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High-performance liquid chromatography

High-performance liquid chromatography

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High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

 and analytical chemistry
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...

 to identify, quantify and purify the individual components of the mixture.

HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column, and a detector to provide a characteristic retention time for the analyte. The detector may also provide additional information related to the analyte, (i.e. UV/Vis
Ultraviolet-visible spectroscopy
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent ranges...

 spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. It is a form of liquid chromatography that utilizes smaller column size, smaller media inside the column, and higher mobile phase pressures.

With HPLC, a pump (rather than gravity) provides the higher pressure required to move the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.

Operation


The sample to be analyzed or separated is introduced, in small volumes, into the stream of mobile phase. The solution moved through the column is slowed by specific chemical or physical interactions with the stationary phase present within the column. The velocity of the solution depends on the nature of the sample and on the compositions of the stationary (column) phase. The time at which a specific sample elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered an identifying characteristic of a given sample. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity
Velocity
In physics, velocity is speed in a given direction. Speed describes only how fast an object is moving, whereas velocity gives both the speed and direction of the object's motion. To have a constant velocity, an object must have a constant speed and motion in a constant direction. Constant ...

 giving the components less time to diffuse
Diffusion
Molecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...

 within the column, improving the chromatogram
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 resolution. Common solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

s used include any miscible combination of water or various organic liquids (the most common are methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...

 and acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

). Water may contain buffers or salts to assist in the separation of the sample components, or compounds such as trifluoroacetic acid
Trifluoroacetic acid
Trifluoroacetic acid is the simplest stable perfluorinated carboxylic acid chemical compound, with the formula CF3CO2H. It is a strong carboxylic acid due to the influence of the electronegative trifluoromethyl group. TFA is almost 100,000-fold more acidic than acetic acid...

 which acts as an ion pairing agent.

A further refinement of HPLC is to vary the mobile phase composition during the analysis; gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes; the gradient depends on how hydrophobic the sample is. The gradient separates the sample mixtures as a function of the affinity. This partitioning process is similar to that which occurs during a liquid-liquid extraction
Liquid-liquid extraction
Liquid–liquid extraction, also known as solvent extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid phase into another liquid...

 but is continuous, not step-wise. In this example, using a water/methanol gradient, more hydrophobic components will elute (come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase).

The choice of solvents, additives and gradient depend on the nature of the column and sample. Often a series of tests are performed on the sample together with a number of trial runs in order to find the HPLC method which gives the best peak separation.

Partition chromatography



Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient
Partition coefficient
In chemistry and the pharmaceutical sciences, a partition- or distribution coefficient is the ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. The terms "gas/liquid partition coefficient" and "air/water partition coefficient" are...

 principle has been applied in paper chromatography
Paper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...

, thin layer chromatography
Thin layer chromatography
Thin layer chromatography is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose...

, gas phase and liquid-liquid applications. The 1952 Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...

 in chemistry was earned by Archer John Porter Martin
Archer John Porter Martin
Archer John Porter Martin, FRS was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge....

 and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as with paper chromatography
Paper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...

; or takes advantage of some coulombic and/or hydrogen donor interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC, this method separates analytes based on polar differences. HILIC most often uses a bonded polar stationary phase and a non-polar, water miscible, mobile phase. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences, however, HILIC has the advantage of separating acidic, basic
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

 and neutral solutes in a single chromatogram.

The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. Retention strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends on the functional groups in the analyte molecule which promote partitioning but can also include coulombic (electrostatic) interaction and hydrogen donor capability.
Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.

Normal-phase chromatography



Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity. It was one of the first kinds of HPLC that chemists developed. NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends not only on the functional groups in the analyte molecule, but also on steric factors. The effect of sterics on interaction strength allows this method to resolve (separate) structural isomers.

The use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times. Very polar solvents in a mixture tend to deactivate the stationary phase by creating a stationary bound water layer on the stationary phase surface. This behavior is somewhat peculiar to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface).

Partition and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC
Hilic
Hydrophilic interaction chromatography is a version of normal phase liquid chromatography. The name was suggested by Dr. Andrew Alpert in his 1990 paper on the subject...

 bonded phases which improve reproducibility.

