INTRODUCTION:

High Pressure Liquid Chromatography, also referred to as High Performance Liquid Chromatography It is a well-liked analytical technique used to separate, recognise, and quantify each component of a mixture. A more sophisticated kind of column liquid chromatography is HPLC. Normally, a column's solvent flows through it with the aid of gravity, but the HPLC process forces the solvent at high pressures of up to 400 atmospheres so that the sample can be divided into its many elements using differences in relative affinities.^1-7

One of the most effective tools in analytical chemistry nowadays is HPLC. Any material that can dissolve in a liquid can have its constituents separated, identified, and quantified using this technique. The most precise analytical techniques, such as HPLC, are frequently employed to assess the stability of pharmacological products as well as their quantitative and qualitative composition.^8-9

Because HPLC operates at considerably greater pressures (50 bar to 350 bar) than conventional ("low weight") liquid chromatography, it can be distinguished from the latter. Conventional liquid chromatography frequently relies on gravity to move the portable stage through the segment. Scientific HPLC isolates very small amounts of sample, hence column section measurements range from 2.1mm to 4.6mm in width and 30 mm to 250mm in length. Additionally, smaller sorbent particles (2m to 50m in normal molecule size) are used to create HPLC segments. This makes HPLC a popular chromatographic technique by giving it great determining or resolving power (the ability to detect components when isolating mixtures).^10-19

Types of HPLC:

Normal Phase HPLC:

This technique uses polarity to separate objects. Hexane, chloroform, and diethyl ether are employed as the non-polar stationary phase while silica serves as the primary polar stationary phase. On the column, the polar samples are kept.

Reverse Phase HPLC:

HPLC is used in reverse to normal phase. The stationary phase is hydrophobic or non-polar while the mobile phase is polar. The non-polar character will be kept more the more of it there is.

Size-exclusion HPLC:

The substrate molecules will be added to the column in a precisely regulated manner. The separation of constituents will take place based on the variation in molecular sizes.

Ion-exchange HPLC:

The ionically charged surface of the stationary phase is the opposite of the charge on the sample. Aqueous buffer is utilised as the mobile phase and will regulate the pH and ionic strength.²⁰

Characteristic of HPLC:

1. Excellent resolution

2. Columns made of stainless steel, glass, or titanium with a 4.6 mm small diameter.

3. Very tiny (3, 5 and 10m) particle column packing.

4. Fairly high inlet pressures and managed mobile phase flow.

5. Constant flow detectors that can handle low flow rates and pick up very minute quantities.

6. Quick evaluation.^21-23

Mechanism:

The mobile phase stream that is permeating the column is introduced with a discrete small volume (typically microliters) of the sample blend that needs to be isolated and dissected. Sample segments move through the segment at varying speeds as a result of specific physical contacts with the adsorbent (likewise called stationary stage). Every component's velocity is dependent on its chemical makeup and mobile phase. The retention time of a specific analyte is the time at which it elutes (rises up out of the column). For a given analyte, the retention time measured under particular circumstances serves as a distinguishing normal.^24-34

There are numerous columns available that are filled with adsorbents with different molecular sizes and surface characteristics ("surface science"). The use of packing materials for tiny molecules necessitates the use of higher operational pressure ("backpressure") and frequently improves chromatographic resolution (i.e., The degree of division between sequential analytes rising up out of the column). The nature of sorbent particles may be polar or hydrophobic. The most common mobile phases are any miscible mixtures of water and several natural solvents (the most widely recognised are acetonitrile and methanol). Some HPLC systems employ mobile phases devoid of water.

To aid in the separation of the sample components, the aqueous portion of the mobile phase may contain salts or acids (such as formic, phosphoric, or trifluoroacetic corrosive).

During the chromatographic analysis, the composition of the mobile phase may be maintained constant ("isocratic elution mode") or altered ("inclination elution mode"). Isocratic elution typically succeeds in separating sample components whose propensities for the stationary stage are not significantly different. The structure of the mobile phase fluctuates typically from low to high eluting quality in gradient elution. Analyte maintenance times reflect the eluting quality of the mobile phase, with high eluting quality producing rapid elution. The stationary stage and several example components ("analytes") are connected by a force that determines the structure of the mobile phase (also known as eluent) (e.g., hydrophobic connections in turned around stage HPLC). Analytes divide between the stationary and mobile stages according to their partiality for each. When the sample's detachment process was taking place. This process is similar to what occurs during a liquid-liquid extraction, however it is continuous rather than stepwise. More hydrophobic components will elute (fall off the column) in this instance using a water/acetonitrile angle later, as the mobile stage becomes more packed in acetonitrile (i.e. in a flexible time of better eluting quality).^36-42

Instrumentation:

Pump:

Compared to gravity-flow columns, a pump propels the mobile phase through the column at a significantly higher speed. Even when the mobile phase's composition changes, the pump is built to maintain a steady flow rate and prevent pulsations.⁴³.

