Introduction Abbreviated for;High-performance liquid chromatographyORHigh-pressure liquid chromatography Defined as;“A chromatographic technique used to separate components ofmixture for the purpose to identify, quantify or purify theindividual components of the mixture “. Widely used in field of biochemistry and analytical chemistry.
History Invented by Martin and Synge as extension of gaschromatography. The main idea was that;“Small sorbent particles and pressure could produce fastliquid chromatography techniques”. The method was used practically in late 1960s for separationamino acids.
Instrumentation HPLC involves following functional instruments.1. Mobile phase reservoir2. Pump3. Injector4. Stationary phase ( Column)5. Detector6. Computer
1. Mobile phase reservoir:• Commonly glass bottles with caps are used.• Teflon tubings and filters are connected to purge gas (helium) fordegassing.• Vaccum for 5-10 min is also used for degassing.2. Pump:• It forces the mobile phase to pass through column.• Flow rate is 1-2 ml/ min.• Trypical pressure is 6000 – 9000psi.3. Injector:• Can be manually (syringe) or automated.• Sample volume 5-20µl.• Ideal to stand pressure of mobile phase.• Autosampler is used for analysis of many samples automatically.
4. Stationary phase (Column):• Heart of HPLC.• Separate sample components on basis of physical and chemicalparameters.• Lenght 10-30cm.• Diameter 4-10nm.• Packing material 5-10nm thick.5. Detector:• Detection of elutes from column.• Quantitative analysis of sample components.• Output transferred to recorder/ computer.6. Computer:• Data system that controls modules of HPLC.• Signals from detector are interpreted to determine elution time,quantitative and qualitative analysis of sample.
Figure displaying complex instrumentation of HPLC
Operation• Operation strategies are explained as follows;1. Sample: The sample to be analyzed is introduced into stream of mobilephase in minute quantity, mostly in microliters. The components of sample moves through different velocitiesreaching the column where it physically interacts with thesorbent. Velocity of sample components depends on;• Chemical nature of sample• Nature of column• Composition of mobile phase
2. Column:• Also known as stationary phase.• Composed of sorbent material varying in particle size, and surfacechemistry.• Size of particles can be smaller which improves chromatographicresolution.• In terms of surface chemistry, the column can be hydrophobic andpolar in nature.
A BA. Plant extracts separated by HPLC by passing through stationary phase (column)B. Columns used in HPLC device
3. Eluent:• Also known as mobile phase. Type of solvent used:• Commonly it is composed of water miscible with organic solvents. Forexample methanol.• Some HPLC utilizes water free mobile phase and uses acids such asformic acid or phosphoric acid.• Many mobile phase works well by using salts such as sodiumphosphate.
Composition of eluent:• It depends on;• Intensity of interactions between analytes and stationary phase. Forexample;i. Hydrophobic interactions in reversed-phase HPLCii. Polar interactions in normal phase HPLCiii. Ionic interaction in ion exchange HPLC• Often a series of trial runs are performed with the sample inorder to find the HPLC method which gives adequate separation.
Advantages• Advantages of HPLC is;• Seapartion of voltile and non- voltile components.• Thermally unstable compounds isolated.• Quick analysis• High resolution• Less cumbersome• More reproducibility
Disadvantages• Disadvantages of HPLC are;• Tedious to detect co-elution.• High cost.• Complex to operate.