Review on method development and validation of pharmaceutical dosage form by using UV visible spectroscopy and HPLC

 Corresponding author: Aishwarya Mukund Jadhav Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Review on method development and validation of pharmaceutical dosage form by using UV visible spectroscopy and HPLC Aishwarya Mukund Jadhav *, Sagar Eknath Tambe, Anamika Balasaheb Kakad and Radhika Sanjay Katpale Student, Samarth institute of pharmacy, Belhe, Maharashtra. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 Publication history: Received 08 October 2025; revised on 17 November 2025; accepted on 19 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.1018 Abstract UV visible Spectroscopy is a form of absorption spectroscopy; Spectroscopy is often used in physical and analytical chemistry for the identification of substance through the spectrum emitted from or absorbed by them. Spectroscopy is also heavily used in astronomy and remote sensing. Most large telescopes have spectrometer, which are used either to measure the chemical composition and physical properties of astronomical objects of to measure the velocities from the Doppler shift of their spectral lines. High performance liquid chromatography (HPLC) is an important qualitative and quantitative technique, generally used for the estimation of pharmaceutical and biological sample. It is the safest versatile, dependable and fastest chromatographic technique for the quality control of the drug component. Keywords: High Performance Liquid Chromatography; Principle; Instrumentation; Method Validation; Method Development; Application; Mobile Phase 1. Introduction The spectrophotometer also called as workhorse of the modern laboratory. Ultraviolet and visible spectrophotometer are the methods used for identification and measurement of organic and inorganic compounds. In branch of medicine, life science, molecular biology the essential tool is spectrophotometer. HPLCHigh Performance Liquid Chromatography or High-Pressure Liquid Chromatography. HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separation during shorter period of time, utilizing very small particles, small column diameters, and very high fluid pressures. 2. UV visible spectroscopy 2.1. History of UV visible spectroscopy The spectrophotometer was invented in 1940, by Arnold J. Beckman and his colleagues at National Technologies Laboratories, the company Beckman had started in 1935. They were led by project leader Howard H. Cary. The spectrophotometer was the company's greatest discovery. Once through the sample, the light collected in phototube for measurement. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 451 2.2. Introduction of UV visible spectroscopy: UV-Visible spectroscopy is an analytical technique that measures the number of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. The light plays an important role in this spectroscopy. Light has certain amount of energy which is inversely proportional to its wavelength. Thus, greater wavelength of light carries less energy and longer wavelength carry more energy. Humans are able to see a spectrum of visible light, from approximately 380 nm, which we see as violet, to 780 nm, which we see as red. 2.2.1. Spectroscopy Spectroscopy is the study of the properties of matter, through its interaction various types of radiation (mainly electromagnetic radiation) of the electromagnetic spectrum. Spectroscopy is an analytical technique. 2.2.2. Spectrometric Techniques Spectrometric Techniques is analytical methods that are based on atomic and molecular spectroscopy. Spectrometry and spectrometric methods refer to the measurement of the intensity of radiation with a photoelectric transducer or other types of electronic device. Electromagnetic radiation interacts with matter at specific energy levels. 2.2.3. The UV-Visible Spectrometry It is one of the oldest instrumental techniques of analysis. UV Visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy. It is the ideal methods for the determination of micro and semi micro quantities of analytes in a sample. Electromagnetic radiations in the UV and/or visible region with the absorbing species like, atoms, molecules or ions. It is analytical method that measures the amount of discrete wavelength of UV or visible light. This property will affect by sample composition. This spectroscopy technique relies on the use of light. Light has certain amount of energy which is inversely proportional to its wavelength. Thus, greater wavelength of light carries less energy and longer wavelength carry more energy. Humans are able to see a spectrum of visible light, from approximately 380 nm. The electron in different bonding environment in a substance require different specific amount of energy to promote the electron to higher energy state. 