Fig 1: Pump for HPLC⁴⁴

Injector:

The injector is generally utilised to inject liquid samples. There are two different types of injectors: automated and manual.⁴³.

Fig 2:- Pump for HPLC⁴⁵

Columns:

It is necessary to explain the significance of the column because it is a crucial part of HPLC. The most popular and often used system is an HPLC column, which is made of packing made of silica.⁴⁶.

Fig 3:- Columns for HPLC⁴⁷

Detector:

As each separated component exits the column, the HPLC detector measures the eluent and generates an electrical signal proportionate to its concentration.⁴⁸

The detectors with the greatest use: Fluorescence, conductivity, evaporative light scattering, refractive index detectors are examples of detectors.

Fig 4:- Detector for HPLC⁴⁹

Data recording:

The detected signal is transformed to an electrical signal when detection is complete, which is then amplified by an amplifier and recorded as a chromatogram in data points. Then, use the programme as the display format in accordance with the manual or software conversion requirements.^50-52.

Fig 5 :- Data Recording For HPLC [53]

Application of HPLC

To survey food and drug products,

To identify confiscated narcotics,

To analyse complex mixtures,

To purify chemical compounds,

To identify the number of chemical compounds found in new drugs in pharmaceutics^54–56.

Pharmaceutical applications:

Research on the pharmaceutical dosage form's tablet dissolving. Drug stability control and shelf-life estimation. Identifying the components that are active. Control of pharmaceutical quality pharmaceutical dosage form dissolving in tablet form. study on the dissolving of tablet doses for prescription drugs. stability studies and shelf-life estimations. Identification of the dosage forms' active medicinal components. Analyzing contaminants in pharmaceutical formulations and conducting assays. quality assurance. development and research.⁵⁷

Environmental applications:

The presence of phenol chemicals in water. Diphenhydramine identification in sedimented samples. pollution biomonitoring. HPLC allows for the quick isolation and identification of carbonyl compounds. Pharmaceuticals and personal care items can be detected using an HPLC/MS/MS solution in water, sediment, soil, and biosolids. HPLC 3-mercaptopropionic acid determination.⁵⁸

Industrial application:

Applications in the HPLC industry3 The process of developing a novel drug involves a wide range of applications, from drug discovery to the production of prepared goods that will be given to patients. This Process to create a new drug can be divided into 3 main stages 1. Finding new drugs 2. Drug creation 3. Drug production The greatest tool for identifying and characterising compounds is LC-MS. As a measurement tool for high throughput screening, it is possible to use it. To isolate and purify hits and lead compounds as needed, preparative HPLC is also used. For instance, a combinatorial synthesis. The capacity to establish International Journal of Pharmaceutical Science and Research, 59(1), November-December 2019, Article No. 20, Pages: 117-124 ISSN 0976 - 044X Review and Research in International Journal of Pharmaceutical Sciences At www.globalresearchonline.net, which is a website. Copyright is reserved. This document may not be modified in any way without the express written consent of the original author(s). Pharmaceutical experiments, which HPLC is appropriate for, have an enantiomeric purity standard of 122.⁵⁹

Clinical:

Catecholamines, including epinephrine and dopamine, are crucial for a variety of biological processes. It is possible to diagnose conditions including Parkinson's disease, heart disease, and muscular dystrophy by analysing their precursors and metabolites. analysis of blood plasma for the presence of antibiotics and ion concentrations in human urine. estimation of biliverdin and bilirubin in blood plasma in the event of hepatic diseases. Finding endogenous neuropeptides in the brain's extracellular fluids.^60-61

Food and Flavour:

Rapid component screening and analysis for non-alcoholic beverages. evaluation of the purity of water and soft medicines. analysis of sugar in fruit juices. Vegetable polycyclic compound analysis. analysis of preservation. LC triple quadrupole MS multiresidue analysis of many pesticides in food samples.⁶²

Advantages and disadvantages of HPLC:

Advantages:

HPLC offers a highly specific, reassuringly accurate, and reasonably quick analytical approach for a variety of complex substances.

HPLC has the ability to handle macromolecules.

Compound resolution and separation speed are both quite high.

A wide range of stationary phases and columns can be used to accommodate various application scenarios

Disadvantages:

Difficulty in separating certain protein-specific antibodies

It is challenging when some chemicals have low sensitivity to the stationary phase in the columns.

