# Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/High performance liquid chromatography

High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column.

## Operation

The basic operating principle of HPLC is to force the analyte through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations 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, depending 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) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. 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 generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.

## Types of HPLC

### Normal phase chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes based on polarity. This method uses a polar stationary phase and a nonpolar mobile phase, and is used when the analyte of interest is fairly polar in nature. The polar analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increase in 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 not only depends on the functional groups in the analyte molecule, but also on steric factors and structural isomers are often resolved from one another. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes while more hydrophobic solvents tend to increase retention times. Particularly polar solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat particular 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 had fallen out of favor in the 1970's 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 utilize a partition mechanism which provides reproducibility.

### Reversed phase chromatography

Reversed phase HPLC (RP-HPLC) consists of 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. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. Retention time is increased by the addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent. Reversed phase chromatography (RPC) is so commonly used that it is not uncommon for it to be 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 interactions, which result from repulsive forces between a polar eluent, the relatively non-polar analyte, and the non-polar stationary phase. 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: 73 erg/cm2, methanol: 22 erg/cm2) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding less-polar solvent (MeOH, ACN)into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically changing the polarity of the 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 nonpolar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's nonpolar 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 ligands alkyl chains and can have problems entering the pores of the stationary phase.

Retention time increases with hydrophobic - nonpolar - 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 the ones 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 erg/cm2 pro Mol for NaCl, 2.5 erg/cm2 pro 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 proteines 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. A volatile organic acid such as formic acid or most commonly trifluoroacetic acid is often added to the mobile phase, if mass spectrometry is applied to the eluent fractions. 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. The effect varies depending on use 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 backbone. 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. 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 it is normal that when a metal ion is present on the surface of the silica the shape of the peak for the 2,2'-bipy will be distorted, tailing will be seen on this distorted peak.

### Size exclusion chromatography

For more details on this topic, see 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, and is the primary technique for determining the average molecular weight of natural and synthetic polymers.

### Ion exchange chromatography

For more details on this topic, see 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. Some types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. (2) Cellulose and dextran ion exchangers (gels)-These possess larger pore sizes and low charge densities making them suitable for protein separation.(3)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: In purifying water, preconcentration of trace components, Ligand-exchange chromatography, Ion-exchange chromatography of proteins, High-pH anion-exchange chromatography of carbohydrates and oligosaccharides, etc.

### 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 Waal's 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.

### Isocratic flow and gradient elution

With regard to the mobile phase, a composition that remains constant throughout the procedure is termed isocratic.

In contrast to this is the so called "gradient elution", which is a separation where the mobile phase changes its composition during a separation process. One example is a gradient in 20 min starting from 10% Methanol and ending up with 30% Methanol. Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it helps speed up elution by allowing components that elute more quickly to come off the column under different conditions than components which are more readily retained by the column. By changing the composition of the solvent, components that are to be resolved can be selectively more or less associated with the mobile phase as a result at equilibrium they spend more time in the solvent and less in the stationary phase therefore they elute faster.

## Parameters

### Internal diameter

The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of analyte that can be loaded onto the column and also influences sensitivity. 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) which 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${\displaystyle \mu }$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.[1][2][3] This means that changing to particles that are half as big in the same size of column will double the performance, but increase the required pressure by a factor of four. Larger particles are more often used in 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 analyses. For example a protein which is only slightly smaller than a pore might enter the pore but 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 6000 lbf/in² (~40 MPa, or about 400 atmospheres). Modern HPLC systems have been improved to work at much higher pressures, and therefore be able to use much smaller particle sizes in the columns (< 2 micrometres). These "Ultra High Performance Liquid Chromatography" systems or UHPLCs can work at up to 15,000 lbf/in² (~100 MPa or about 1000 atmospheres). Note that the term "UPLC", sometimes found instead is a trademark of Waters Corporation and not the name for the technique in general.

## References

1. http://www.lcgceurope.com/lcgceurope/article/articleDetail.jsp?id=333246&pageID=3#
2. Y. Xiang, Y. Liu and M. L. Lee; J. Chrom. A, Vol. 1104, Issues 1-2, (2006), Pages 198-202
3. Cs.Horváth, B.A. Preiss and S.R. Lipsky; Anal. Chem., 39, 1422–1428 (1967).