2.2.4. Beer’s Lambert’s Law When light is incident on homogeneous medium a part of incident light is absorbed, a part is reflected and remaining is allow to transmit. 𝑃𝑜 = 𝑃𝑎 + 𝑃𝑡 + 𝑃𝑟 2.2.5. Lambert’s Law A beam of monochromatic light is allowed to pass through a transparent medium the rate of decrease in intensity of light with thickness of medium is directly proportional to intensity of incident light. − − 𝑑𝐼/ 𝑑𝑡 ∝ 𝐼 2.2.6. Beer’s Law When a beam of monochromatic light is allowed to pass through a transparent medium the intensity of beam of monochromatic light decrease exponentially with concentration absorbance substance arithmetically. 𝐴 = 𝑎. 𝑏. 𝑐. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 452 3. How does UV visible spectrometer work? There are many variations on the UV-Visible spectrophotometer, to gain a better understanding of how an UV Visible spectrophotometer works, let us consider the main components. 3.1. Instrumentation of UV visible spectrophotometer It consists of important instrumental component are as follows: • Radiation Source • Wavelength selector • Sample holder • Detector • Amplifier • Recorder 3.2. Radiation source • It should possess following properties • It must be sufficient intensity for the transmitted energy to be detected. • It must be stable. • It must cover entire wavelength region in which it is used. Following is some examples of radiation sources: • Tungsten lamp • Hydrogen discharge lamp • Deuterium lamp • Xenon discharge lamp • Mercury arc lamp 3.3. wavelength selector Wavelength selector is used to select desired wavelength of radiations. The essential elements of wavelength selector are entrance slit, monochromator and exist slit. 3.3.1. Monochromator Monochromator select spectral band nearby to the desired wavelength. • There are 2 types of monochromators o Prism o Grating 3.4. Sample holder It also called as cuvette. The sample cell used to hold liquid solution. • The sample cell should fulfill 3 requirements: • Must be uniform of construction. • Must transmit light of wavelength used. • Must be inert to the solvent. 3.5. Detectors / Transducer Detectors are used to detect transmitted light which fall on them. When UV visible beam passes through sample holder. Detectors have 4 types World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 453 • Photovoltaic cell • Phototube • Photo multiplier tube • Silicon photodiode 3.6. Amplifier It enhanced the intensity of signal transfer by detector. 3.7. Recorder Used to record the signals There are 3 types of recorders • Pen recorder • Chart recorder • Now day electronic recorders are used. 4. Method development by UV visible spectroscopy 4.1. Quantitative techniques Single component and multi component analysis In single component analysis there is determination of single drug or API. 4.1.1. Methods used for single component analysis are • Use of absorptivity value • Calibration curve method • Single point standardization • Double point standardization 4.2. Use of absorptivity This procedure is adopted by official compendia, for stable substances which are practically unaffected by variation of instrumental parameters. The use of standard A (1%, 1cm) or € values avoids the need to prepare a standard solution of the reference substances in order to determine its absorptivity 𝑨 = 𝒂 𝒃 𝒄 Were, • A= Absorbance of the solution • a = Standard Absorptivity value • b = Path length of sample cell • c = Concentration of solution 4.3. Calibration curve method • A calibration curve is plotted using concentration Vs absorbance value of 5 standard solutions. • In this procedure the absorbances of a number 4-6 of standard solutions of the reference solutions at concentrations encompassing the sample concentration are measured and a calibration graph is constructed. • Calibration data is essential in concentration and absorbance show linear relationship. