High-performance liquid chromatography is an analytical technique used to separate, identify and quantify each component of 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 flow out of the column. Say no to plagiarism. Get a tailor-made essay on “Why violent video games should not be banned”?Get the original essay HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and biological products), legal (for for example to detect performance-enhancing drugs in urine), for research purposes (e.g. separating components of a complex biological sample or similar synthetic chemicals from each other) and for medical purposes (e.g. example, 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 the separation of the sample components. The active component of the column, the adsorbent, is usually a granular material consisting of solid particles (e.g. silica, polymers, etc.), 2 to 50 micrometers in size. The components of the sample mixture are separated according to their different degrees of interaction with the adsorbent particles. The liquid under pressure is usually a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is called 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 differs from traditional ("low pressure") liquid chromatography because operating pressures are significantly higher (50 to 350 bar), while ordinary liquid chromatography generally relies on the force of gravity to move the mobile phase to through the column. Due to the small amount of samples separated in analytical HPLC, typical column dimensions are 2.1 to 4.6 mm in diameter and 30 to 250 mm in length. Additionally, HPLC columns are manufactured with smaller sorbent particles (2 to 50 micrometers average particle size). This gives HPLC superior resolving power (the ability to distinguish 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. The 1952 archer John Porter Martin and Richard Laurence Millington Synge won the Nobel Prize in Chemistry for their development of the technique used for the separation of amino acids. Much like hydrophilic interaction chromatography (HILIC; a subtechnique of HPLC), this method separates analytes based on differences in their polarity. HILIC uses a bound polar stationary phase and a mobile phase composed primarily of acetonitrile with water as the strong component. Partition HPLC was used on unbound silica or alumina supports. It effectively separates analytes byrelative polar differences. HILIC bonded phases separate acidic, basic and neutral solutes in a single chromatographic operation. 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 strength of interaction 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 forming hydrogen bonding inducing more retention. Retention also increases by Coulomb (electrostatic) interactions. The retention time of the analytes decreased with the use of more polar solvents in the mobile phase. while the retention time was increased for more hydrophobic solvents. Normal phase chromatography[edit] 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. It is therefore based on the ability of the analyte to engage in polar interactions (such as hydrogen bonding or dipolar bonding). dipolar type interactions) with the surface of the sorbent. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. chloroform) and works effectively to separate analytes that are easily soluble in non-polar solvents. The analyte associates and is retained by the polar stationary phase. Adsorption forces increase with increasing 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. Using more polar solvents in the mobile phase will decrease analyte retention time, while more hydrophobic solvents tend to induce slower elution (increased retention times). Highly polar solvents such as traces of water in the mobile phase tend to adsorb onto the solid surface of the stationary phase, forming a stationary bound layer (water) which is considered to play an active role in retention. This behavior is somewhat unique 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 sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still widely used for structural separations of isomers in column and thin layer chromatography formats on activated (dried) silica or alumina supports. Separation HPLC and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC due to poor reproducibility of retention times due to the presence of a water or solvent layer protic organic on the surface of the silica or alumina chromatographic support. . This layer changes with any change in the composition of the mobile phase (e.g. humidity level), causing retention times to drift. Recently, partition chromatography has become popular again through the development of hilic-bound phases that demonstrate improved reproducibility, and through a better understanding of the range of utility of the technique. Displacement ChromatographyThe basic principle of displacement chromatography is that a molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites and thus displace all molecules with lower affinities.