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High-performance liquid chromatography is an analytical technique used to separate, identify, and quantify each component in a mixture. The liquid solvent containing the sample mixture passes through a column filled with a solid adsorbent material. Each component in the sample interacts differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and biological products), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g. detecting vitamin D levels in blood serum) purposes.
Chromatography can be described as a mass transfer adsorption. HPLC contains pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2–50 micrometers in size. The components of the sample mixture are separated by their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a “mobile phase”. Its composition and temperature play a major role in the separation process. These interactions are physical in nature, such as hydrophobic (dispersive), dipole-dipole and ionic, most often a combination.
HPLC is differentiated from traditional (“low pressure”) liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm in diameter, and 30–250 mm length. Also, HPLC columns are made with smaller sorbent particles (2–50 micrometer in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.
HILIC Partition Technique Useful Range Partition chromatography was one of the first kinds of chromatography that was developed by chemists. The principle of partition coefficient was applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid separation applications. The 1952 Archer John Porter Martin and Richard Laurence Millington Synge earned Nobel Prize in chemistry for their development of the technique, which was used for their separation of amino acids. Just as in Hydrophilic Interaction Chromatography (HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition HPLC has been used on unbonded silica or alumina supports. It effectively separates analytes by relative polar differences. HILIC bonded phases separate acidic, basic and neutral solutes in a single chromatographic run.
The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Retention also increases by Coulombic (electrostatic) interactions. The retention time of the analytes was decreased with the use of more polar solvents in the mobile phase. whereas retention time was increased for more hydrophobic solvents.
Normal–phase chromatography was one of the first kinds of HPLC developed by chemists. Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity from a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), 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. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule but also on steric factors. The effect of steric hindrance 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 analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase chromatography because it is governed almost exclusively by an adsorptive mechanism (i.e. analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still widely used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.
Partition- and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina chromatographic media. This layer changes with any changes in the composition of the mobile phase (e.g. moisture level) causing drifting retention times.
Recently, partition chromatography has become popular again with the development of Hilic bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique.
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. 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 concentration.
Reversed-phase chromatography (RPC)
A chromatogram of the complex mixture (perfume water) obtained by reversed phase HPLC
For more details on this topic, see Reversed-phase chromatography.
Reversed-phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times 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. RP-HPLC is so commonly used that it is often incorrectly referred to as “HPLC” without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.
RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC 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 on the stationary phase. 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/cm?, methanol: 2.2?10-6 J/cm?) 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) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with the higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO- or -NH3+ in their structure) are less retained as they are better integrated into the water. Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.
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/cm? per Mol for NaCl, 2.5?10-7 J/cm? 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 factor is the mobile phase pH since it can change the hydrophobic character of the analyte. For this reason, most methods use a buffering agent, such as sodium phosphate, to control the pH. Buffers serve multiple purposes: control of pH, neutralize the charge on the silica surface of the stationary phase and act as ion pairing agents to neutralize analyte charge. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-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 effluent. 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 a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution.
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 the 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.
Size-exclusion chromatography (SEC), also called as gel permeation chromatography or gel filtration chromatography separates particles on the basis of molecular size (actually by a particle’s Stokes radius). It is usually a low-resolution chromatography and thus it is often reserved for the final, “polishing” phase of the purification. It is also used to determine the tertiary structure and quaternary structure of purified proteins. SEC is used widely for the analysis of large molecules such as proteins or polymers. SEC traps these smaller molecules in the pores of a particle. The larger molecules pass by the pores as they are too large to enter the pores. Larger molecules flow through the column quicker than smaller molecules, ie., smaller the molecule, longer the retention time.
This technique is usually used for determination of molecular weight of polysaccharides. SEC is the official technique (suggested by European Pharmacopeia) for the molecular weight comparison of different commercially available low-molecular-weight heparins.
For more details on this topic, see 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. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions (e.g. increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc…).
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. A decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation exchange column, for instance, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations.
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.
For more details on this topic, see Affinity 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 reversed-phase solvents.
Isocratic and gradient elution
At the ARS Natural Products Utilization Research Unit in Oxford, MS., a support scientist (r) extracts plant pigments that will be analyzed by a plant physiologist (l) using an HPLC system.
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 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 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 reversed-phase chromatography, solvent A is often water or an aqueous buffer, 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.
A schematic of gradient elution. Increasing mobile phase strength sequentially elutes analytes having varying interaction strength with the stationary phase.
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.
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