Liquid Chromatography Principles
Liquid chromatography (LC) is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a solvent.
Chromatography is used to separate proteins, nucleic acids, or small molecules in complex
mixtures. Liquid chromatography (LC) separates molecules in a liquid mobile phase using a
solid stationary phase. Liquid chromatography can be used for analytical or preparative
applications. Here, we restrict our discussion to column liquid chromatography techniques and
considerations.
Related Topics: Chromatography, Column Chromatography Methods and Instrumentation
Page Contents
Liquid Chromatography Technique
Liquid Chromatography Workflow
Liquid Chromatography Considerations
Related Content
Liquid Chromatography Technique
In column liquid chromatography, as the liquid mobile phase passes through the column,
components in the mobile phase interact to varying degrees with the solid stationary phase, also
known as the chromatography media or resin. Molecules of interest in the mobile phase are
separated based on their differing physicochemical interactions with the stationary and mobile
phases.
These interactions can be based on molecular size (size exclusion chromatography), charge
(ion exchange chromatography), hydrophobicity (hydrophobic interaction
chromatography), specific binding interactions (affinity chromatography), or a combination
of these (multimodal or mixed-mode chromatography).
The compostion of the mobile phase is typically changed during a separation run so as to alter
the strengths of the interactions of the compounds of interest, that is, to change the phase
partitioning of each compound between the stationary and mobile phases. Each compound then
elutes from the column in a particular order depending on the relative strengths of its interaction
with the resin and the mobile phase.
As the mobile phase continues to flow through the column, the column effluent, or eluate, is
typically collected in fractions while monitoring the concentrations of the compounds eluted
from the column over time to yield an elution curve, or chromatogram. The mode of detection
varies with the analyte to be detected. For protein separations by column chromatography,
protein concentration can be monitored manually using a dye-based protein assay such as the
Bradford assay; however, such manual monitoring is labor intensive.
More commonly, a UV detector or spectrophotometer is attached to the chromatography system
to continuously monitor protein elution from the column by measuring light absorption at 280
nm (A280) by the amino acid tryptophan. The resulting chromatogram is then analyzed to
quantitate proteins in the eluate. Each distinct peak represents a unique component resolved by
the column, and the area under the curve corresponds to the amount of that compound eluted
from the column. It is important to note that a single peak may contain more than one protein
species; therefore, further analysis of the eluted fractions may be required, for example, by gel
electrophoresis.
A typical chromatogram displays an initial broad peak of eluted protein that interacts weakly
with the resin or, in some cases, not at all. In most cases, the column is washed with binding
buffer until this first flowthrough peak (labeled “Binding Conditions” in Figure 1) has
completely eluted, and the A280 reading returns to baseline.
Fig. 1. Example chromatogram showing a linear gradient elution.
Proteins that interact strongly with the resin are then eluted by changing the composition of the
elution buffer. The specific composition of the elution buffer depends on the physicochemical
properties of the molecules to be separated and the chromatography media used, often referred to
as different media chemistries. Ion exchange chromatography, for example, which relies on
interactions based on the net charges of the molecules, uses elution buffers of increasing ionic
strength.
The red line in Figure 1 shows a linear gradient elution, but proteins can also be eluted using
stepwise isocratic elution, where buffer conditions are changed in a stepwise fashion. By
altering the elution gradient, flow rate, column length, and resin particle size, the protein
separation ability (or resolution) of a given resin can be changed. Chromatography methods are
optimized to yield sharp, tall peaks that are well separated (Figure 2).
Fig. 2. Optimization of resolution by changing flow rate. Optimal peak separation is achieved
at a flow rate of 229 cm/hr.
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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
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.
Sample can be loaded manually or using a sample pump. Some types of chromatography limit
the volume of sample that can be loaded onto the column. Another important sample loading
consideration is that most resins have a finite capacity to bind protein; overloading a column by
applying too much sample can adversely affect separation.
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.
Proteins bound to a hydrophobic interaction resin, conversely, are eluted by lowering the ionic
strength of the buffer. In affinity chromatography, proteins are commonly eluted from the
column by the introduction of a competing ligand or by cleaving the affinity tag and may also be
eluted using high-salt buffers or altering pH. Other elution protocols may involve mixing
solvents of varying polarity to tune the solubility of each component in the mobile phase.
Fig. 3: Example buffer composition diagrams for isocratic (top) and gradient (bottom)
elution protocols.
Elution conditions can either be changed in a linear gradient fashion or in a stepwise fashion.
