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 of the sample interacts differently with the adsorbent material, causing different flow rates for the different components and leading to separation of the components as they exit the column. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an Original Assay HPLC has been used for manufacturing (e.g. during the manufacturing process of pharmaceuticals and biologics), legal (e.g. to detect performance-enhancing drugs in urine), research (e.g. to separate components of a complex biological sample or similar synthetic chemicals from each other) and for medical purposes (e.g. for the detection of vitamin D levels in blood serum). Chromatography can be described as mass transfer adsorption. HPLC contains pumps to pass a pressurized liquid and sample mixture through an adsorbent-filled column, leading to separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made up of solid particles (e.g. silica, polymers, etc.), with dimensions between 2 and 50 micrometers. 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 an important role in the separation process. These interactions are physical in nature, such as hydrophobic (dispersive), dipole-dipole, and ionic, most often a combination. HPLC differs from traditional (“low-pressure”) liquid chromatography because the operating pressures are significantly higher (50–350 bar), whereas ordinary liquid chromatography typically relies on the force of gravity to push the mobile phase through the column. Due to the small amount of sample separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm in diameter and 30–250 mm in length. Additionally, HPLC columns are made with smaller absorbent particles (2–50 micrometers average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, making it a popular chromatographic technique. HILIC Partition Technique Useful Range Partition chromatography was one of the first types of chromatography developed by chemists. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid separation applications. In 1952 archer John Porter Martin and Richard Laurence Millington Synge won the Nobel Prize in Chemistry for their development of the technique, which was used for the separation of amino acids. Just as in hydrophilic interaction chromatography (HILIC; a subtechnique of HPLC), this method separates analytes based on differences in their polarity. HILIC uses a bonded polar stationary phase and a mobile phase consisting primarily of acetonitrile with water as the strong component. Partitioned HPLC was used on unbonded silica or alumina supports. Effectively separates analytes based on their polar differences. HILIC bonded phases separate acidic, basic and neutral solutes into onechromatographic analysis. Polar analytes diffuse into a layer of stationary water associated with the polar stationary phase and are then retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase), the longer the elution time will be. The interaction strength depends on the functional groups that are part of the molecular structure of the analyte, with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing greater retention. Retention also increases through Coulomb (electrostatic) interactions. The retention time of the analytes was reduced by the use of more polar solvents in the mobile phase. while the retention time was increased for more hydrophobic solvents. Normal-phase chromatography Normal-phase chromatography was one of the first types of HPLC developed by chemists. Also known as normal-phase HPLC (NP-HPLC), this method separates analytes based on their affinity to a stationary polar surface such as silica, then relies on the analyte's ability to engage in polar interactions (such as hydrogen bonds or dipoles - dipolar type interactions) with the absorbing surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. chloroform) and works effectively to separate analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. The adsorption strength increases with increasing polarity of the analyte. The strength of the interaction 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 the interaction strength allows this method to resolve (separated) structural isomers. The use of more polar solvents in the mobile phase will decrease the retention time of the analytes, while more hydrophobic solvents tend to induce slower elution (longer retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb onto the solid surface of the stationary phase forming a bound stationary layer (water) which is believed to play an active role in retention. This behavior is somewhat peculiar to normal-phase chromatography because it is governed almost exclusively by an adsorption mechanism (i.e. the analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the surface of the sorbent; see also HPLC in reverse phase below). Adsorption chromatography is still widely used for separations of structural isomers in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports. Partitioned HPLC and NP-HPLC fell out of use in the 1970s with the development of reversed-phase HPLC due to poor reproducibility of retention times due to the presence of a layer of water or protic organic solvent on the surface of the silica or alumina chromatographic medium. This layer changes with any change in mobile phase composition (e.g. moisture level) causing retention times to drift. Recently, partition chromatography has become popular again with the development of Hilic bonded phases demonstrating improved reproducibility and due to a better understanding of the range of utility of the technique. Displacement Chromatography The basic principle of displacement chromatography is: a molecule with a high affinity for the chromatographic matrix (the displacer) will compete effectively for binding sites and therefore replace all molecules with lower affinities.[11] There are distinct differences between displacement and elution chromatography. In the elution mode, the substancesthey typically emerge from a column in narrow Gaussian peaks. Wide peak separation, preferably relative to baseline, is desirable to achieve maximum purification. The rate 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 therefore be resolved, there must be substantial differences in some interactions between the biomolecules and the chromatographic matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of peaks can only be achieved with gradient elution and low column loadings. Therefore, two disadvantages of elution mode chromatography, especially on a preparative scale, are operational complexity, due to gradient solvent pumping, and low productivity, 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 isotherms, on a given column a larger column feed can be separated with the purified components recovered at a 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 nonpolar stationary phase and an aqueous, moderately polar mobile phase. A common stationary phase is a silica that has been surface modified with RMe2SiCl, where R is a straight-chain alkyl group such as C18H37 or C8H17. With such stationary phases, the retention time is longer for less polar molecules, while polar molecules elute more easily (at the beginning of the analysis). A researcher can increase retention times by adding more water to the mobile phase; thus making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger than for the now more hydrophilic mobile phase. Likewise, a researcher can reduce retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often mistakenly called "HPLC" without further specification. The pharmaceutical industry regularly uses RP-HPLC to qualify drugs prior to release. RP-HPLC works on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar structure of water and plays the most important role in all processes in life sciences. 