A Brief Review on Dual Wavelength Spectrophotometry:

The Simultaneous Estimation Method and its Application

 

Amitkumar J. Vyas¹, Dhara U. Desai¹*, A. B. Patel¹, A. I. Patel¹, S.R. Shah¹, D. B. Sheth²

¹Department of Pharmaceutical Quality Assurance, B.K. Mody Government Pharmacy College Polytechnic Campus, Near Aji Dem, Bhavnagar Road, Rajkot - 360003 Gujarat, India.

²L. M. College of Pharmacy, Navrangpura, Ahmedabad - 380009 Gujarat, India.

 

ABSTRACT:

The dual wavelength spectrophotometry is developed and validated for simultaneous estimation of various combinations of two drugs. Dual wavelength method is used to eliminate interference due to absorbance of other drug at sampling wavelengths for one drug. Two wavelengths are selected for each drug in such a way that the difference in absorbance is zero for the second drug. The principle for dual wavelength method is the absorbance difference between two points on the mixture spectra is directly proportional to the concentration of the component of interest and independent of interfering component. The Following review article contains a brief regarding dual wavelength spectrophotometry introduction, principle, examples and application of different pharmaceutical, organic and inorganic compounds.

 

 

 

INTRODUCTION:

Dual-wavelength spectrophotometry methods can be used to determine an unknown concentration of a component of interest that is present in a mixture containing both the component of interest and an unwanted interfering component by determining the difference in absorbance between two points in the spectrum of the mixture. This method has been useful for the measurement of small changes in absorbance in systems containing high-absorbing backgrounds. It was also a promising analytical technique for suspension analysis.

 

The prerequisite for the application of a dual-wavelength method is the selection of two wavelengths such that the interfering component shows the same absorbance while the component of interest shows a significant difference in absorbance with changes in concentration. The technique consists of using a reference wavelength to correct for a highly absorbing background. In addition, dual wavelength spectrophotometry requires extremely small quantities of sample for analysis.¹ The purpose of stability and related substance study is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors^2-4. HPLC, UV Spectrophotometric methods, and LC-MS/MS methods are essential in ensuring quality control in pharmaceuticals, where substance stability and integrity are crucial for safety and effectiveness^5-11. Presence of impurities critically affects the stability and pharmacological action of pharmaceutical API and drug product.^12-16. Analytical quality by design [AQbD] and CCD help in regulatory compliance for RP-HPLC method development, stress testing or stability indicating methods^17-20.

 

PRINCIPLE:

Since the dual-wavelength technique is not yet very familiar in analytical chemistry, we shall first of all describe the principle involved.

 

In dual-wavelength measurement, light from a highly stabilized tungsten-iodide or deuterium lamp is divided into two beams in two grating monochromators. The light beams of different wavelength from the two gratings are time-shared through a single cell by means of a rotating sector, and the difference ∆A between the absorbance at wavelength λ₁ and λ₂ is measured. Even a very slight change in the absorbance of a sample can thus be determined accurately on using maximum scale expansion range of absorbance because the various sample and cell errors which may occur and its classical spectrophotometry are eliminated: only one cell is used and the radiation at λ₁ and λ₂ is incident on the same position of the cell.²¹

 

 

Figure 1: Principle of dual wavelength spectrophotometry, L-light source, G1andG2-gratings, P-PMT, r-rotating sector, C- cell

 

Dual wavelength method "also known a two wavelengths method" facilitates analyzing a component in presence of an interfering component by measuring the absorbance difference (∆A) between two points in the mixture spectrum.

 

Figure 2: Selection of wavelengths for dual wavelength method.

 

In dual-wavelength spectrophotometry, the absorbance difference ∆A between two wavelengths λ₁ and λ₂ is measured as shown in Figure, so that no reference solution is required. The most important prerequisite is a linearity or proportionality between measured signals and concentration of the component under investigation. Thus, many types of background can be eliminated by selecting a suitable combination of two wavelengths.²¹

 

In this method one of the drugs is considered as a component of interest and the other drug is considered as an interfering component and vice-versa. The basis for such method is the selection of two wavelengths where the interfering component shows the same absorbance (∆A equals zero) whereas the component of interest shows significant difference in absorbance with concentration. ∆A between two points on the mixture spectra is directly proportional to the concentration of the component of interest independent of interfering component.²²

 

 

INSTRUMENTAL ASPECTS (ANALYSIS OF MIXTURE):²³

Equi-absorbance method:

Suppose that a two-component sample contains x and y. The measured absorbances at each wavelength λ₁ and λ₂ are

 

 

where, ε_x and ε_y are the molar absorptivities of the x and y components at λ_l, respectively ε_x´ and ε_y´ are the molar absorptivities of the two components at λ₂ and c, are the concentrations (mol/L) of the components.

 

Then, the absorbance obtained in the dual-wavelength measurement is

 

 

 

If the second component y shows the same absorbance at both wavelengths λ₁ and λ₂ i.e., if A_λ2 – A_λ1 =0, equation simplifies to

 

 

Since the difference absorbance ∆A is now no longer dependent on the concentration of the component y, it is possible to eliminate the influence due to component y.

