Hplc (high performance liquid chromatography) is one of the types of chromatography.
It is highly sophisticated and expensive analytical tools in the present science.
It is given prominent importance due to its attributes like
- High sensitivity i.e. ability to evaluate samples of very minute concentrations like in nano-gram and picogram
- Detect precisely the closely similar molecules and also
- Highest accuracy in the identification of components of complex mixtures.
This is possible in hplc analysis due to efficient separation of molecules under pressure over a large surface area. Besides there is also availability of highly sensitive detectors like UV-visible and florescence spectrophotometers, electrochemical detectors etc.
But unlike other analytical techniques, HPLC analysis is time taking easy and trouble shooting is very important to run the test smoothly. So one is advised to learn it from an expert in hplc operation.
Applications of HPLC
This method of chromatography finds vast use in
1. Clinical diagnosis of diseases, disorders.
2. In scientific research for discovery.
3. In pharmaceutical labs for analysis.
4. In food industry for quality control.
5. For standards control by government.
6. For separation of similar molecules.
HPLC analysis in clinical diagnosis and health industry:
Many disorders related to body metabolism, those related to endocrine and exocrine gland secretion, alteration in body fluids are diagnosed by HPLC analysis of concerned fluids.
For example estimation of metabolites of purines, pyrimidines or other metabolites from plasma, cerebrospinal fluid and urine samples in patients.
Estimation of corticoids from plasma in disorders of adrenal gland which secretes an endocrine hormone.
Because of time factor, most of the diagnostic methds are replaced by Elisa, electrophoresis and RIA methods. But still for a new a rare problem, HPLC method is preferred to pin point the cause of disorders (i.e. any change in some biochemistry).
HPLC application in scientific research:
HPLC system is a mandatory tool in most of the labs involved in research. The fields of research include medical, biological, chemical, biochemical, phytochemical (plant chemical research.
When a research is taken up, the scientists are not sure of the actual which need attention in a body fluid or drug sample etc. Then they have to screen every possible molecule to point out the altered change (component). Then HPLC is much suited as it can analyse each and every molecule in the mixture.
It finds it application to analyze and quantify the molecules. Components with similar chemistry and properties are easily distinguished by this method. Due to the principle of separation in HPLC similar molecules get separated and hence their detection, identification and quantification becomes easier.
HPLC applications in pharmaceutical industry:
In pharmaceutical industry, hplc analysis is prominently used in quality control. Beside it also used R& D wings (research and development).
- In quality control, it is used to check if the prepared or manufactured products are in compliance with the specified standards as per the pharmacopoeia and other drug regulating bodies. For quality control, the manufactured product must comply with the quality guidelines referring to pharmacopoeia will give an idea of how the peak of the drug in the formulation should look, at he specified hplc solvents and mobile phases used. If the peaks do not correspond to those shown in pharmacopeia, the batch cannot be passed for quality check.
- In R & D as discussed in research before, it is used to identify the specific molecule or component in mixture under research. Further it is used for bioavailabilty studies, drug release from the formulation, dissolution studies etc. After a formulation is designed, the drug release over a period of time is tested in bioavailabilty studies. Then the sample released is taken and injected into HPLC system to note the individual molecules released in terms of quantity. Since the molecules might be similar, their separation is easier over the column under pressure. Further their detection becomes easier as the system is connected UV-visible detector or other specified detectors.
For this the formulation like injections, solutions, dissolved form of solid dosage forms are injected into HPLC to record the peaks of the individual constituents.
- Also any new molecule under development or preclinical trial, are analyzed to see their concentration in the blood after certain intervals of administration. This helps to evaluate the metabolic profile, plasma concentration, bio-availability etc of the formulation or chemical moieties under development.
- In plant constituents, there are many molecules with similarity in chemistry like isoflavones, glycosides, saponins etc. but different activity or nutritional value. These compound can’t be precisely determined by other methods, hence they are determined by HPLC analysis by means of separation into individual components and there by identification.
For standards control by governments: The pharmacopeia making bodies like United states Pharmacopeia (USP), Indian Pharmacopeia (I.P), British Pharmacopeia (B.P), use HPLC extensively. They fix standards of control for any drug formulation the industry makes. The companies send the formulation to the pharmacopeia bodies for standardization. Most formulations are estimated by HPLC to see the peaks of active ingredients (drug molecules). The peaks are then published in the official volumes of USP, IP or BP for reference by the industries for quality control. This gives an idea of how the peaks for active ingredient in a formulation appear under the specified mobile phase solvents.
The effectiveness and use hplc application in recent days is further enhanced due to coupling with detectors like Mass Spectroscopy, Nuclear Magnetic Resonance etc.