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