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 nonpolar segment of the analyte molecule after binding with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water to “cavity reduce” around the analyte and the C18 chain compared to 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 ligand respectively. Retention can be decreased by adding a less polar solvent (methanol, acetonitrile) in the mobile phase to reduce the surface tension of the water. Gradient elution takes advantage of this effect by automatically reducing the polarity and surface tension of the aqueous mobile phase during the analysis. The structural properties of the analyte molecule play an important role in its retention characteristics. In general, aanalyte with a larger hydrophobic surface area (C–H, C–C, and generally nonpolar atomic bonds, such as SS and others) is retained longer because it does not interact with the water structure. On the other hand, analytes with the largest polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO- or -NH3+ in their structure) are retained less as they are better integrated into water. Such interactions are subject to steric effects as very large molecules can only have limited access to the pores of the stationary phase, where interactions with surface ligands (alkyl chains) take place. Such surface obstruction typically results in less retention. 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 small. Similarly, organic compounds with single C–C bonds elute later than those with a C=C or C–C triple bond, because the double or triple bond is shorter than a single C–C bond. Aside from mobile phase surface tension (organizing force in the 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? for Mol for ( NH4)2SO4), and since the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tends to increase the retention time. This technique is used for the gentle separation and recovery of proteins and for the protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC). Another important factor is the pH of the mobile phase as 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: pH control, charge neutralization on the silica surface of the stationary phase, and act as ion pairing agents to neutralize the charge of the analyte. Ammonium formate is commonly added in mass spectrometry to improve the detection of certain analytes by forming analyte-ammonium adducts. A volatile organic acid such as acetic acid, or more commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent. Trifluoroacetic acid is rarely used in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but it can be effective in improving the retention of analytes such as carboxylic acids in applications using other detectors, because it is a fairly strong organic acid. The effects of acids and buffers vary depending on the application but generally improve chromatographic resolution. Reversed-phase columns are quite difficult to damage compared to regular silica columns; however, many reversed phase columns are made 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 may corrode the metal parts of the HPLC equipment. RP-HPLC columns should be washed with clean solvent after use to remove residual acids or buffers and stored in an appropriate solvent composition. The metal content of the columnsHPLC must be kept low if the best possible ability to separate substances is to be maintained. A good test for the metal content of a column is to inject a sample that is a mixture of 2,2'- and 4,4'- bipyridine. Size Exclusion Chromatography Size exclusion chromatography (SEC), also called gel permeation chromatography or gel filtration chromatography, separates particles based on molecular size (actually based on the Stokes radius of the particle). This is usually low resolution chromatography and is therefore often reserved for the final "polishing" stage of purification. It is also used to determine the tertiary structure and quaternary structure of purified proteins. SEC is widely used for the analysis of large molecules such as proteins or polymers. SEC traps these smaller molecules in the pores of a particle. Larger molecules pass through the pores as they are too large to fit into the pores. Larger molecules flow through the column faster than smaller molecules, i.e. the smaller the molecule, the longer the retention time. This technique is usually used for the determination of the molecular weight of polysaccharides. SEC is the official technique (suggested by the European Pharmacopoeia) for comparing the molecular weight of various commercially available low molecular weight heparins. Ion Exchange Chromatography For more details on this topic, see Ion Exchange Chromatography. In ion exchange (IC) chromatography, retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions with the same charge as the charged sites on the column are excluded from binding, while solute ions with the opposite charge as the charged sites on the column are retained on the column. Solutic ions are retained on the column can be eluted from the column by changing the conditions of the solvent (e.g. increasing the ionic 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: Allow cross-linking which increases chain stability. Higher cross-linking reduces deviations, which increases equilibration time and ultimately improves selectivity. • Cellulose and dextran ion exchangers (gels) – Have 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 with higher charge and smaller radius. An increase in the concentration of counterions (relative to the functional groups in the resins) 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 example, more hydrogen ions are available to compete for positions on the anionic stationary phase, thus eluting loosely 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. Bioaffinity chromatography. For more details on this topic, see Affinity Chromatography. This chromatographic process is based on the property of biologically active substances to form stable, specific and reversible complexes. The training of these.