Often, a gradient elution protocol, in which the composition of the mobile phase changes
linearly over time, is chosen to determine the elution profile and the elution buffer concentration
at which the protein of interest is freed from the resin. Once this concentration has been
determined, to save time, a stepwise isocratic elution protocol, in which the composition of the
mobile phase is constant at each step, can be designed for future purifications.
Note: Size exclusion chromatography does not require buffer changes since it does not depend
on specific interactions between the mobile phase and the stationary phase. There are no true
wash and elution steps, as SEC relies solely on the fact that large molecules are retarded by
porous beads, whereas small molecules pass through the resin with minimal resin interaction.
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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Liquid Chromatography Considerations
Four factors are important when designing protein purification workflows: resolution, yield,
sample integrity, and sample purity.
Resolution refers to the separation of peaks in a chromatogram. The purpose of chromatography
is to separate molecules of interest. Resolution is affected by the selective properties of the resin,
by equilibration, wash, and elution buffer composition, flow rate, and by the sample volume.
Compounds that elute as distinct peaks with a particular column and elution protocol may co-
elute as a single peak with another chromatographic technique.
If the goal of chromatographic separation is purification of a protein of interest for downstream
applications (that is, preparative chromatography), then yield, defined as the amount of the
desired protein fraction recovered, is an important consideration.
Sample integrity is another key consideration for preparative chromatography. Applications
such as crystallography require full-length, correctly folded protein. If activity of the protein is to
be assessed in vitro, the purified protein must retain its enzymatic activity. Buffer choice, the
addition of appropriate protease inhibitors, and speed are common, but not always sufficient,
measures to maintain sample integrity.
Lastly, sample purity is an important consideration. In the case of co-eluting compounds, the
detection of a single peak in a chromatogram does not ensure pure sample. It is therefore
necessary to assess sample purity by gel electrophoresis (SDS-PAGE) or other analytic
techniques.
When developing a purification workflow it is wise to consider the sample purity that is required
for the intended downstream applications because sample purity, integrity, and yield often
display an inverse relationship. For example, a five-column workflow may yield exquisitely pure
protein, but because of the length of time required to separate the protein of interest from
contaminating proteins and proteases, the protein may be completely inactive. In addition, since
some of the protein of interest is lost at each column fractionation step, the total amount of
protein recovered after five columns may be insufficient for the desired downstream
applications.
Absolute sample purity is essential in certain applications such as antibody production for
diagnostic or therapeutic applications. Some enzymatic studies, however, may require only
functional purity; proteins that do not interfere with or enhance the protein of interest’s activity
may be tolerated as contaminants to maximize sample integrity and yield.
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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.
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.
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. The following figure shows the migration of
two components within a mixture:
In the first step, the mixture of components sits atop the wet column. As the mobile phase passes
through the column, the two components begin to separate into bands. In this example, the red
component has a stronger affinity for the mobile phase while the blue component remains
relatively fixed in the stationary phase. As each component is eluted from the column, each can
be collected separately and analyzed by whatever method is favored. The relative polarities of
these two compounds are determined based on the polarities of the stationary and mobile phases.
If this experiment were done as normal phase chromatography, the red component would be less
polar than the blue component. On the other hand, this result yielded from reverse phase
chromatography would show that the red component is more polar than the blue component.
Column Chromatography
The stationary phase in column chromatography is most typically a fine adsorbent solid; a solid
that is able hold onto gas or liquid particles on its outer surface. The column typically used in
column chromatography looks similar to a Pasteur pipette (Pasteur pipettes are used as columns
in small scale column chromatography). The narrow exit of the column is first plugged with
glass wool or a porous plate in order to support the column packing material and keep it from
escaping the tube. Then the adsorbent solid (usually silica) is tightly packed into the glass tube to
make the separating column. The packing of the stationary phase into the glass column must be
done carefully to create a uniform distribution of material. A uniform distribution of adsorbent is
important to minimize the presence of air bubbles and/or channels within the column. To finish
preparing the column, the solvent to be used as the mobile phase is passed through the dry
column. Then the column is said to be "wetted" and the column must remain wet throughout the
entire experiment. Once the column is correctly prepared, the sample to be separated is placed at
the top of the wet column. A photo of a packed separating column can be found in the links.
Components
Chromatography is effective because different components within a mixture are attracted to
the adsorbent surface of the stationary phase with varying degrees depending on each
components polarity and its unique structural characteristics, and also its interaction with the
mobile phase. The separation that is achieved using column chromatography is based on factors
that are associated with the sample. So, a component that is more attracted to the stationary phase
will migrate down the separating column at a slower rate than a component that has a higher
affinity for the mobile phase. Also, the efficacy of the separation is dependent on the nature of
the adsorbent solid used and the polarity of the mobile phase solvent.