If mass spectrometry and HPLC are not interfaced, qualitative analysis might be constrained.

Very complex samples have a limited ability to resolve.⁶³

CONCLUSION:

One of the most popular analytical techniques is HPLC. It has a number of benefits over traditional chromatographic methods. To ensure that all of the components are accurately recognised, the selection of the detection strategy is essential. Chromatography is a separation process that uses a stationary and mobile phase to separate distinct compounds from a mixture. Accuracy, precision, and specificity can all be improved with HPLC. The cost of HPLC is its lone drawback.

REFERENCE:

Rogatsky E. Modern high performance liquid chromatography and HPLC 2016 International Symposium. J Chromatogr Sep Tech. 2016; 7:135.

Mulubwa M, et al. Development and validation of high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for determination of tenofovir in small volumes of human plasma. J Chromatogr Sep Tech. 2015; 6:300.

Santini DA, et al. Development of a high performance liquid chromatography method for the determination of tedizolid in human plasma, human serum, saline and mouse plasma. J Chromatogr Sep Tech. 2015; 6:270.

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. 2015; 6:383.

AL-Jammal MKH, et al. Development and validation of micro emulsion high performance liquid chromatography (MELC) method for the determination of nifedipine in pharmaceutical preparation. Pharm Anal Acta. 2015; 6:347.

Myron P, et al. Tributylamine facilitated separations of fucosylated chondroitin sulfate (fucs) by high performance liquid chromatography (HPLC) into its component using 1-phenyl- 3-methyl-5-pyrazolone (pmp) derivatization. J Chromatogr Sep Tech. 2015; 6:256.

Tang M, et al. HPLC analysis of monomer release from conventionally and high temperature high-pressure polymerised urethane dimethacrylate intended for biomedical applications. J Chromatograph Separat Techniq. 2014; 5:227.

Rao, B.V.; Sowjanya, G.N.; Ajitha, A.; Rao, V.U. A review on stability indicating HPLC method development. World Journal of Pharmacy and Pharmaceutical Sciences. 2015; 4(8):405-23.

Ahuja, S.; W dong, M. Handbook of Pharmaceutical analysis by HLPC, Elsevier: New York, 2005; 6

Akan JC, et al. Determination of organochlorine, organophosphorus and pyrethroid pesticide residues in water and sediment samples by high performance liquid chromatography (HPLC) with UV/visible detector. J Anal Bioanal Tech. 2014; 5:226

Parbhunath OL, et al. Optimization and validation of a reverse-phase high performance liquid chromatography assay with ultra-violet detection for measuring total l-ascorbic acid in food and beverage products. J Anal Bioanal Tech. 2014; 5:201

Szterk A, et al. Comparison of various detection systems coupled to high performance liquid chromatography for determination of tocopherols in meat. The influence and comparison of the most popular sample preparation method. J Anal Bioanal Tech. 2013; S2:005.

Lories IB, et al. High performance liquid chromatography, TLC densitometry, first-derivative and first-derivative ratio spectrophotometry for de-termination of rivaroxaban and its alkaline degradates in bulk powder and its tablets. J Chromatograph Separat Techniq. 2013; 4:202.

Chierentin L and Nunes Salgado HR. Development and validation of a simple, rapid and stability-indicating high performance liquid chromatography method for quantification of norfloxacin in a pharmaceutical product. J Chromat Separation Techniq. 2013; 4:171.

Srinivasarao K, et al. Validated method development for estimation of formoterol fumarate and mometasone furoate in metered dose inhalation form by high performance liquid chromatography. J Anal Bioanal Tech. 2012; 3:153.

Sun H, et al. A Rapid and effective method for simultaneous determination of residual sulfonamides and sarafloxacin in pork and chicken muscle by high performance liquid chromatography with accelerated solvent extraction–solid phase extraction cleanup. J Chromat Separation Techniq. 2012; 3:154.

Virkar PS, et al. Development and validation of a high performance liquid chromatography method for determination of telmisartan in rabbit plasma and its application to a pharmacokinetic study. J Anal Bioanal Tech. 2012; 3:133.

Gugulothu DB, et al. A versatile high performance liquid chromatography method for simultaneous determination of three curcuminoids in pharmaceutical dosage forms. Pharmaceut Anal Acta. 2012; 3:156.

Devika GS, et al. Simultaneous determination of eprosartan mesylate and hydrochlorthiazide in pharmaceutical dosage form by reverse phase high performance liquid chromatography. Pharm Anal Acta. 2011; 2:122.

