The HPLC is an effective technique for the separation and quantification of several biological compounds based on their size or charge. It is superior to gas chromatography in that it can be used for separation of thermally unstable and non-volatile substances also. Amino acids, proteins, nucleic acids, hydrocarbons, fatty acids, carbohydrates, phenols, pesticides, antibiotics, inorganic elements and others can be separated by employing HPLC.
Principle
When a sample containing different substances is injected onto a separating column and some pressure is applied over it, the solvent drives the substances along the column. Based on their size or charge, different substances are eluted out at different rates. After separation, the substance can be detected with the help of a detector and recorder. If the separating column is composed of highly polar materials and the solvent is non-polar, Least polar substances are eluted from the column very rapidly. This type of HPLC is called Normal phase chromatography.
If the separating column is composed of non-polar materials and the solvent is polar, highly polar substances are eluted first. This type of HPLC is called reverse phase chromatography. The detector measures the quantity of substances in the sample based on overall change in the physical property of the mobile phase or physical properties of the substances in the solvent.
Instrument
The HPLC setup consists of four basic components.
They are –
- Solvent loading system
- Stationary phase
- Sample loading system
- Detector and recorder
- Solvent Loading System: The solvent (mobile phase) is continuously applied to the separating column using HPLC pumps. Reciprocating piston pump, syringe type pump or constant pressure pump is attached to top of the column to inject the mobile phase and apply enough pressure. These pumps pull the mobile phase from the solvent reservoir and add it onto the separating column. A vacuum pressure is applied to the base of separating column to facilitate rapid movement of substances through that column. The solvent to be used as a mobile phase is chosen based on its polarity, eluting power, boiling point, viscosity, detector compatibility and toxicity. It should not interact with the column materials. If the mobile phase composition is same throughout separation, the separation process is called isocratic elution. If the strength of the mobile phase is increased step by step, the separation is called gradient elution. If the separating column contains hydrophobic resins, it is called polytypic elution.
- Stationary phase: The stationary phase is the solid support which constitutes the separating column. It is formed of inert materials such as silica, styrene divinyl benzene polymers and other matrices. Liquid-liquid column, liquid-solid column, ion exchange column and affinity column are used depending upon the substance to be separated. The mobile phase pass through the pores in the stationary phase and derives the solutes.
- Sample Loading System: Sample is injected into the HPLC via an injection port which is equipped with an injection valve and sample loop. The sample is injected before loading the mobile phase. Stopped-flow injection method or continuous method or syringe pump method is used for loading the sample into the HPLC.
- Detector and Recorder: The detector is attached to the posterior part of the separating column to detect the eluted substances. Band width, peaks of absorption, etc. are adjusted to zero before going to analyze the substances.
- The detector may work on the principle of refractive index (RI) or UV light absorption and fluorescence or radiochemical methods or mass spectroscopy or NMR or light scattering (LS) properties. The refractive index is measured with a photodetector. These detectors may detect the sequential changes in the properties of mobile phase o physical properties of the substance to be analyzed.
Procedure or HPLC involves the following steps:
- Column material is filled to 10-30 cm height in the metallic tube.
- Sample is loaded on to the top of the column material.
- Mobile phase is injected on to the column using pumps and valves.
- Pressure is applied over the separating column. Meantime, vacuum pressure is applied to the base of the separating column.
- Mobile phase derives the substances through the separating column. Because of size Difference and charge, the substances are eluted at different times.
- The eluted substances are analyzed by the detector and the result is recorded. It is fully automated.
Applications
HPLC is used for the isolation and purification of several compounds. Egs. Amino acids, proteins, mucleic acids, carbohydrates, amoxycillin, ampicillin anisomycin, chloramphenicol cycloheximide, erythromycin, levorin, neomycin, penicillin, streptomycin, etc.
It is used in the identification of the following substances:
- Inorganic substances such as cyanide, fluoride, etc.
- Formic acid (an organic acid)
- Additive drugs such as heroin, opium alkaloids.
- Choline-esterase inhibitors
- CO2 level in liquids
- Alcohols
- Water soluble and fatty acid soluble vitamins
- Heat labile poisonous substances
- It is used in forensic science to detect criminals by analyzing lipstick smears in cloths, glasses and papers.
- It is used in the differential analysis of explosives such as TNT, TETRYC, RDS, HMX, PETN, EGDN and NG.
- It is used to quantify preservatives and antioxidants in foods. Egs. Sorbic acid, benzoic acid, methyl esters, biphenyls, PHB esters and biphenyls.
- HPLC is used to detect the amount of oestrogen and androgen in the blood. This is very Useful for the disease management.