Stationary Phase
The type of adsorbent material used as the stationary phase is vital for efficient separation of
components in a mixture. Several different solid may be employed. Adsorbent material can be
chosen based on particle size and activity of the solid. The activity of the adsorbent is
represented by its activity grade, which is a measure of an adsorbent's attraction for solutes in the
sample solution. The solids with the highest activity grading are those that are completely
anhydrous. Silica gel and alumina are among the most popular adsorbents used. Alumina caters
well to samples that that require specific conditions to adequately separate. However, the use of
non-neutral stationary phases should be done with great caution, an increase or decrease of pH in
the alumina stationary phase may allow chemical reactions within the components of the
mixture. Silica gel, however, is less active than alumina and can generally be used as an all-
around adsorbent for most components in solution. Silica is also preferred because of its high
sample capacity, making it one of the most popular adsorbent materials.
Mobile Phase
The proper mobile phase must also be chosen for the best separation of the components in an
unknown mixture. This eluent will be chosen based on its polarity relative to the sample and
the stationary phase. With a strong polar adsorbent stationary phase like alumina, a polar solvent
used as the mobile phase will be adsorbed by the stationary phase, which may displace molecules
of sample in the mixture and may cause the sample components to elute vary quickly. This will
provide little separation of the sample, so it is best to start elution with a solvent of lower polarity
to elute the components that are weakly adsorbed to the stationary phase first. The solvent
may also be changed during separation in order to change the polarity and therefore elute the
various components separately in a more timely manner. This method is very similar to the
gradient method of separation used in High Performance Liquid Chromatography (HPLC).
Types of Chromatography
Normal Phase Chromatography
The components in a mixture will elute at different rates depending on each one's polarity
relative to the next. When the column to be used for the separation is more polar than the mobile
phase, the experiment is said to be a normal phase method. In normal phase chromatography,
the stationary phase is polar, and so the more polar solutes being separated will adhere more to
the stationary adsorbent phase. When the solvent or gradient of solvents is passed through the
column, the less polar components will be eluted faster than the more polar ones. The
components can then be collected separately, assuming adequate separation was achieved, in
order of increasing polarity. This method of chromatography is not unique to liquid-solid
column chromatography and is often used when performing High Performance Liquid
Chromatography (HPLC). Although HPLC is an example of liquid-liquid chromatography, in
which both the stationary and mobile phases are liquid, normal phase elution is achieved by
coating the solid adsorbent column with a polar liquid.
Reverse Phase Chromatography
In reverse phase chromatography, the polarities of the mobile and stationary phases are opposite
to what they were when performing normal phase chromatography. Instead of choosing a non-
polar mobile phase solvent, a polar solvent wil be chosen. Or, if the experiment requires a
solvent polarity gradient, the gradient must be carried out with the most polar solvent first and
the least polar solvent last (reverse order of normal phase chromatography). Common polar
solvents mixtures of solvents include water, methanol, and acetonitrile. It is slightly more
difficult and expensive to obtain a column where the stationary phase is non polar, as all solid
adsorbents are polar by nature. The non polar stationary phase can be prepared by coating
silanized silica gel with a non polar liquid. Silanizing the silica gel reduces the silica gel's ability
to adsorb polar molecules. Common non polar liquid phases include silicone and various
hydrocarbons. An alternative to this type of column is used in HPLC, in which a bonded liquid
phase is used as the stationary phase. The less polar liquid is chemically bonded to the polar
silica gel in the column. So using reverse phase, the most polar compounds in the sample
solution will be eluted first, with the components following having decreasing polarities.
