2.High Performance Liquid Chromatography HPLCThe chromatography term is derived from the greek words namely chroma (colour) and graphein (to write). High Performance Liquid Chromatography or High Pressure Liquid Chromatography, HPLC is a separation technique in which liquid is used to force the sample at high pressure through a column packed with stationary phase in order to identify and/or quantify the components.
It.is characterize as a set of techniques which is used for the separation of constituents in a mixture. HPLC separation technique provides high speed, efficiency and high sensitivity as compared to liquid chromatography used for for separation ,identification,quantification, and purifications in a variety of areas including pharmaceuticals, biotechnology, environmental, polymer and food industries. HPLC is an advanced technique of column liquid chromatography A very small volume of sample is injected into a tube packed with a stationary phase (3-5 microns particles). ²(1,2,3).
History 2.1 HLPC s develop in the late 1960s and early 1970 , it is now widely application for separation and purification . used standard liquid chromatographic techniques. Gas chromatography (GC) in happen was more powerful than liquid chromatography (LC), but, it was believed that(GS)separation and analysis of very polar high molecular weight biopolymers was impossible.
it found Two materials useful is calcium carbonate and alumina.GC was ineffectual for numerous biochemists owing to of the thermal instability of the solutes, As a result, alternative methods were hypothesized. (4) 2.2.OperationThe sample to be separated in dissolved in microtiter volume and injected in a stream of mobile phase traveling through the column.The component of sample travel through colum by different speed depending on the physical connection with stationary phase. The velocity of every component depend on the its compound nature and composition of mobile phase.
The time at which a particular analyte elutes (rises up out of the column) is called its retention time. The retention time measured under specific conditions is a distinguishing normal for a given analyte . Different type of columns are available and packed with stationary phase varying in molecular size and nature of their surface.
The utilization of small molecule size packing phase requires the utilization of higher operational pressure (“backpressure”) and regularly enhances chromatographic resolution.Sorbent particles might be hydrophobic or polar in nature. Basic mobile phases utilized incorporate any miscible mixture of water with different natural solvents (the most widely recognized are acetonitrile and methanol).
Some HPLC systems use without water mobile phases. The aqueous segment of the mobile phase may contain acids, (for example, formic, phosphoric or trifluoroacetic corrosive) or salts to help with the seperation of the sample components.The mobile phase composition of constant (isocratic elution mode) or changed (inclination elution mode)during HPLC examination(5,6,) .Isocratic elution is normally successful in the separate of sample.Mobile phase solvent composition remains constant with time.
Best for simple separation ,often used in quality control application that support and are close proximity to a manufacturing process. Gradient mobile phase solvent composition increase with time .Best for the analysis of complex samples,often used in method development for unknown mixture.Linear gradient are most popular.
The quality of the mobile phase by analyte maintenance times with high eluting quality delivering quick elution. The structure of the mobile phase (additionally called eluent) depend on the force of connections between different parts and stationary stage (7,8) INSTRUMENTATION2.3 The instrumentation of HPLC include pump, injector, column, detector, integrator and display system. In the column the separation occurs include:2.
3.1Solvent ReservoirThe parts of mobile phase are present in glass container. In HPLC the mobile phase is a mixture of polar and non-polar liquid components Depending on the composition of sample.(9)2.3.2.
Pump:The pump suctions the mobile phase from solvent reservoir and forces it to column and then passes to detector. This operating pressure depends on the column dimensions, particle size, flow rate and composition of mobile phase. 2.
3.3.Sample InjectorThe injector can may be solitary or computerized infusion framework.2.3.4. columns are typically fashioned of cleaned stainless steel, and have an inward distance across of somewhere around 2 and 5 mm. They are generally loaded with a stationary phase with a molecule size of 3 ?m to 10 ?m.
Columns with inner diameters of <2 mm are regularly alluded to as microbore segments. Preferably the temperature of the mobile phase and the column should be kept consistent during investigation.(10)2.3.5.
Detector:located toward the end point of the column distinguishes the analytes as they elute from the chromatographic column. take advantage of detectors are UV-spectroscopy, fluorescence, massspectrometric and electrochemical identifiers.2.3.6..
Integrator: data collection on graph recorders or electronic integrators that fluctuate in many-sided quality and in their capacity to process, store and reprocess chromatographic information. 2.4.TYPES OF HPLC.-Normal phases of HPLC2.4.1the separates analytes based on polarityis . The stationary phase is polar, mainly silica is used and the non-polar phase used is hexane, chloroform and diethyl a polar staonary phase, which retains polar analytes, and a nonRpolar mobile phaseand, and Retenontion time(adsorpon strength)increases with increased analyte polarity.
(11) 2.4.2.- Reverse Phaseof HPLCa non -polar stationary phase and an aqueous ,moderately polar mobile phase ,Operates on the principle of hydrophobic interactions ,The mobile phase is polar and the stationary phase is non polar or hydrophobic. The more is the non-polar nature the more it will be retained.- Size exclusion chromatography (SEC,gel filtration chromatography)this type use Separates particles on the basis of size,and the separation of polymers such as proteins and polysaccharides.(12)-Ion-exchange HPLC2.
4.3Widely distributed in purifying water, ligand exchange, purification of proteins.Changing the pH of mobile phase enables the elution of proteins with different pI,Retention is based on the attraction between solute ions and charged sites bound to the staonary phase.(13) Bioaffinity chromatography-2.4.
4separation is based on specific reversible interaction between proteins and ligands.??. 2.5.APPLICATIONS OF HPLCPharmaceutical Applications The include controlling of drug stability and dissolution studies and quality control.Environmental Applications Identification of diphenhydramine in sedimented samples, Detection of phenolic compounds in water and pollutants.Applications in Forensics in the Determination of anabolic steroids ,Determination of cocaine and metabolites in blood.
Food and Flavor use in ensuring the quality of carbonated drink and drinking water in human . Analysis of beer ,Sugar fruit juices,polycyclic compounds in vegetables and military high explosives in agricultural crops.Applications in Clinical Tests use in Urine analysis, Analysis of bilirubin, and Detection of endogenous Neuropeptides.(14.
15,16) 2.6.PARAMETERS affect HPLC separation For the accurate analysis of a compound, there is a change occurs in the parameters the result may be affected greatly. The a lot commonly used parameters are internal diameter, particle size, pore size, pump pressure (17). Internal diameter -2.6.1that determines quantity of analyte that can be loaded into the column and also affected in sensitivity. Larger columns are seen in industrial applications like as the purification of a drug product for later use.
-Particle size2.6.2 Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles . 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.2.6.3.-Pore sizeMany stationary phases are porous to provide greater surface area.
Pore size defines an ability of the analyte molecules to penetrate inside the particle and interact with its inner surface.2.6.4.Pump pressurePumps s different in pressure capacity,and measured performance is on their ability to yield a consistent and frequens flow rate. Referance :1: Lin G, et al. Determination of sodium tanshinone iia sulfonate in rat plasma by high performance liquid chromatography and its application to pharmacokinetics studies. Pharm Anal Acta.
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