Dr. Subhash Technical Campus, Junagadh.
Spectroscopy is a scientific method that analyses the interaction of matter with light or electromagnetic radiation. It can be divided into atomic, molecular, absorption, and electronic levels, with UV spectroscopy being the most widely used for analysing various compounds. UV spectrophotometric methods use the principle of additivity and absorbance to record and mathematically process absorption spectra of standard solutions and sample solutions. This process leads to electron excitation, causing transitions between energy levels. Multicomponent analysis is a growing challenge in modern chemistry, with multimolecular techniques being popular for determining drug components and reducing pollution in clinical drug analysis. Combination drug products and fixed-combination drugs play crucial roles in therapeutics, and multicomponent analysis can be applied to overlapping drug spectra. This article includes simultaneous determination method like Simultaneous equation method, Q-Ratio method, Dual wavelength method Derivative spectroscopy, Dual wavelength, Difference spectroscopy, Area under curve etc.
Spectroscopy is the branch of science that deals with interaction of matter with light or electromagnetic radiation. Spectroscopy is made up of two words: spectrum and spokien. In spectroscopy, band of different colors formed due to difference in wavelength is known as spectrum. The most important consequence of such interaction is that energy is absorbed or emitted by the matter indiscrete amount called quanta. Spectroscopy is used for qualitative and quantitative analysis. [1]
Spectroscopy can be conveniently divided based on:
Table:1 Whether the study is made at atomic or molecular level:
|
Atomic spectroscopy |
Atomic Absorbance, Flame photometry |
|
Molecular spectroscopy |
UV Spectroscopy, Colorimetry, IR Spectroscopy |
Table:2 Whether the study is based upon absorption or emission:
|
Molecular spectroscopy |
UV Spectroscopy, Colorimetry, IR Spectroscopy |
|
Absorbance spectroscopy |
UV Spectroscopy, IR Spectroscopy, NMR |
Table:3 Whether the study is at magnetic or electronic level:
|
Electronic spectroscopy |
UV spectroscopy, Colorimetry, Fluorimetry |
|
Magnetic spectroscopy |
NMR, ESR |
UV-VIS spectroscopy is considered as the most important spectrophotometric technique that is most widely used for the analysis of variety of compounds. Among the various spectroscopic techniques, UV spectroscopy emerges as a powerful analytical tool, utilizing light within the UV or visible region with wavelengths ranging from 200 to 800 nm. This technique stands versatile, capable of analyzing both colorless compounds in the UV range (400-200 nm) and colored compounds in the visible range (800-400 nm). [2,3]
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Figure:1. Region in Spectroscopy [2]
Table:4 Wavelength of Visible Region
|
colors |
Wavelength region |
|
Violet |
400-420nm |
|
Indigo |
420-440nm |
|
Blue |
440-490nm |
|
Green |
490-570nm |
|
Yellow |
570-585nm |
|
Orange |
585-620nm |
|
Red |
620-780nm |
The basic principle behind the UV spectroscopy is absorption of visible and UV radiation (200 400 nm) is associated with excitation of electrons, in both atoms and molecules, from lower to higher energy levels. Since the energy levels of matter are quantized, only light with the precise amount of energy can cause transitions from one level to another will be absorbed. UV spectrophotometric methods based on principle of additivity and absorbance, recording and mathematical processing absorption spectra of standard solutions and sample solutions in same way or differently. [4] In routine practice, analyst has to perform rapid analysis of multicomponent formulations, biotherapeutic products and samples of a complex matrix. Number of ultraviolet (UV) spectrophotometric methods used for these purposes. However, among all of these methods, UV spectrophotometry is favorite tool. [4] UV spectrophotometric techniques are mainly used for multicomponent analysis thus minimizing the cumbersome task of separating interferents and allowing the determination of an increasing number of analytes, consequently reducing analysis time and cost Analysis of samples with numerous components presents a major challenge in modern analysis. Multicomponent analysis has become one of the most appealing topics for analytical chemists in the last few years, in fields as clinical chemistry, drug analysis, analysis of drugs in mixtures, pharmaceutical preparations, pollution control…. etc. Multicomponent spectrometric methods involve the simultaneous determination of compounds in a multicomponent system. [5]
Terms:
Chromophores:
Many organic molecules absorb ultraviolet/visible radiation and this is usually because of the presence of a particular functional group. The groups that actually absorb the radiation are called chromophores. It may or may not impart any color to the compound. [5]
Auxochrome:
The Color of a molecule may be intensified by groups called Auxochrome which generally do not absorb significantly in the 200-800nm region, but will affect the spectrum of the chromophore to which it is attached. The most important Auxochrome groups are OH, NH2, CH3 and NO2 and their properties are acidic (phenolic) or basic. [6]
Bathochromic shift or red shift:
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Figure:2 Spectroscopy Terms [6]
It involves shift of absorption maximum towards longer wavelength because of [7]