Flash Chromatography
Because the elution rate of the mobile phase in regular column chromatography as described
above is controlled primarily by gravity, chromatographic runs can potentially take a very long
time to complete. Flash chromatography is a modified method of column chromatography in
which the mobile phase moves faster through the column with the help of either pressurized air
or a vacuum. A vacuum line is attached to the bottom of the separating column, this pulls the
mobile phase solvent, and the components in the mobile phase, through the column at a faster
rate than gravity does. A figure of this set-up can be seen in the links section. Flash
chromatography is powered by compressed air or air pumps works by pushing the mobile phase
through the column and achieves faster flow rates of the mobile phase just as vacuum facilitated
flash chromatography does. For this method, a pressurized air line is attached to the top of the
separating column. It is for this reason that flash chromatography is also referred to as medium
pressure chromatography. An inert gas is used as to not interact with the mobile or stationary
phase or the component mixture. Nitrogen gas is commonly used for this method of
chromatography. Many instruments are available to perform flash chromatography as efficiently
as possible: expensive columns, pumps, and flow controllers. This maintains a constant and
precise air pressure or vacuum to the column in order to obtain steady flow rate of the mobile
phase and favorable separation of the samples in solution. However, less expensive alternatives
are available, as flow controllers can be made so that pressurized air can be used to facilitate
flash chromatography:
By using the above apparatus, purchasing expensive air pumps can be avoided. This method is
useful to an extent. Since the flow rate of the pressurized gas is controlled manually by the flow
rate controller, it is more difficult to quantify the flow rate and keep that flow rate constant.
Instruments available for flash chromatography are able to set flow rates digitally and keep flow
rate constant.
Flash chromatography is similar to HPLC in that the mobile phase is moved through the column
by applying pressure to the solvent in order to achieve a quicker result. However, in flash
chromatography, only medium pressure is applied to the system within the solution. In HPLC,
pressures as high as 5000 psi can be applied in the column by high performance pumps.
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 equilibria of ion exchange between the
ions in solution and the counter ions to pair with the oppositely charged ions that are fixed to the
stationary phase. This sttionary phase would either have positive of negative functional groups
affixed to it, usually sulfonate (-SO3-) or a quaternary amine (-N(CH3)3
+), being a cation and
anion exchanger respectively.
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.
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. The stationary phase is usually agrose or
a porous glass bead that is able to immobilize the bonded molecule. It is possible to change the
elution conditions by manipulating the pH or the ionic strength of the binding ligand. This
method is often used in biochemistry in the purification of proteins. The ligand tag is bonded and
after separation the tag is then removed and the and the pure protein is obtained.
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.
Plate Theory and Rate Theory
Plate theory and Rate theory are two theories that are applicable to chromatography. Plate theory
describes a chromatography system as being in equilibrium between the stationary and mobile
phases. This views the column as divided into a number of imaginary theoretical plates. This is
significant because as the number of plates in a column increases or the height equivelant
theoretical plates or HETP increases, so does the separation of components. It also provides an
equation that describes the elution curve or the chromatogram of a solute it can also be used to
find the volume and the column efficiency.
HETP = L/N ; where L= column length and N= number of theoretical plates
The Rate theory on the other hand describes the migration of molecules in a column. This
included band shape, broadening, and the diffusion of a solute. Rate theory follows the Van
Deemter equation, which is the most appropriate for prediction of dispersion in liquid
chromatography columns. It does this by taking into account the various pathways that a sample
must travel through a column. Using the Van Deemter equation, it is possible to find the
optimum velocity and and a minimunm plate height.
H=A+Bu=Cu
Where:
A = Eddy-Diffusion, B = Longitudinal Diffusion, C = mass transfer, u = linear velocity
Instrumentation
This schematic is of the basic instrumentation of a liquid-solid chromatograph. The solvent inlet
brings in the mobile phase which is then pumped through the inline solvent filter and passed
through the injection valve. This is where the mobile phase will mix with the injected sample. It
then gets passed through another filter and then passed through the column where the sample
will be separated into its components. The detector detects the separation of the analytes and the
recorder, or usually a computer will record this information. The sample then goes through a
backpressure filter and into waste.
A basic LC system consists of (a) a solvent inlet filter, (b) pump, (c) inline solvent filter,
(d) injection valve, (e) precolumn filter, (f) column, (g) detector, (h) recorder, (i) backpressure
regulator, and a (j) waste reservoir.
Advantages / Disadvantages
Liquid-solid column chromatography is an effective separation technique when all appropriate
parameters and equipment are used. This method is especially effective when the compounds
within the mixture are colored, as this gives the scientist the ability to see the separation of the
bands for the components in the sample solution. Even if the bands are not visible, certain
components can be observed by other visualization methods. One method that may work for
some compounds is irradiation with ultraviolet light. This makes it relatively easy to collect
samples one after another. However, if the components within the solution are not visible by any
of these methods, it can be difficult to determine the efficacy of the separation that was
performed. In this case, separate collections from the column are taken at specified time
intervals. Since the human eye is the primary detector for this procedure, it is most effective
when the bands of the distinct compounds are visible.