Qiao G, et al. Modified a colony forming unit microbial adherence to hydrocarbons assay and evaluated cell surface hydrophobicity and biofilm production of vibrio scophthalmi. J Bacteriol Parasitol. 2012; 3:130

Types of HPLC. http://hplc.chem.shu.edu/NEW/HPLC_Book/Introduction/int_t yps.html. (Accessed Feb 12, 2017)

Jeffery, G.H.; Bassett, J.; Mendham, J.; Denney, R.C. Titrimetric analysis. Vogel’s textbook of quantitative chemical analysis, 5th edition; Longman Scientific and Technical, Essex: UK, 1989; 372-3.

Connors, A.K. A Text Book of Pharmaceutical Analysis, 3rd ed.; A Wiley Interscience publication: New York, 2005; 373- 400.

Harmita, et al. Optimation and validation of analytical method of cotrimoxazole in tablet and plasma in vitro by high performance liquid chromatography. J Bioanal Biomed. 2012; 4:26-29.

Nardulli P, et al. A combined HPLC and LC-MS approach for evaluating drug stability in elastomeric devices: a challenge for the sustainability in pharmacoeconomics. J Pharmacovigilance. 2014; 2:157.

Hafez HM, et al. Development of a stability-indicating HPLC method for simultaneous determination of amlodipine besylate and atorvastatin calcium in bulk and pharmaceutical dosage form. Pharm Anal Acta. 2014; 5:316.

Shintani H. Immobilized enzyme column combined with HPLC and column switching method for the analysis of complicated matrix such as body fluids. PharmaceutReg Affairs. 2014; 3:e142.

Murthy TGK and Geethanjali J. Development of a validated RP-HPLC method for simultaneous estimation of metformin hydrochloride and rosuvastatin calcium in bulk and in-house formulation. J Chromatogr Sep Tech. 2014; 5:252.

Suresh Babu VV, et al. Validated HPLC method for determining related substances in compatibility studies and novel extended-release formulation for ranolazine. J Chromatograph SeparatTechniq. 2014; 5:209.

Arayne MS, et al. Monitoring of pregabalin in pharmaceutical formulations and human serum using UV and RPHPLC techniques: application to dissolution test method. Pharm Anal Acta. 2014; 5:287.

Praveen C, et al. Method development and validation for simultaneous estimation of ethinyl estradiol and drospirenone and forced degradation behavior by HPLC in combined dosage form. Pharmaceut Anal Acta. 2013; 4:231.

Abdulla SA, et al. Validated HPLC method for the determination of nisoldipine. Pharm Anal Acta. 2013; S1:004.

Sawsan Mohammed AH, et al. Effects of blood collection tubes on determination vitamin-A by HPLC. J Chromat Separation Techniq. 2013; 4:184.

Subbaiah PR, et al. Method development and validation for estimation of moxifloxacin HCl in tablet dosage form by RP-HPLC method. Pharm Anal Acta. 2010; 1:109.

https://images.app.goo.gl/FQ4D1ngdU2VJyWha

Ahir KB, et al. Simultaneous estimation of metformin hydrochloride and repaglinide in pharmaceutical formulation by HPTLC-Densitometry method. J Chromat Separation Techniq. 2013; 4:166.

Khodadoust S, et al. A QSRR study of liquid chromatography retention time of pesticides using linear and nonlinear chemometric models. J Chromat Separation Techniq. 2012; 3:149.

Vali SJ, et al. Separation and quantification of octahydro-1h-indole-2-carboxilic acid and its three isomers by HPLC using refractive index detector. J Chromat Separation Techniq. 2012; 3:136.

Ndorbor T, et al. Chromatographic and molecular simulation study on the chiral recognition of atracuriumbesylate positional isomers on cellulose tri- 3, 5-dimethylphenycarbamate (CDMPC) column and its recognition mechanism. J Chromat Separation Techniq. 2013; 4:176.

Hua Z, et al. Extraction and purification of anthocyanins from the fruit residues of Vacciniumuliginosum Linn. J Chromat Separation Technique. 2013; 4:167.

Rogatsky E. 2D or Not 2D. Column-switching techniques, multidimensional separations and chromatography: approaches and definitions. J Chromat Separation Techniq. 2012; 3:159.