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 454 4.4. Single point standardization The single point procedure involves the measurement of the absorbance of a sample solution and of a standard solution of the reference substance. The standard and sample solutions are prepared in a similar manner; ideally, the concentration of the standard solution should be close to that of the sample solution. The concentration of the substance in the sample is calculated from the proportional relationship that exists between absorbance and concentration. 𝐶2 = 𝐶1 ∗ (𝐴 2 / 𝐴 1) Whereas, • C1 and C2 = Concentration of Standard and sample • A 2 and A 1 = Absorbance of standard and sample 4.5. Double point standardization Occasionally a linear but non-proportional relationship between concentration and absorbance occurs, which is indicated by a significant positive or negative intercept in a Beer’s law plot. A 'two-point bracketing’ standardization is therefore required to determine to concentration of the sample solutions. The concentration of one of the standard solutions is greater than that of the sample while the other standard solutions have a lower concentration than the sample. The concentration of the substance in the sample solution is given by the equation 𝐶2 = 𝐴1 − 𝐴2[(𝐴1 − 𝐴2) (𝐶2 − 𝐶3) + 𝐶1(𝐴1 − 𝐴2) ] 4.6. Multicomponent analysis It includes determination of two or more drug/API in sample. • Assay as a single component sample. • Assay using absorbance corrected of interference. • Assay after solvent extraction of sample • Simultaneous equation method. • Absorbance ratio method/Q method. • Geometric correction method. • Orthogonal polynomial method. • Different spectrophotometry. • Derivation spectrophotometry. • Simultaneous equation method If a sample contains two absorbing drugs (X and Y) each of which absorbs at the lambda max of other, it may be possible to determine both drugs by the technique of simultaneous equations. (Vierodt's method) provided that certain criteria apply. The information required is • The Absorptivity of X at lambda 1 and lambda 2, ax1 and ax2 respectively • The Absorptivity of Y at lambda 1 and lambda 2, ay1 and ay2 respectively • The absorbance’s of the diluted sample at lambda 1 and lambda 2, A1 andA2 respectively Let Cx and CY be the conc. Of X and Y respectively in the diluted sample. Two eq. are constructed based upon the fact that at lambda 1 and lambda 2 the absorbance of the mixture is the sum of the individual absorbances of X and Y. At lambda 1 𝐴1 = 𝑎𝑥1𝑏𝑐𝑥 + 𝑎𝑦1𝑏𝑐𝑦 …..... (1) At lambda 2 𝐴2 = 𝑎𝑥2𝑏𝑐𝑥 + 𝑎𝑦2𝑏𝑐𝑦 ……… (2) For measurements in 1cm cells, b=1. Rearrange eq. (2) World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 455 𝐶𝑦 = 𝐴2 − 𝑎𝑥2𝑐𝑥/𝑎𝑦2 Substituting for Cy in eq. (1) and rearranging gives 𝐶𝑥 = 𝐴2 ∗ 𝑎𝑦1 − 𝐴1 ∗ 𝑎𝑦2/𝑎𝑥2 ∗ 𝑎𝑦1 − (𝑎𝑥2 ∗ 𝑎𝑦2) 𝐶𝑦 = 𝐴1 ∗ 𝑎𝑥2 − 𝐴2 ∗ 𝑎𝑥1/𝑎𝑥2 ∗ 𝑎𝑦1 − (𝑎𝑥2 ∗ 𝑎𝑦2) 4.6.1. Absorbance ratio method The absorbance ratio method is a modification of the simultaneous procedure. It depends on the property that, for a substance which obeys Beer's Law at all wavelength, the ratio of absorbance at any two wavelengths is a constant value independent of concentration or pathlength. It also called as Q method. 𝐶𝑥 = {(𝑄𝑀 −𝑄𝑦)/ (𝑄𝑥 −𝑄𝑦)} − (𝐴1/𝑎𝑥1) 𝐶𝑦 = {(𝑄𝑀 −𝑄𝑥)/ (𝑄𝑦 −𝑄𝑥)} − (𝐴1/𝑎𝑦1) Other Applications- • Detection of conjugation. • Detection of geometrical isomer. • Detection of functional group. • Detection of impurities. • Molecular weight determination. • Dissociation constant determination. • Determination of chemical kinetics. • Determination of charge transfer transition. • Determination of tautomeric equlibrium. 4.7. HPLC 4.7.1. History of HPLC Liquid chromatography was initially discovered as an analytical technique in the early twentieth century and was first used as a method of separating colored compounds. This is where the name chromatography chroma means color, graphy means writing, was derived. A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic separation to purify mixtures of plant pigments into the pure constituents. He separated the pigments based on their interaction with a stationary phase, which is essential to any chromatographic separation. The stationary phase he used was powdered chalk and aluminia , the mobile phase in his separation was the solvent. After the solid stationary phase was packed into a glass column, he poured the mixture of plant pigments and solvent in the top of the column. He then poured additional solvent into the column until the samples were eluted at the bottom of the column. The result of this process most crucial to his investigation was that the plant pigments separated into bands of pure components as they passed through the stationary phase. Modern high performance liquid chromatography or HPLC has its roots in this separation, the first form of liquid chromatography. The chromatographic process has been significantly improved over the last hundred years, yielding greater separation efficiency, versatility and speed. 