were able to establish the basics of partition chromatography and also develop Plate
theory.

LIQUID CHROMATOGRAPHY
Liquid chromatography (LC) is a
separation technique in which the mobile phase is a liquid, where sample ions or molecules are
dissolved.

It is carried out either in a column or a plane. The sample with the mobile liquid will pass through
the column or the plane, which is packed with a stationary phase composed of irregularly or
spherically shaped particles. Due to the differences in ion-exchange, adsorption, partitioning, or size,
different solutes will interact with the stationary phase to different degrees, and therefore the
separation of the compounds can be achieved and the transit time of the solutes through the column
can be determined by utilising these differences.

Conventional LC is commonly used in preparative scale work to purify and isolate some components
of a mixture. Nowadays liquid chromatography generally utilizes very small packing particles and a
relatively high pressure for analytical separations of solutions, detection & quantification, referred to

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as high performance liquid chromatography (HPLC). HPLC can provide a very high resolution (up
to parts per trillion) and a fast analysis time.

General Scheme

Components within a mixture are separated in a column based on each component's affinity for the
mobile phase. So, if the components are of different polarities and a mobile phase of a distinct
polarity is passed through the column, one component will migrate through the column faster than
the other. Because molecules of the same compound will generally move in groups, the compounds
are separated into distinct bands within the column. If the components being separated are colored,
their corresponding bands can be seen. Otherwise as in high performance liquid chromatography
(HPLC), the presence of the bands are detected using other instrumental analysis techniques such as
UV-VIS spectroscopy1.

Other Varieties of Liquid Chromatography

 Partition Chromatography: In this method, both the stationary phase and the mobile phase
are liquid. The stationary phase liquid would be an immiscible liquid with the mobile phase.
 Liquid-Solid Chromatography

- This method is similar to partition chromatography only that the stationary phase has been
replaced with a bonded rigid silica or silica based component onto the inside of the column.
Sometimes the stationary phase may be alumina.

- The analytes that are in the mobile phase that have an affinity for the stationary phase will be
adsorbed onto it and those that do not will pass through having shorter retention times. Both normal
and reverse phases of this method are applicable.

 Ion Exchange or Ion Chromatography

- This is a type of chromatography that is applied to separate and determine ions on columns that
have a low ion exchange capacity.

- This is based on the equilibrium of ion exchange between the ions in solution and the counter
ions to pair with the oppositely charged ions thatare fixed to the stationary phase.

 Size Exclusion Chromatography

- Size exclusion chromatography separates molecules by their size. This is done by having the
stationary phase be packed with small particles of silica or polymer to form uniform pores.

- The smaller molecules will get trapped in the silica particles and will elude from the column at
a rate that is greater than that of larger molecules. Thus, the retention time depends on the size of the
molecules. Larger molecules will be swept away in the mobile phase, therefore having a smaller
retention time.

- Also notice that in this type of chromatography there isn’t any interaction, being physical or
chemical, between the analyte and the stationary phase.

 Affinity Chromatography

- This type of chromatography involves binding a reagent to the analyte molecules in a sample.

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- After the binding, only the molecules that have this ligand are retained in the column, the
unbound analyte is passed through in the mobile phase.

 Chiral Chromatography

- Chiral chromatography enables the use of liquid chromatography to separate a racemic mixture
into its enantiomeric parts.

- A chiral additive can be added to the mobile phase, or a stationary phase that has chiral
properties can be used. A chiral stationary phase is the most popular option.

- The stationary phase has to be chiral in order to recognize the chirality of the analyte, this will
create attractive forces between the bonds and also form inclusion complexes.

Liquid Chromatography Workflow

Regardless of the interactions that are being exploited, liquid column chromatography is carried out
in six steps:

· Column equilibration

· Sample loading

· Washing

· Elution

· Final column washing

· Column regeneration

Column Equilibration

Most liquid chromatography protocols begin with a resin equilibration step. A buffer that is
compatible with the protein of interest and the resin of choice is passed over the column. A common
practice is to equilibrate the column with 5–10 column volumes (CVs) of equilibration buffer.

For example, binding of proteins to hydrophobic interaction resins is most efficient at high ionic
strength. Prior to sample application, the resin is therefore equilibrated in a buffer of high ionic
strength.

The properties of the protein of interest are also considered in equilibration buffer selection, as buffer
factors such as ionic strength are limited by protein stability; typically, one would avoid equilibration
buffer conditions that would denature the protein of interest or prevent it from interacting with the
stationary phase.

Sample Loading

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After equilibration, the sample is loaded onto the column. The sample is generally loaded in a buffer
with the same composition as the equilibration buffer to maximize protein interaction with the
stationary phase.

Column Washing

Once proteins have been immobilized on the stationary phase, proteins that interact only weakly or
nonspecifically with the resin are removed by washing the column with several column volumes of
wash buffer. This wash buffer can have the same composition as the equilibration buffer or contain
components that disrupt weak specific interactions.

For example, immobilized-metal affinity chromatography (IMAC) elutes proteins bound to the resin
with a high concentration of immidazole. A common practice is to use a wash buffer that includes an
intermediate concentration of immidazole to eliminate contaminating proteins that are only weakly
bound to the resin.

The column is washed until no protein is detected in the eluate. When using a chromatography
system with a UV detector, the column is washed until the 280 nm absorption reading returns to the
baseline.

Sample Elution

After all nonspecifically and weakly interacting proteins have been washed off of the resin, proteins
that interact strongly with the resin are eluted from the column by changing the composition of the
buffer that is passed over the resin.

In ion exchange chromatography, proteins are eluted with high–ionic-strength buffers or with a
change in pH to disrupt the electrostatic interactions that immobilized the protein of interest.

Final Column Washing

After the protein of interest has been eluted from the resin, any proteins that remain bound to the
resin are eluted by increasing the strength of the elution buffer. This step permits columns to be
reused for future separations.

Column Regeneration

After stripping the remaining compounds bound to the media, the column is then either saturated
with equilibration buffer for subsequent reuse or filled with a storage buffer.

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