I. Presence of certain groups such as -OH and -NH2 called Auxochrome or
II. By change of solvent.
E.g. Decreasing the polarity of solvent causes red shift in n* absorption of carbonyl compounds.
III. When two or more chromophores are present in conjugation in a molecule.
E.g. Ethylene shows T’T* transition at 170 nm whereas 1,3 butadiene shows at 217 nm.
Hypochromic shift or Blue shift:
It involves shift of absorption maximum towards shorter wavelength because of removal of conjugation in the system or by changing polarity of solvent. [8]
Hyperchromic effect:
It involves increase in intensity of absorption. It is caused by introduction of an Auxochrome. [9]
Hypochromic effect:
It involves decrease in intensity of absorption. It is caused by groups which are able to distort geometry of the molecule. [9]
Isosbestic point:
In spectroscopy, an isosbestic point is a specific wavelength, wavenumber or frequency at which the total absorbance of a sample does not change during a chemical reaction or a physical change of the sample. The word derives from two Greek words: "iso", meaning '"equal", and "sbestos", meaning "extinguishable”. [10] Isosbestic point is the wavelength where the molar absorptivity is the same for two substances that are interconvertible. It is used to determine the concentration of a substance present along with impurities. When isosbestic point is used, irrelevant absorption due to impurities is eliminated. It can be determined by recording the absorption curves of same substance at different pH on the same paper and noting the point of intersection.
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Figure:3 Isosbestic Point
Multicomponent analysis:
It refers to a mixture containing multiple components, where the conc. Of all component are determined simultaneously using a single spectrum or set of spectrums. These methods were applied to solve different complex pharmaceuticals mixtures. These developed methods were simple and cost-effective. [11] Combination drug products occupy a time-honored and important role in therapeutics. When rationally formulated, fixed-combination drugs may produce greater convenience, lower cost, and sometimes greater efficacy and safety. The spectrophotometric multi-component analysis can be applied where the spectra of drugs overlap. In such cases of overlapping spectra, multi-component analysis can be applied to any degree of spectral overlap provided that two or more spectra are not similar. [12]
Principle of multicomponent spectroscopy: If a system is containing several absorbing components, it follows the principle of additivity of absorbances, i.e. the absorbance of a system containing several components is equal to the sum of absorbances of all individual components at a particular wavelength, and if no mutual interaction takes place between them, then Where is the molar absorptivity of the component ‘i’ at the λ wavelength and “L” is the path length. [12]
Advantages of Multicomponent Analysis:
Limitation:
Methods of Multicomponent Analysis:
Simultaneous Equation Method:
It also called Vierodt’s method. One of the most common and easiest methods employed for Spectrophotometric multi-component analysis in which concentration of several components present in the given mixture can be determined by solving a set of simultaneous equation even if their spectra overlap. [13,14] This method derives its principal from the additive nature of absorbance of individual components in any mixture. If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λmax of the other, it may be possible to determine both drugs by the technique of simultaneous equation.