Al-Sagar KA and Smyth MR. Multi-Dimensional column chromatographic method with uv detection, for the determination of propranolol at therapeutic levels in human plasma. Pharmaceut Anal Acta. 2012; 3:197

Flores HE and Galston AW. Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol. 1982; 69:701-706

Euerby MR, Petersson P. Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis. J Chromatogr. 2003; 994(1-2):13-36.

https://www.google.com/imgres?imgurl=https%3A%2F%2F3.imimg.com%2Fdata3%2FKO%2FUA%2FMY-2624684%2Fhplc-pump-500x500.jpg&imgrefurl=https%3A%2F%2Fwww.indiamart.com%2Fmsp-lab-instruments%2Fsp930d-hplc-column-solvent-delivery pump.html&tbnid=HN0bANBp_nEoxM&vet=1&docid=aV0zIrAjS2agOM&w=500&h=333&source=sh%2Fx%2Film

https://images.app.goo.gl/afp8DZPb14KwAcWS8

Tyagi A, Nitin Sharma, Mittal K, Mashru R, Bhardwaj T, Malik J. HPTLC-densitometric and RP-HPLC method development and validation for determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in tablet dosage forms. Pharmaceutica Analytica Acta. 2015; 6(3):350.

https://images.app.goo.gl/u6ZnXtxw19h5zxW36

Dare M, Jain R, Pandey A. Method validation for stability indicating method of related substance in active pharmaceutical ingredients dabigatran etexilate mesylate by reverse phase chromatography. J Chromatogr A. 2015:6(2); 263

https://www.google.com/imgres?imgurl=https%3A%2F%2F4.imimg.com%2Fdata4%2FOD%2FTA%2FMY-2481501%2Fhplc-uv-detector-500x500.jpg&imgrefurl=https%3A%2F%2Fwww.indiamart.com%2Fproddetail%2Fhplc-uv-detector-1947969088.html&tbnid=vJ-l1qazmGYmnM&vet=1&docid=qjygK0sdCMJIyM&w=500&h=500&source=sh%2Fx%2Fim

Erk N. Rapid and sensitive HPLC method for the simultaneous determination of dorzolamide hydrochloride and timolol maleate in eye drops with diode-array and UV detection. Pharmazie. 2003 Jul; 58(7):491-493.

Donald DH, Shah M. Development and validation of HPLC stability indicating assays, in: sens t. carstensen, c.t.rhodes, editors drug stability-principle & practice. 3rd Edition. New York. Marcel Dekker Inc.332; 2008.

Lindholm J. Development and Validation of HPLC Method for Analytical and Preparati ve Purpose, Acta Universities Upsaliensis Uppsala 2004; 13-14.

https://images.app.goo.gl/EX6gfnp9nZaC6bWy6

Korodi T, Bukowski K and Lachmann B. Evaluation of a short stability indicating HPLC method for diclofenac sodium gels. Pharmazie.2012:67(12); 980-983

Mulgund S, Phoujdar M, Londhe S, Mallade P, Kulkarni T, Deshpande A, et al. Stability indicating HPLC method for simultaneous determination of mephenesin and diclofenac diethylamine. Indian J Pharm Sci. 2009:71(1); 35-45.

Vincent JB. Is chromium pharmacologically relevant, J Trace Elem. Med Biol. 2014:28(4); 397-405.

Heewon Lee, Pharmaceutical applications of liquid chromatography coupled with mass spectrometry (LC/MS), Journal of liquid chromatography & related technologies, 28, 2005, 7-8.

Salgado P, Visnevschi-Necrasov T, Kiene R.P, Azevedo I, Rocha A.C.S, Almeida C.M.R, Magalhaes C, Determination of 3-mercaptopropionic acid by HPLC: A sensitive method for environmental applications, Journal of Chromatography B, 992, 2015, 103-108.

Bailey F, Applications of high-performance liquid chromatography in the pharmaceutical industry, Journal of Chromatography A, 122, 1976, 73-84.

Ian M Bird, High-performance liquid chromatography: principles and clinical applications, BrMedJ, 299, 1989, 783- 7.

Ante M. Krstulovic, Investigation of catecholamine metabolism using high-performance liquid chromatography: Analytical methodology and clinical applications, Journal of chromatography B: Biomedical Sciences and Applications, 229, 1982, 1-34.

Ma Bokai, Gou XinLei, Zhao XinYing, Application of highperformance liquid chromatography in food and drug safety analysis, Journal of Food Safety and Quality, 7, 2016, 4295- 4298.

Shine sudev, Dr. Sree Janardhanan.V Review on HPLC Development Validation and Optimization: Int. J. Pharm. Sci. Rev. Res, 56(2), 2019; Article No. 05, Pages:28-43.

Received on 21.10.2022 Modified on 17.11.2022

Asian J. Pharm. Ana. 2023; 13(1):61-65.

DOI: 10.52711/2231-5675.2023.00011