Liquid-solid column chromatography is also a less expensive procedure than other methods of
separation (HPLC, GC, etc.). This is because the most basic forms of column chromatography do
not require the help of expensive machinery like high pressure solvent pumps used in HPLC. In
methods besides flash chromatography, the flow of the mobile phase, the detection of each
separation band, and the collection of each component, are all done manually by the scientist.
Although this introduces many potential instances of experimental error, this method of
separation can be very effective when done correctly. Also, the glass wear used for liquid-solid
column chromatography is relatively inexpensive and readily available in many laboratories.
Burets are commonly used as the separating column, which in many cases will work just as well
as an expensive pre-prepared column. For smaller scale chromatography, Pasteur pipettes are
often used.
Flash chromatography has the potential to be more costly than the previous methods of
separation, especially when sophisticated air pumps and vacuum pumps are needed. When these
pieces of machinery are not needed, however, a vacuum line can be instead connected to an
aspirator2 on a water faucet. Also, home-made pressurized air flow controllers can be made as
shown previously.
Definition of Liquid chromatography (LC)
Liquid chromatography (LC) is an analytical chromatographic technique that is useful for
separating ions or molecules that are dissolved in a solvent. If the sample solution is in contact
with a second solid or liquid phase, the different solutes will interact with the other phase to
differing degrees due to differences in adsorption, ion-exchange, partitioning , or size. These
differences allow the mixture components to be separated from each other by using these
differences to determine the transit time of the solutes through a column. Instrumentation Simple
liquid chromatography consists of a column with a fritted bottom that holds a stationary phase in
equilibrium with a solvent. Typical stationary phases (and their interactions with the solutes) are:
solids (adsorption), ionic groups on a resin (ion-exchange), liquids on an inert solid support
(partitioning), and porous inert particles (size-exclusion). The mixture to be separated is loaded
onto the top of the column followed by more solvent. The different components in the sample
mixture pass through the column at different rates due to differences in their partioning behavior
between the mobile liquid phase and the stationary phase. The compounds are separated by
collecting aliquots of the column effuent as a function of time.
Schematic of a simple liquid chromatographic separation
Conventional LC is most commonly used in preparative scale work to purify and isolate some
components of a mixture. It is also used in ultratrace separations where small disposable columns
are used once and then discarded. Analytical separations of solutions for detection or
quantification typically use more sophisticated high-performance liquid chromatography
instruments. HPLC instruments use a pump to force the mobile phase through and provide higher
resolution and faster analysis time.
Book Description:
The versatility of liquid chromatography (LC) allows its applicability to countless areas. From the quality
control of various industries, such as pharmaceutical, alimentary or chemistry, passing through health,
environmental, toxicological or forensic activities, and also in the area of genetics or R&D; liquid
chromatography applications are used worldwide. This book presents key support for everyone that
works or intends to work in analytical fields, from students to senior researchers.
The principles of liquid chromatography, the new fluorinated stationary phases or how to achieve
robustness, are examples of fundamental liquid chromatography issues that are discussed in the book.
Furthermore reviews about the latest developments on the LC-MS/MS determination of antibiotic
residues in food-producing animals or of emerging pollutants in environmental samples are presented,
as well as liquid chromatography applications for the determination of vitamin E isomers in foods.
Preparative liquid chromatography is also discussed, as is the role of liquid chromatography to evaluate
food authenticity, namely milk and dairy products.
Last but not least, metabolomic and proteomic analysis, as well as serendipity are important issues that
also benefit liquid chromatography utilization. The present book is truly innovating and, certainly, will be
an important tool for those that are engaged in analytical science in all of the different areas of interest.
types
There are a variety of types of liquid chromatography. There is liquid adsorption
chromatography in which an adsorbent is used. This method is used in large-scale applications
since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is
analogous to gas-liquid chromatography. The three types that will be considered here fall under
the category of modern liquid chromatography. They are reverse phase, high performance and
size exclusion liquid chromatography, along with supercritical fluid chromatography.
Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low
polarity stationary phase which is chemically bonded to an inert solid such as silica. The
separation is essentially an extraction operation and is useful for separating non-volatile
components.
High performance liquid chromatography (HPLC) is similar to reverse phase, only in this
method, the process is conducted at a high velocity and pressure drop. The column is shorter and
has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.
Size exclusion chromatography, also known as gel permeation or filtration chromatography does
not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of
separating large molecules from smaller ones. The larger molecules elute first since they cannot
penetrate the pores. This method is common in protein separation and purification.
Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier
is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids,
supercritical fluids have solubilities and densities have as large, and they have diffusivities and
viscosities quite a bit larger. This type of chromatography has not yet been implemented on a
large scale.
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column
packed with solid particles which may or may not be
coated with another liquid.
With the proper solvents, packing conditions, some
components in the sample will travel the column
more slowly than others resulting in the desired
separation.
The 4 basic liquid chromatography modes are named according to t
he mechanism
of separation involved:
1.
Liquid/Solid Chromatography
(adsorption chromatography)
2.
Liquid/Liquid Chromatography
(partition chromatography)
3.
Ion Exchange Chromatography
4.
Gel Permeation Chromatography
(exclusion chromatography)
FOUR BASIC LIQUID CHROMA
Liquid Chromatography Mohammad Azam Mansoor, Central Hospital in Rogaland, Stavanger, Norway
Liquid chromatography, also called high-performance liquid chromatography, is a popular quantitative analytical technique applied in many areas of chemical, biomedical and pharmaceutical sciences. Liquid chromatography currently accounts for about 60% of separation technology applied in life sciences around the world.
Introduction Chromatographic separations in liquid chromatography
(LC) (except size exclusion chromatography) are the
consequences of interactions developed between the
functional groupsofsolute molecules,solventmolecules
andthestationaryphase.TheinteractionspresentinLC
arehydrogenbonding,vanderWaalsforcesandelectro-
staticforces,andthemodesofLCareclassifiedaccording
to the nature of these interactions. The mode of
chromatography best suited for a particular separation
depends upon molecular mass, polarity and ionic char-
actersofasolute.
Modes of Liquid Chromatography Different modes of liquid chromatographyhave evolved
foranalysesofavarietyofcompoundsindiversetypesof
matrices. The modes of liquid chromatography include:
normal-phaseliquidchromatography(NPLC),reversed-
phase liquid chromatography (RPLC), ion-exchange
liquid chromatography (IELC) and size-exclusion chro-
matography (SELC). Selection of a liquid chromato-
graphic mode for a particular analysis also requires
selectionofacolumn(stationaryphase)andsolventsfor
the mobile phase. Whichever mode is selected for devel-
opmentofaparticularmethod,itisimportantthatcertain
criteriaformethodvalidationarefulfilled.
Normal-phase liquid chromatography (NPLC) InNPLCoradsorptionchromatography,thestationary
phaseisapolarsolidadsorbentbasedonparticlesofsilica
gel,aluminaorcarbon.Silicagelandcarbonparticlesare
also modified covalently with polar groups, for instance
aminopropyl(–NH 2
),cyanopropyl(–CN)anddiol(2OH)
functional groups. The adsorbent particles may be fully
porousorsphericalglassbeadscoveredwithanirregular
layerofsilicagel.
Themobilephasein NPLCis composedofnonpolar,
organic solvents (dehydrated), for example methanol,
ethanol, 2-propanol, acetonitrile, ethyl acetate, tetrahy-
drofuran,carbontetrachlorideorhexane,orabinaryora
tertiary solvent system based on two or three nonpolar
organicsolvents.
InNPLC,polarinteractionsareexhibitedbythepolar
functionalgroups(–OH,–NH 2
,etc.)onsolutemolecules
and polar groups present on the adsorbent molecules.
Solventmoleculesalsocompetewithsolutemoleculesto
form interactions with adsorbent molecules (for adsorp-
tion sites on the stationary phase). Solutes are eluted in
order of increasing polarity; thus retention of solute
moleculesdecreaseswithincreasingpolarityofasolvent
inamobilephase.
Thecompositionofamobilephasecanbeoptimizedfor
aparticularseparationbyselectingsuitablesolventswith
‘therightsolventstrength’.Thesolventstrengthparameter
(
e o
) for
n
-pentane is 0; those for other organic solvents
increasingintheordermethanol
4
ethanol
4
2-propano-
l
4
acetonitrile
4
ethyl acetate
4
tetrahydrofuran
4
car-
bontetrachloride
4
hexane.
ThesurfaceofsilicagelusedinNPLCiscoveredwith
freelowenergylevelhydroxylgroups(OH),(Si–OH)and
reactive hydroxyl groups with high energy level (HOH),
(Si–OH).TheHOHarestrongbondingagentsandadsorb
bothpolarcomponentsofasoluteandwatertothegel.
Thisdoubleactivityofthehydroxylgroupisresponsible
forbroadsplitpeaks.Toavoidthisproblem,wateroran
alcoholisaddedinthemobilephasetodeactivatehydroxyl
groupsonthegel.