Displacement chromatography


The basic principle of displacement chromatography
Displacement Chromatography
Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column and is then displaced by a solute that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive ...

 is:
A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.
There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.

Reversed-phase chromatography (RPC)



Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With these stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily. An investigator can increase retention time by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release.

RPC operates on the principle of hydrophobic forces, which originate from the high symmetry in the dipolar water structure and play the most important role in all processes in life science. RPC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand in the aqueous eluent. This solvophobic
Solvophobic
Solvophobic theory/ solvophobicity attempts to explain interactions between polar solvents and non-polar solutes. The molecular structure of the solvent molecule is driven by hydrogen bonding between similarly structured solvent molecules but is perturbed by non-polar solute molecules...

 effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension
Surface tension
Surface tension is a property of the surface of a liquid that allows it to resist an external force. It is revealed, for example, in floating of some objects on the surface of water, even though they are denser than water, and in the ability of some insects to run on the water surface...

 of the eluent (water: 7.3 J/cm², methanol: 2.2 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.

Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is non-interacting with the water structure. On the other hand, polar groups, such as -OH, -NH2, COO or -NH3+ reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligand's alkyl chains and can have problems entering the pores of the stationary phase.

Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 J/cm² per Mol for NaCl, 2.5 J/cm² per Mol for (NH4)2SO4), and because the entropy
Entropy
Entropy is a thermodynamic property that can be used to determine the energy available for useful work in a thermodynamic process, such as in energy conversion devices, engines, or machines. Such devices can only be driven by convertible energy, and have a theoretical maximum efficiency when...

 of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).

Another important component is the influence of the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent
Buffering agent
A buffering agent is a weak acid or base used to maintain the acidity of a solution at a chosen value. The function of a buffering agent is to prevent a rapid change in pH when acids or bases are added to the solution. Buffering agents have variable properties—some are more soluble than others;...

, such as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Ammonium formate
Ammonium formate
Ammonium formate, NH4HCO2, is the ammonium salt of formic acid. It is a colorless, hygroscopic, crystalline solid.-Uses:Pure ammonium formate decomposes into formamide and water when heated, and this is its primary use in industry...

 is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts. A volatile organic acid such as acetic acid
Acetic acid
Acetic acid is an organic compound with the chemical formula CH3CO2H . It is a colourless liquid that when undiluted is also called glacial acetic acid. Acetic acid is the main component of vinegar , and has a distinctive sour taste and pungent smell...

, or most commonly formic acid
Formic acid
Formic acid is the simplest carboxylic acid. Its chemical formula is HCOOH or HCO2H. It is an important intermediate in chemical synthesis and occurs naturally, most notably in the venom of bee and ant stings. In fact, its name comes from the Latin word for ant, formica, referring to its early...

, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent. Trifluoroacetic acid
Trifluoroacetic acid
Trifluoroacetic acid is the simplest stable perfluorinated carboxylic acid chemical compound, with the formula CF3CO2H. It is a strong carboxylic acid due to the influence of the electronegative trifluoromethyl group. TFA is almost 100,000-fold more acidic than acetic acid...

 is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids. The effects of acids and buffers vary by application but generally improve the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

 as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture
Mixture
In chemistry, a mixture is a material system made up by two or more different substances which are mixed together but are not combined chemically...

 of 2,2'- and 4,4'- bipyridine
Bipyridine
Bipyridines are a family of chemical compounds with the formula 2, which are formed by the coupling of two pyridine rings. Six isomers of bipyridine exist, but two isomers are prominent: 2,2'-bipyridine is a popular ligand in coordination chemistry and 4,4'-bipyridine is a precursor to the...

. Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal
Metal
A metal , is an element, compound, or alloy that is a good conductor of both electricity and heat. Metals are usually malleable and shiny, that is they reflect most of incident light...

 ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...

s are present on the surface of the silica...

Size-exclusion chromatography



Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

 and quaternary structure
Quaternary structure
In biochemistry, quaternary structure is the arrangement of multiple folded protein or coiling protein molecules in a multi-subunit complex.-Description and examples:...

 of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparin
Heparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...

s.

Ion-exchange chromatography



In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. Types of ion exchangers include:
  • Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity.
  • Cellulose and dextran
    Dextran
    Dextran is a complex, branched glucan composed of chains of varying lengths...

     ion exchangers (gels) – These possess larger pore sizes and low charge densities making them suitable for protein separation.
  • Controlled-pore glass or porous silica


In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. An increase in pH reduces the retention time in cation exchange while a decrease in pH reduces the retention time in anion exchange.