4.7.2. Introduction of HPLC The early problem with a liquid chromatography was the slow rate at which analysis take place. This problem was largely overcome by advent of high-performance liquid chromatography (HPLC). In this system pressure is applied to column, forcing the mobile phase at much higher rate. Factor affecting separation of liquid chromatography is : • Plate height • The sample distribution between the stationary and mobile phase. • And the selection of stationary and mobile phase. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 456 It has found that smaller the particle size, better will be the resolution. Pressure use normally ranges from 30 to 200 atm, a depending on the column use. 4.8. Principle of HPLC It is known that the resolving power of chromatographic column increases with column length and number of theoretical rates, although there is the limit to the length of the column due to the problem of peak broadening. The smaller the particle size of the stationary phases, better the resolution. Unfortunately, smaller the particle size, greater the resistance to eluent flow. 4.9. Theory of HPLC The particle size of the stationary phase material plays a very vital and crucial role in HPLC. In fact, high efficiency stationary phase materials have been researched and developed exclusively for HPLC having progressively smaller particle size termed as micro particulate column packings. These silica particles are mostly uniform, porous, with spherical or irregular shape, and having diameter ranging from 3.5 to 10 micrometer. High performance liquid chromatography is a chemistry technique is used to separate compounds of interested from a liquid mixture based on chemical and physical properties. When HPLC is coupled with a spectrophotometric detector, it can be used quantify analytes to provide detailed information on chemical composition of a sample. HPLC is commonly used in quality control or research laboratories in the chemical, agriculture, biotechnology, pharmaceutical, dietary supplement, and cosmetic industries. 4.9.1. Instrumentation Solvent reservoir and degassing system • Pump • Precolumn • Sample injection system • Chromatographic column • Column packing materials • Detectors Figure 1 Solvent reservoir and degassing system World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 457 4.10. Solvent reservoir and degassing system Degassing of these solvents should be done to remove air or oxygen dissolved in them, which if present in mobile phase, can ruptured the packing of the column or can produce unwanted peaks in chromatogram. Degassing of mobile phase can be done by • By filtration under vacuumMillipore filters are used for filtration of the mobile phase. • By distillation of mobile phase – when the required mobile phase is distilled under vacuum, the distillate becomes free of dissolved air or oxygen. • By ultrasonication - Ultrasonication is the sonication using ultrasound i.e. sound with the frequency greater than the upper limit of human hearing (approx 20 KHz). • By sparging of an inert gas of low solubilitysparging (gas flushing), which involve bubbling a chemically inert gas of low solubility such as Ar or He through the mobile phase. This can be used to remove oxygen and nitrogen that interfere by forming bubbles in the column. 4.10.1. Pumps The pumps are used to pass mobile phase through the column at high pressure and at control flow rate. The pumps are categorized into • Displacement pumps or syringe pumps • Reciprocating pumps • Pneumatic pumps • Constant displacement pumps or syringe pumps It consist of a large, syringe like chamber equipped with a plunger that is activated by a screw driver mechanism powered by a stepping motor. 