Reversed-phase liquid chromatography (RPLC) Reversed-phaseLCdiffersfromNPLCinthatitisbased
onanonpolarstationaryphase.Themostpopularcolumn
packing material is octadecylsilyl silica (ODS-C18), in
whichsilicaiscovalentlymodifiedbyC 18
functionalgroup.
Octadecyl(C 18
)octyl(C 8
),hexyl(C 6
),propyl(C 3
),ethyl
(C 2
),methyl(C 1
),phenylandcyclohexylfunctionalgroups
bondedtosilicasurfacerendersilica(stationarysurface)
nonpolarandhydrophobic.Morethan250reversed-phase
column packing materials have been introduced since
1970.
InRPLCthemobilephaseismorepolarthanstationary
phase;waterandwater-miscibleorganicsolventssuchas
Article Contents
Introductory article .
Introduction .
Modes of Liquid Chromatography .
Derivatization .
Applications .
Limits and Possibilities
1 ENCYCLOPEDIA OF LIFE SCIENCES / &
2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
methanol,acetonitrileandtetrahydrofuranarecommonly
used.
Since most organic molecules have some nonpolar
regions, retention in RPLC arises because water in the
mobile phase repels the nonpolar regions of solute
molecules and facilitates their interaction with the non-
polarfunctionalgroupsofthesilica(stationaryphase).
Solute molecules are eluted in order of increasing
hydrophobicityordecreasingpolarity.
Separation of ionic species by RPLC Severalapproacheshavebeendevelopedfortheseparation
ofionicspeciesbyRPLC.Inthefirstapproach,ion-pairing
reagents (counter-ions) are added in a mobile phase to
neutralizeionicspecies.Neutralizedspeciessoformedare
separated by common reversed-phase columns. For
cations (bases) heptanesulfonic acid and related com-
poundsareused,andforanions(acids)tetrabutylammo-
niumphosphateandrelatedcompoundsareusedasion-
pairreagents.
Partialionizationofsolutesduetoweakacidicorweak
basic character may create problems during chromato-
graphicseparationofpeaks.Additionofstrongacids(for
examplephosphoricorsulfuricacid)orstrongbases(for
example ammonium carbonate) may help to sharpen
peaks.
RPLC is a popular mode of liquid chromatography
because it utilizes an aqueous mobile phase that is
compatible with most biological samples. This mode
constitutesmorethan70%ofallliquidchromatographic
applications. Figure 1
shows a typical high-performance liquid
chromatographysystem,and
Figure 2
showsanapplication
ofLCinbiomedicine.
Ion Exchange Chromatography (IELC) In this mode, the stationary phase typically consists of
silicaonwhichanionicorcationicgroupsareimmobilized.
The anions and cations are classified according to their
abilitytoretainoppositelychargedmolecules;quaternary
salts are strong and amines are weak anion exchangers,
whereassulfonicacidsarestrongandcarboxylicacidsare
weakcationexchangers.
Chromatographicseparationtakesplaceonthebasisof
theionicchargeofasolute,andisinfluencedtotheextent
to which opposite charges are retained on the column.
Retentionofionicsoluteonthestationaryphaseisaffected C–G Cys Hcy 8.04 ER 0 14.95 11.0 9.67 12.72 13.73 14.28
Cys C–G Hcy Inject 11.0 9.69 12.79 13.80 14.44 15.11 ER 0 8.07
Cys Hcy C–G 8.03 9.71 11.0 Inject 14.92 12.65 ER 0 13.64 14.18
(a) Relative fluorescence (b) (c) Figure 2 A typical chromatogram of a monobromobimane (mBrB)- derivatized plasma sample of (a) a healthy control subject; (b) an epilepsy patient on anti-epileptic drug, (c) a patient with kidney disease. Samples were analysed according to the method of Mansoor et al . (1992). Cys, cysteine, CG, cysteinylglycine, HCY, homocysteine. Column, 150 mm 4 mm i.d.; stationary phase, particles ODS 3; mobile phase contains ion- pairing agent tetrabutylammonium phosphate; column temperature 25 8
C. Column Heater Integrator Autosampler Detector Pump
Degasser Figure 1 A typical high-performance liquid chromatography system consisting of a degasser, pump, mobile phase delivery system (isocratic or gradient) detector (UV, fluorescence or electrochemical), autosampler and integrator (computer aided). The column may be placed in an oven for constant temperature.
