High-performance liquid chromatography

High-performance liquid chromatography

An HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance.
Acronym	HPLC
Classification	Chromatography
Analytes	organic molecules biomolecules ions polymers
Manufacturers	Agilent Technologies Beckman Coulter, Inc. Cecil Instruments Dionex Corp Hitachi, Ltd. PerkinElmer, Inc. Shimadzu Scientific Instruments Thermo Fisher Scientific Varian, Inc. Waters Corporation
Other techniques
Related	Chromatography Aqueous Normal Phase Chromatography Ion exchange chromatography Size exclusion chromatography Micellar liquid chromatography
Hyphenated	Liquid chromatography-mass spectrometry

HPLC apparatus.

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds, and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.

HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis 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.

With HPLC, a pump (rather than gravity) provides the higher pressure required to propel 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 is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. How much the analyte is slowed depends on the nature of the analyte and on the compositions of the stationary and mobile phases. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as 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 chosen depends on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase). The more hydrophilic compounds will elute under conditions of relatively low methanol/high water.

The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of trial runs may be processed in order to find the HPLC method which gives the best separation of peaks.

Types

Partition chromatography

A modern self contained HPLC. In this picture; an Agilent 1200 connected to a PC

Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin 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; or takes advantage of some additional 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 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.

Partition and NP-HPLC had fallen 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 bonded phases which improve reproducibility.

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).

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 bonded phases which improve reproducibility.

Displacement chromatography

The basic principle of displacement chromatography 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.^[1] 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)

A chromatogram of complex mixture (perfume water) obtained by reversed phase HPLC

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 more non-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 is allowing 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 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 of the eluent (water: 7.3 × 10⁻⁶ J/cm², methanol: 2.2 × 10⁻⁶ 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) 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 × 10⁻⁷ J/cm² per Mol for NaCl, 2.5 × 10⁻⁷ J/cm² per Mol for (NH₄)₂SO₄), and because the entropy 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 since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent, 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 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, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. Trifluoroacetic 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 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 of 2,2'- and 4,4'- bipyridine. 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 ions 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 and quaternary structure 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 heparins.

Ion-exchange chromatography

In ion-exchange chromatography, 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 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 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 from Yale University, 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.^[2] One example is a gradient starting at 10% methanol 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. Solvent A is often water, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, 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.

Parameters

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 detection or with other detection methods like liquid chromatography-mass spectrometry
• Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. 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 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.^[3][4][5]

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

Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure 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, but is sometimes used to refer to the more general technique.

Manufacturers of HPLC chromatographs

• Agilent Technologies
• Shimadzu Scientific Instruments
• Beckman Coulter
• Cecil Instruments
• Dionex Corp
• Hitachi
• PerkinElmer, Inc.
• Thermo Fisher Scientific
• Varian, Inc.
• Waters Corporation

Manufacturers of HPLC columns and accessories

• Agilent Technologies
• Shimadzu Scientific Instruments
• AkzoNobel
• Beckman Coulter
• Dionex Corp
• Merck KGaA
• Phenomenex
• SGE Analytical Science
• Shodex
• Sigma-Aldrich
• Thermo Fisher Scientific
• Tosoh Corporation
• Varian, Inc.
• Waters Corporation
• W. R. Grace and Company

See also

• Csaba Horváth
• Ion chromatography
• Column Chromatography

References

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

• Brief Guide to HPLC Instruments – A video that describes the different parts of a HPLC instrument and the role that each performs
• HPLC Primer – Beginners Guide to Liquid Chromatography, J.C. Arsenault and P.D. McDonald [2009] - A detailed primer with history, glossary, and technology sections
• A History of Chromatography - P.D. McDonald, The Quest for Ultra Performance in Liquid Chromatography - Origins of UPLC Technology, P.D. McDonald [2009] - a history of the 100 years of chromatography, with glossary and detailed notes
• Beginners Guide to UPLC Technology - Beginners Guide to UPLC (Ultra-Performance Liquid Chromatography), E.S. Grumbach, J.C. Arsenault, and D.R. McCabe [2009] - a good introduction to UPLC Technology
• Scientific Calculator for HPLC