HPLC (high-performance liquid chromatography) is one of the advanced types of chromatography.
It is highly sophisticated and expensive tools in the present analytical chemistry.
It is given prominent importance due to its attributes like
- High sensitivity, i.e., the ability to evaluate samples of very minute concentrations like in nano-gram and picogram
- Detect precisely chemically similar molecules like monoamines and also
- Ability to identify compounds with complex chemistry.
This is possible in HPLC chromatography due to efficient separation under pressure over a large surface area. Besides, the HPLC system is also connected to highly sensitive detectors like UV-visible and fluorescence spectrometers, electrochemical detectors, etc.
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 the food industry for quality control.
5. For standards control by the government.
6. For separation of similar molecules.
HPLC analysis in the 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 the adrenal gland which secretes an endocrine hormone.
Because of the time factor, most of the diagnostic methods are replaced by Elisa, electrophoresis, and RIA methods. But still for a new a rare problem, the HPLC method is preferred to pinpoint the cause of disorders (i.e., any change in some biochemistry).
HPLC applications in scientific research
HPLC system is a mandatory tool in most of the labs involved in the research. The fields of study include medical, biological, chemical, biochemical, phytochemicals (plant chemical research).
When 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 analyze every molecule in the mixture.
It finds its 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 become easier.
HPLC applications in the pharmaceutical industry:
In the pharmaceutical industry, the qualitative type of HPLC analysis is widely used. In the research and development wing, both qualitative and quantitative methods are employed.
- In quality control, it is used to check if the manufactured products comply with the specified standards. These specific standards are fixed by the pharmacopeias and other drug regulating bodies. The guidelines mentioned in the pharmacopeia will give an idea of how the peak of the drug in the formulation should look, when run with specified HPLC mobile phases are used. If the peaks do not correspond to those shown in the pharmacopeia, the batch cannot be passed for quality check.
- In R & D, it is used to identify the specific molecule or component in the mixture under research. Further, it is used for bioavailability studies, drug release from the formulation, dissolution studies, etc. After a formulation is designed, the drug release over some time is tested in bioavailability studies. Then the sample released is taken and injected into the 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 drug formulations like injections, solutions, a dissolved form of solid dosage forms are injected into the HPLC injector to record the peaks of the individual constituents.
- Also, any new molecule under development or in a 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, bioavailability, 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 the different activity or nutritional value. These compounds can’t be precisely determined by other methods. Hence they are determined by HPLC analysis through separation into individual components and thereby identification.
For standards control by governments: The pharmacopeia making bodies like United States Pharmacopeia (USP), British Pharmacopeia (B.P) and others 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 the active ingredient in a formulation appear under the specified mobile phase solvents.
However, unlike other analytical techniques, HPLC analysis is time taking consuming process. A test run could run from a few hours to days together. It also requires expert troubleshooting in case of system failure, improper chromatogram peaks, etc. So one is advised to learn it from an expert in HPLC operation.