Liquid Chromatography 2 ENCYCLOPEDIA OF LIFE SCIENCES / &
2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
bytheionicstrength,pHandconcentrationoftheorganic
modifierinthemobilephase.
The ionic strength is related to the concentration and
chargeofdissolvedionsinthemobilephase.Buffersalts,
for example ammonium phosphate, sodium acetate or
sodium borate, are commonly used for this purpose.
Organicmodifiers,forexamplemethanol,acetonitrileor
tetrahydrofuran,areaddedtothemobilephaseforelution
ofionizablecompoundsthatareinsolubleinwaterandare
boundverystronglytothestationaryphase.
Size exclusion liquid chromatography (SELC) In this mode, chromatographic elution of compounds
depend upon the molecular size of molecules and their
abilitytopenetratetheporesofthestationaryphase.The
stationaryphaseconsistsofdifferentformsofporoussilica
gelorpolystyrenethatbehavelikeasieve.Thisallowsthe
smallest molecules to penetrate the pores whereas larger
molecules are excluded; consequently, larger molecules
elutefirstandsmallermoleculeselutelater.
Themobilephaseisasolventusedmerelytodissolvethe
solutemolecules;itservessolelyasacarrierforthesolute
andtherearenointeractionsbetweenthestationaryphase,
solutemoleculesandmobilephase.
SELCisusuallyappliedforseparationanddetermina-
tionofthemolecularmassofacompoundorthemolecular
massdistributionofcompoundsinasample.Thismodeis
rarelyusedforanalyticalpurposes.
Derivatization Derivatization takes two forms: precolumn and postcol-
umn derivatization. For precolumn derivatization, un-
desiredcomponentsinasampleareremovedandsensitive
detectablegroupsareaddedtoincreasethedetectabilityof
a particular class of compound before the samples are
injected onto a column. For postcolumn derivatization,
thesestepsaretakenafterseparationofthecomponentsin
asamplehastakenplace.
Applications .
Biomedicalmatrices:RPLCandIELCmayberecom-
mendedforanalysesofaminoacids,peptides,proteins,
lipids and other compounds in serum, plasma, blood,
urineandspinalfluid.
.
Dairy products: RPLC, IELC and NPLC may be
recommended for milk, cheese and other related
samples.
.
Oilsandfats:NPLCmayberecommendedforvegetable
oils,animalfatsandfat-solublevitaminsandotherlipid
compounds.
Derivatizationprocedureswillbedifferentforthedifferent
matrices.
Limits and Possibilities Liquidchromatographyisarobustanalyticaltechnology,
but modifications are required to develop methods that
reduce consumption of organic solvents, decrease costs
andsavetime.
Higherspeedandimprovedresolutioncanbeachieved
byreducingthelengthandinnerdiameterofacolumn,and
theaveragediameterofparticlesofthestationaryphase,
and increasing the flow rate of the mobile phase. An
unwantedincreaseinthepressuredevelopedinthesystem
owingtothepresenceofsmallerparticlesandahigherflow
rate can be reduced by raising the temperature of the
column.Anincreaseinthetemperatureby1
8
Cdecreases
theretentiontimeby1–3%foraparticularsolute.
Methodology development in liquid chromatography
specifically aimed at biomedical and pharmaceutical
applications should have a focus on automation of the
instrumentation.
Further Reading DolanJW(2000)Startingoutright,partI.Selectingthetools.
LC-GC 13
:
12–15.[www.lcgcmag.com]
DolanJW(2000)Startingoutright,partII.Measuringsatisfaction.
LC-
GC 13
:72–76.[www.lcgcmag.com]
DorseyJG,FoleyJP,CooperWT,BarfordRAandBarthHG(1990)
Liquidchromatography:theoryandmethodology.
AnalyticalChem-
istry 62
:324R–356R.
LimCK(1986)
HPLCofSmallMolecules,aPracticalApproach
.Oxford:
IRL.
Lough WJ and Wainer IW (eds) (1996)
HighPerformanceLiquid
Chromatography,FundamentalPrinciplesandPractice
. London:
BlackieAcademicandProfessional.
MansoorMA,SvardalAMandUelandPM(1992)Determinationofthe
invivoredoxstatusof cysteine,cysteinylglycine, homocysteine and
glutathioneinhumanplasma.
AnalyticalBiochemistry 200
:218–229.
SnyderLR(2000)HPLC:pastandpresent.
AnalyticalBiochemistry 72
:
412A–420A.
Liquid Chromatography 3 ENCYCLOPEDIA OF LIFE SCIENCES / &
2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net