 

APPLICATION:

Pharmaceutical Application:

Table 1: Analytical Characteristics of Dual Wavelength Procedure for Simultaneous Determination of Pharmaceutical Compound

S. No	Compound Name	Wavelength(λ) (nm)	Solvent	Linearity	Ref
1	Ambroxol HCL and Desloratadine HCL	253.2 and 258.5 and 301.2 and 314	0.1 N HCl	5-75 μg/ml 5-75 μg/ml	24
2	Amoxicillin and potassium clavulanate	261 and 226 and 279 and 225	0.1M NaOH	10-50 µg/ml 2-10 µg/ml	25
3	Atenolol and Indapamide	266 and 270. 2 and 246.4 and 252.2	Methanol	100-350 µg/ml 5-17.5 µg/ml	26
4	Cilnidipine and Olmesartan Medoxomil	352.92 and 282.99 and 337.85	Methanol	10-60 μg/ml 20-120 μg/ml	27
5	Drotaverine HCL and Aceclofenac	301.5 and 311.0 and 271.5 and 280.0	Methanol	8‐32 μg/ml 10‐40 μg/ml	28
6	Hydrochlorothiazide and Telmisartan	258 and 299 and 316 and 326	0.1 N NaOH	2-20 μg/ml 2-20 μg/ml	29
7	Olmesartan Medoxomil and Hydrochlorothiazide	242 and 263 and 253 and 284	Methanol	4 -32 μg/ml 2.5-20 μg/ml	30
8	Amlodipine and Atorvastatin	238 and 243.8 and 246.6 and 243.8	Methanol	4 -40 µg/ml 8-32 µg/ml	31
9	Cinnarizine and Domperidone	240.2 and 273.2 and 230.8 and 259.2	Methanol	40–180 µg/ml 60–120 µg/ml	32
10	Clotrimazole and Beclomethasone Dipropionate	237 and 241 and 259 and 264	Methanol	100-450 µg/ml 6-34 µg/ml	33
11	Nitazoxanide and Ofloxacin	271.5 and 359.5 and 300.5 and 365.5	Mixture of dichloromethane and n-Hexane (60:40)	5-25 μg/ml 2-10 μg/ml	34
12	Elbasvir and Grazoprevir	351 and 315 and 375 and 334.5	Methanol	-	35
13	Isbesartan and hydrochlorothiazide	263.4 and 281 and 243.4 and 247.6	0.1 N NaOH	5-15 μg/ml 4-12 μg/ml	36
14	Atorvastatin and Ezetimibe	228.8 and 240.8 and 232 and 259.2	Methanol	5-50 μg/ml 5-30 μg/ml	37
15	Atorvastatin and Fenofibrate	238 and 256 and 259 and 298	Methanol	5-30 μg/ml 5-30 μg/ml	38
16	Ofloxacin and Cefpodoxime proxeil	224 and 247.4 and 278.2 and 320	Methanol	2-12 μg/ml 4-24 μg/ml	39
17	Alogliptin and Pioglitazone	270 and 265 and 280 and 271	Methanol	-	40
18	Avanafil and Dapoxetine hydrochloride	206.80 and 214.80 and 241.20 and 251.60	Methanol	1-6 μg/ml 1-3.5 μg/ml	41
19	Empagliflozin Metformin	232 and 244 and 224 and 238	Methanol	2–10 μg/ml 4–20 μg/ml	42
20	Diclofenac Esomeprazole	281.7 and 315 262.6 and 301.6	Methanol	5-25 μg/ml 2-10 μg/ml	43
21	Paracetamoland Nabumetone	237.4 and 214.4and 332.6	Methanol	4 – 20 μg/ml 2 – 20 μg/ml	44
22	Ethamsylate and Mefenamic acid	274.4 and 301.2 and 304.8 and 320.4	Methanol	0-50 μg/ml 0-50 μg/ml	45
23	Pyrimethamine and Sulphadoxine	278 and 295and 246 and 298	Methanol	3-18 µg/ml 7-42 µg/ml	46
24	Paracetamoland Diclofenac sodium	245 and 249and 257 and 294	Urea + distilled water	5-35 mcg/ml 5-40 mcg/ml	47
25	Amlodipine besylateand Metoprolol succinate	365and 226 and 248.7	Water	2–25 µg/ml 2–30 µg/ml	48
26	Naproxenand Esomeprazole Magnesiumtrihydrate	312 and 298and 314 and 334.2	0.1M NaOH	15-75 µg/ml 2.8-6.0 µg/ml	49
27	Metoprolol succinateand Telmisartan	282.4 and 284.6and 330	Methanol	3-20 µg/ml 4-16 µg/ml	50
28	Carvediloland Hydrochlorothiazide	238 and 248.8and 266 and 289.4	0.1N HCl	1–10 μg/ml 1–10 μg/ml	51
29	Thiocolchicosideand Diclofenac Sodium	371and 248 and 268 248 nm and 26	Methanol	2–16 μg/ml   12.5–100 μg/ml	52
30	Alprazolam and Propranolol Hydrochloride	258.2 nmand 319.4 nm	Methanol	1–40 μg/ml 80–200 µg/mL	53
31	Norfloxacinand Tinidazole	277.2 and 336.2and 309 and 254.6	0.01 M HCl	2-10 μg/ml 4-40 μg/ml	54
32	Rivaroxabanand Aspirin	250 and 286.44and 243.53 and 259.2	Acetonitrile	2 – 12 μg/ml 40-240 μg/ml	55
33	Meropenemand sulbactam sodium	242.0 and 274.0and 285.0 and 306.0	0.1N NaOH	4–24μg/ml 2–12 μg/ml	56
34	Amlodipine Besylate and Olmesartan Medoximil	232 and 241.6and 244 and 255.4	Methanol	2.5-12.5 μg/ml 5-25 μg/ml	57
35	Sofosbuvir and Ledipasvir	252 and 310.70and 332 and 310.70	Methanol	10- 30 µg/ml 2.25 – 6.75 µg/ml	58
36	Amlodipine Besylateand Indapamide	232 and 242.0and 235 and 245	Methanol	5-25 μg/ml 2-10 μg/ml	59
37	Efonidipine hydrochloride ethanolate and Telmisartan	242.5 and 257.5and 244.5 and 287	Methanol	8-20 μg/ml 16-40 μg/ml	60
38	Atorvastatin calciumand Amlodipine besylate	259.9 and 354and 363	50% Methanol	0-40 µg/ml 0-20 µg/ml	61
39	Carbamazepineand Lamotrigine	304 and 313and 282 and 290	Ethanol: Water (2:1)	1-50 µg/ml 1-80 μg/mL	62
40	Emtricitabineand Tenofovir	230.696 and 250and 250 and 268.670	Methanol	4-32 µg/mL 6-48 µg/mL	63
41	Ketotifenand Salbutamol	284 and 267.84and 315 and 284.59	0.1N HCL	5-25 μg/ml 10- 50 μg/ml	64
42	Metoprolol succinate and Olmesartan medoxomil	225.2 and 258.2and 211 and 229.8	-	5-30 μg/ml 5-30 μg/ml	65
43	Losartan potassium and Hydrochlorothiazide	206.6 and 261.4and 270.6	0.1 N HCL	6.4 -19.2 µg/ml 1.6-4.8 µg/ml	66
44	Antipyrine and Benzocaine	254.1 and 309.1and 230.1 and 263.5	Double distilled water	5–50 μg/ml 1–20 μg/ml	67
45	Ciprofloxacin Hydrochloride and Metronidazole	281.5 and 335.5and 313 and 324	Methanol	30-160 mg 30- 160 mg	68
46	Dapoxetine hydrochlorideand Sildenafil citrate	213.2 and 226.09and 242.09 and 271.70	Methanol	2–12 µg/ml 10 - 60 µg/ml	69
47	Cilostazol and Telmisartan	284 and 264.9and 243.4 and 270.6	Methanol	1-40 μg/ml 1-25 μg/ml	70
48	Sulphadoxine and Trimethoprim	274.2 and 299.5and 238.1 and 300.1	Methanol	-	71
49	Phenylephrine hydrochloride and Tropicamide	260.8 and 268.2and 245.4 and 271.8	0.1N HCl	25-125 mg/ml 4-20 mg/ml	72
50	Trimetazidine hydrochloride and Metoprolol succinate	264.20 and 282.60and 280 and 260	Water	40-200 μg/ml 54-270 μg/ml	73