[11] There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column as narrow Gaussian peaks. Wide peak separation, preferably relative to the baseline, is desired in order to achieve maximum purification. The rate at which any component of a mixture moves down the column in elution mode depends on many factors. But for two substances to move at different speeds and thus be resolved, there must be substantial differences in certain interactions between the biomolecules and the chromatographic matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, basic peak separation can only be achieved with gradient elution and low column loadings. Thus, two disadvantages of elution mode chromatography, particularly 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 areas of pure substances rather than "peaks." Since the process takes advantage of the nonlinearity of isotherms, a larger column load can be separated on a given column, with the purified components recovered at a significantly higher concentration. Reversed phase chromatography (RPC) A chromatogram of the complex mixture (eau de parfum) 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 a moderately polar aqueous 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 start of the analysis). An investigator 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 compared to the now more hydrophilic mobile phase. Likewise, an investigator can reduce the retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often mistakenly referred to as "HPLC" without further specification. The pharmaceutical industry regularly uses RP-HPLC to qualify drugs before marketing. RP-HPLC works on the principle of hydrophobic interactions, which arises from the high symmetry of the water dipolar structure and plays the most important role in all life science processes. RP-HPLC allows the measurement of these interactive forces. Binding of the analyte to the stationary phase is proportional to the contact area around the nonpolar 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 the “reduction of cavities” around the analyte and the C18 chain compared to the complex of the two. 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 the surface hydrophobicity of the analyte andligand respectively. Retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of the water. Gradient elution utilizes this effect by automatically reducing the polarity and surface tension of the aqueous mobile phase during analysis. The structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (CH, CC, 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 a higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO- or -NH3+ in their structure) are less retained because they are better integrated into water. Such interactions are subject to steric effects to the extent that very large molecules may have only restricted access to the pores of the stationary phase, where interactions with surface ligands (alkyl chains) take place. Such surface obstruction generally results in less retention. Retention time increases with hydrophobic (non-polar) surface. Branched-chain compounds elute more quickly than their corresponding linear isomers because their overall surface area is decreased. Likewise, organic compounds with single CC bonds elute later than those with a C=C or CC triple bond, because the double or triple bond is shorter than a single CC bond. Besides the surface tension of the mobile phase (organizational force in the eluent structure), other modifiers of the mobile phase can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (approximately 1.5? 10-7 J/cm? per mole for NaCl, 2.5? 10-7 J/cm? per mole 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 gentle separation and recovery of proteins and for protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC). Another important factor is the pH of the mobile phase since it can modify the hydrophobicity of the analyte. For this reason, most methods use a buffering agent, such as sodium phosphate, to control pH. Buffers serve several purposes: controlling pH, neutralizing the charge on the silica surface of the stationary phase, and acting 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 through 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 rarely used in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving the retention of analytes such as carboxylic acids in applications. using 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 to normal silica columns; however, many reversed phase columns are made of silica particles derived from alkyland should never be used with aqueous bases as these will destroy the underlying silica particles. They can be used with aqueous acid, but the column should not be exposed to acid for too long, as this may corrode the metal parts of the HPLC equipment. RP-HPLC columns should be rinsed with a clean solvent after use to remove residual acids or buffers, and stored in an appropriate solvent composition. The metal content of HPLC columns must remain 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 on the basis of molecular size (actually by the Stokes radius d 'a particle). This is generally low resolution chromatography and is therefore often reserved for the final “polishing” phase 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 small molecules in the pores of a particle. Larger molecules pass through the pores because they are too large to enter the pores. Larger molecules flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time. This technique is generally used to determine the molecular weight of polysaccharides. SEC is the official technique (suggested by the European Pharmacopoeia) for comparing the molecular weight of different commercially available low molecular weight heparins. Ion exchange chromatography 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 with the same charge as the charged sites on the column are excluded from binding, while solute ions with the opposite charge to the charged sites on 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 ionic effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, modifying the pH of the solvent, etc.). Types of ion exchangers include: • Polystyrene resins – They provide cross-linking which increases chain stability. Higher cross-linking reduces the gap, which increases equilibration time and ultimately improves selectivity. • Cellulose and dextran ion exchangers (gels) – These have larger pore sizes and low charge densities, making them suitable for protein separation. • Pore-controlled glass or porous silica In general, ion exchangers favor the bonding of ions of 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 phase.