This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others.

Bioaffinity chromatography


This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waals
Van der Waals
-People:* Fransje van der Waals , Dutch medical physician* Johannes Diderik van der Waals , Dutch physicist-Physics:* the Van der Waals force, named after the physicist* the Van der Waals equation, named after the physicist...

 interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.

Aqueous normal-phase chromatography


Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reverse-phase solvents.

Isocratic flow and gradient elution


A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (meaning constant composition). The word was coined by Csaba Horvath
Csaba Horváth (chemical engineer)
Csaba Horváth was a Hungarian-American chemical engineer, particularly noted for building the first high performance liquid chromatograph.-Life:...

 who was one of the pioneers of HPLC.,

The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. One example is a gradient starting at 10% methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...

 and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. In reverse-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

, methanol, THF
Tetrahydrofuran
Tetrahydrofuran is a colorless, water-miscible organic liquid with low viscosity at standard temperature and pressure. This heterocyclic compound has the chemical formula 4O. As one of the most polar ethers with a wide liquid range, it is a useful solvent. Its main use, however, is as a precursor...

, or isopropanol.

In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.

Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.

In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.

The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.

Internal diameter


The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.
  • Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.
  • Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.
  • Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence
    Fluorescence
    Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

     detection or with other detection methods like liquid chromatography-mass spectrometry
    Liquid chromatography-mass spectrometry
    Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...

  • Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry
    Mass spectrometry
    Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

    . They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.

Particle size


Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica
Silicon dioxide
The chemical compound silicon dioxide, also known as silica , is an oxide of silicon with the chemical formula '. It has been known for its hardness since antiquity...

 particles (very small beads). These particles come in a variety of sizes with 5 m beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.

This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.

Pore size


Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.

Pump pressure


Pump
Pump
A pump is a device used to move fluids, such as liquids, gases or slurries.A pump displaces a volume by physical or mechanical action. Pumps fall into three major groups: direct lift, displacement, and gravity pumps...

s vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure
Pressure
Pressure is the force per unit area applied in a direction perpendicular to the surface of an object. Gauge pressure is the pressure relative to the local atmospheric or ambient pressure.- Definition :...

 may reach as high as 40 MPa (6000 lbf/in2), or about 400 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 m). These "Ultra High Performance Liquid Chromatography" systems or RSLC/UHPLCs can work at up to 100 MPa (15,000 lbf/in²), or about 1000 atmospheres. The term "UPLC" is a trademark of the Waters Corporation
Waters Corporation
Waters Corporation is a publicly traded laboratory analytical instrument and software company headquartered in Milford, Massachusetts. The company employs more than 5,000 people, with manufacturing facilities located in Milford, Taunton, Massachusetts; Wexford, Ireland; Manchester, England; and...

, but is sometimes used to refer to the more general technique.

See also

  • Csaba Horváth
    Csaba Horváth (chemical engineer)
    Csaba Horváth was a Hungarian-American chemical engineer, particularly noted for building the first high performance liquid chromatograph.-Life:...

  • Ion chromatography
  • Column Chromatography
    Column chromatography
    Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.The main advantage of column chromatography is the relatively low cost and disposability...


Further reading

  • L. R. Snyder, J.J. Kirkland, and J. W. Dolan, Introduction to Modern Liquid Chromatography, John Wiley & Sons, New York, 2009.
  • M.W. Dong, Modern HPLC for practicing scientists. Wiley, 2006.
  • L. R. Snyder, J.J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, John Wiley & Sons, New York, 1997.
  • S. Ahuja and H. T. Rasmussen (ed), HPLC Method Development for Pharmaceuticals, Academic Press, 2007.
  • S. Ahuja and M.W. Dong (ed), Handbook of Pharmaceutical Analysis by HPLC, Elsevier/Academic Press, 2005.
  • Y. V. Kazakevich and R. LoBrutto (ed.), HPLC for Pharmaceutical Scientists, Wiley, 2007.
  • U. D. Neue, HPLC Columns: Theory, Technology, and Practice, Wiley-VCH, New York, 1997.

External links