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Figure:4 Graph of Simultaneous Equation Method [15]
Criteria:
Equation:
Cx=A1ay1-A1ay2Ax2ay1-ax1ay2
Cy=A1ax2-A2ay1ax2ay1-ax1ay2
Absorption Ratio Method:
Absorption Ratio Method is also known is Q-Ratio method. Absorption ratio method is used for the ratio of the absorption at two selected wavelengths one of which is iso-absorptive point and other being the λmax of one of the two components. [16] It is multi component method of analysis using UV spectrophotometer. There is no need of separation of multi component present in sample or formulation. It is modified version of simultaneous equation method. It depends on the property that, for a substance which obeys Beer’s-Lambert’s law at all wavelength, the ratio of absorbance at any two wavelengths is constant value independent from concentration and pathlength. It involves measurement of absorbance at two different wavelength one being the λmax of one drug and other being an iso-bestic wavelength. Iso-bestic point is defined as a specific wavelength, at which the total absorbance of a sample doesn’t change during chemical or physical change of sample. At iso-bestic point both components shows same absorbance or absorptivity. [17]
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Fig:5 Absorbance Ratio Method [17]
If ratio of A1/A2 and ratio of A3/A4 is constant then the ratio is called as Q-Value. It indicates that it is independent from pathlength and concentration.
Criteria:
Equation:
Cx=Qm-Qy⋅A1Qx-Qy⋅ax1
Derivative Spectroscopy:
Between 1953 and 1955 Hammond et al., Morrison and Giese and French introduced this method, which is becoming increasingly important. [19] Derivative spectroscopy is technique for altering the spectrum data for the aim for of spectral analysis to relate chemical structure to electronic transitions and for analytical circumstances when mixing contributes interfering absorption. It is used for background correction. It involves the mathematical derivative of absorbance with respect to the wavelength of radiation is calculated by instrument itself, electronically or by using microcomputers. It involves the conversions of a normal spectrum to its first, second, third and higher derivative spectrum. In derivative spectroscopy, the normal absorption spectrum is referred to as the fundamental, zero or D° spectrum. [20]
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Fig :6 Derivation from Normal Spectrum [21]
The derivative spectra are more structured than the original spectra, since the number of peaks goes on increasing with the increase in order of derivative. In first order derivative spectrum the original bond splits into two. In second order derivative spectrum, the original band split into three. The number of peaks in spectrum of order ‘n’ would be ‘n+1’.
Equation: [21]
dA /dλ=c.d. dε /dλ [8]
d2A /d2λ =c·d· d2ε / dλn
dnA /dnλ -=c·d. dnε / dλ n
Dual Wavelength:
Dual wavelength also known as two wavelength method. In this method two wavelengths were selected for the estimation of each drug in such a way that the difference in the absorbance was zero for the second drug on the respective wavelength for the first drug. [22] Dual wavelength spectrophotometry can be used to determine an unknown conc. Of a component of interest that is present in mixture containing both the component of interest & an unwanted interfering component by determining the difference in absorption ( ?A ) between two points in spectrum of mixture. The method has been useful for measurement of small changes in absorbance in high absorbing backgrounds. This method is suitable for suspension analysis. In dual wavelength method the selection of two wavelength such as interfering component shows the same absorbance, while the component of interest shows a significant difference in absorbance with change in concentration. The tungsten-iodide or deuterium lamp is used as light source in dual wavelength method. [23] The absorbance difference between two point in the mixture of spectra is directly proportional to concentration of component of interest. In dual wavelength spectrophotometry, the absorbance difference ( ?A ) between two wavelengths γ1 & γ2 is measured. There is no need of reference. In this method the one drug is considered as a component of interest & other is considered as an interfering & vice-versa.
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Fig:7 Dual Wavelength
Criteria:
Equation:
Difference Spectroscopy:
The essential feature of this method is that the measured value is the absorbance difference (ΔA) between two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics. The most straightforward and widely used method of modifying the spectral characteristics is to modulate the PH using an aqueous solution of acid, alkaline and buffer. [25] It provides a sensitive method for detecting small changes in environment of chromophore, it can be used to demonstrate ionization of chromophore leading to identification and quantitation of various component in mixture. The selectivity and accuracy of spectrophotometric analysis of sample containing absorbing interference may be markedly improved by the technique of difference spectrophotometry. Apart from pharmaceutical assay, difference spectroscopy also used in biopharmaceutical formulation development to characterize protein structure and to investigate the response of structure to the formulation composition. [26] This application based on the fact that stable protein conformations provide high real-time physical stability and difference spectra used for characterizing and quantifying changes in protein structure.