4.10.2. Reciprocating pump It consists of small chamber in which the solvent is pushed back and forth with the help of a motor driven piston or pressure may be transmitted by a diaphragm which is hydraulically pumped by reciprocating piston. Pneumatic pumps: In this the mobile phase is contained in a collapsible container housed in a vessel that can be pressurised by a compressed gas. 4.11. Precolumn (Guard column) Precolumn is broader the ananalytical column. Precolumn is placed between pump and sample injection System. So that only mobile phase is passed through the precolumn. The precolumn is mainly used to remove the impurities from the solvent and thus prevent contamination of the analytical column. 4.11.1. Sample injection system • It is used to introduce a fixed volume of the sample solution into the mobile phase. • The sample injectors are of the following types: • Syringe injection: - This is the earliest and simplest technique. Hence the sample is injected through a self sealing elastomeric septum andthe syringes are designed to withstand pressure upto 1500 psi. • Stop flow injection: - In this a fitting at the column head is removed and sample is injected directly onto the head of the column packing at atmospheric pressure. Then the fitting is replaced and the system is again pressurised. • Solvent flowing: -This type of injectors is usually used for injecting sample volumes more than 10 microliter. In the fill position the sample loop is filled at atmospheric pressure. When the valve is actuated the sample in the loop is also actuated. 4.12. Chromatographic column Usually constructed from smooth bore stainless steel tubing or heavy-walled glass tubing. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 450–463 458 The columns are of 2 types 4.12.1. Analytical column Size: Length: 25 to 100 cm with internal diameter: 2 to 6 mm. 4.12.2. Preparative column Size: Length: 25 to 100 cm and internal diameters: 6 mm or more. 4.13. Column packing materials Two basic types of packing shave been used in HPLC Pellicular 4.13.1. Porous Particle • Pellicular: It consist of small spherical glass or polymer beads with a diameter of 30 to 40 uncoated with 1 to 3umlayer of a porous material such as silica gel, alumina or an ion exchange resin. • Porous Particle: These particles have diameter ranging from 3 to 10 um and are composed of silica, alumina or an ion exchange resin. 4.13.2. Detectors A detector is required to sense the pressure and the amount of sample component in the column effluent. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and or the solute. A detector that measures property which is possess by both mobile phase and solute called as Bulk property detector. E.g refractive index detector, conductivity detector. Alternatively, if the property is possessed essentially by the solute e.g. absorption of UV visible light of electro chemical property, the detector is called as Solute property detector. E.g. UV detectors, Fluorescence detector. 4.14. Method development on HPLC A step involved in method development of HPLC is as follows • Understanding the Physicochemical properties of drug molecule • Selection of chromatographic conditions • Developing the approach of analysis • Sample preparations • Method optimization • Method validations 5. Understanding the Physicochemical properties of drug molecule Physicochemical properties of a drug molecule play an important role in method development. For Method development one has to study the physical properties like solubility, polarity, pKa and Ph of the drug molecule. Polarity is a physical property of a compound. It helps an analyst, to decide the solvent and composition of the mobile phase. The solubility of molecules can be explained on the basis of the polarity of molecules. Polar, e.g. water, and non-polar, e.g. benzene, solvents donor mix. In general, like dissolves like i.e., materials with similar polarity are soluble in each other. The selection of mobile phase or diluents is based on the solubility of analyte. The analyte must be soluble in diluents and must not react with any of its component. Ph and pKa plays an important role in HPLC method development. The Ph value is defined as the negative of the logarithm to base 10 of the concentration of the hydrogen ion. 𝑝𝐻 = − 𝑙𝑜𝑔10 [𝐻3𝑂+] Selecting a proper Ph for ionisable analytes often leads to symmetrical and sharp peaks in HPLC.