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Fig:8 Spectrum of compound in A(acid) B(Base) & Difference spectrum of B relative to A[17]
Criteria:
Equation: [28]
Area Under the Curve:
Area under curve method is a newly established spectrophotometric method provides a simple way to determine concentration of the component of interest depending on area of its absorption spectrum. The area under curve approach can be used when a broad spectrum or sharp peak is obtained. [29] It entails figuring out the integrated absorbance value with respect to wavelength between two chosen wavelength λ1 & λ2 . In order to determine the linearity between AUC & concentration, this wavelength range was chosen based on repeated observations Area calculation processing item calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by the entering the wavelength ranges over which area has to be calculated.
Criteria:
Equation:
A1 = ax1 C(x) + ay1 C(y) (λ1−λ2) nm
A2 = ax2 C(x) + ay2 C(y) (λ3−λ4) nm
C (x) = [A2 × aX2−A1 × ay2]/ [aX2 × aY1−aX1 × aY2]
C (y) = A2−aX2 × C(x)/aY2
Absorptivity Factor Method:
This method is a modification of classical absorption method. [31] The ratio between the two absorptivity (ax, aY) at intersection point with the same absorbance value. This point is called the absorptivity factor point Unlike the isoabsorptive point technique, the crossing of spectra might happen at different drug concentrations rather than at the same concentration. In the absorptivity factor approach, absorptivity equals the inverse ratio of the concentrations used at this crossing point. The crossing point is referred to as the absorptivity factor point, and the ratio discovered is known as the absorptivity as absorptivity factor (F). [32]
Criteria:
Equation:
Absorption Factor Method
Absorption factor method, a spectroscopic method, also used for the analysis of binary sample mixtures having overlapping spectra and, in those cases, where it is found that one compound exhibits some interference at the λmax of another compound, while another compound does not exhibit any interference at the λmax of the other compound. [33] This method is applicable for the ex vivo and in vitro characterization of the drugs for topical delivery. It is also applicable for the drugs which are present in the biological samples. Let’s consider a mixture of X, Y having a wavelength maximum at λx and λy where Y shows some interference at λx but X does not show interference at λy. Then, from the binary mixture of X, Y by subtracting the value of absorption Y at λx, the absorption value of X can be calculated quantitatively.
Criteria:
Equation:
Difference between absorption factor method & absorptivity factor method:
Absorption correction method:
Absorption correction method (ACM) is a simple spectrophotometric method which involves simultaneous estimation of both the drugs at their own λmax. ACM is the modification of simultaneous estimation method. Here, quantitative determination of one drug is carried out by A (1%, 1 cm) and quantitation of other drug is carried out by subtracting absorbance of the other drug using absorption factor. [35]
Criteria:
Equation:
Absorbance Subtraction Method:
This is a novel, simple accurate, specific spectrophotometric method which is applied for the simultaneous determination of two drugs and also there is no need of prior separation steps. The principle of this method is similar to the principle of absorption factor method and the method is applied for the analysis of the binary mixture of drugs (X and Y) having overlapped spectra which intersect the isoabsorptive point and also it is found that one compound exhibits some interference at the λmax of another compound, while another compound does not exhibit any interference at the λmax of the other compound. [37] The absorbance values of component X and Y at λiso are calculated by the help of absorbance factor {Aiso/A2} is a constant for the pure component Y which represents the average of the ratio between the absorbance values of different concentrations of pure component Y at λiso (Aiso) at λ2 (A2). [38]
Criteria:
Equation:
REFERENCES
Shivani Herma*, Kiran Rathod, Dr. Chintankumar Tank, Denisha Meghnathi, Himanshu Parmar, Pritesh Odedara, Unraveling Complexity: A Critical Review of UV Spectrophotometric Methods for Multicomponent Analysis, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 588-602. https://doi.org/10.5281/zenodo.15591790
10.5281/zenodo.15591790