Table 2: Analytical characteristics of dual wavelength procedure for individual determination of pharmaceutical compound

No	Compound name	Remarks	Solvent	linearity	Ref
1.	Lovastatin	In fermentation broth of aspergillus terreus 226 and 260 nm	75% ethanol	3.20 to 64.0µg/ml	21
2.	Phenazopyridine hydrochloride	In the presence of its oxidative degradation product 2,3,6-triaminopyridine (TAP) 425 and 256 nm	Water	1-14μg/ml	74

 

FOOD ANALYSIS:

Determination of calcium by dual wavelength spectrophotometry:

Table 3: Determination of calcium by dual wavelength spectrophotometry:

S. No.	Compound	Reagent	Wavelength	Linearity	Ref
1	Calcium in milk	Calcium + Chrome black T – polyethylene glycol in an alkaline buffer	558 and 645nm	0-4.8µg/ml	75
2	Calcium in food	Calcium + arsenazo I + NH3-NH4Cl buffer (PH:9.5) Form a purple complex	480 and 570nm	0-1.3µg/ml	76

 

Determination of citric acid and ascorbic acid in Vit. C tablet:⁷⁷

Acids reacts with copper (2) – ammonium complex

 

Cu⁺² – NH₃ complex            decomposed                         Cu⁺²- citrate complex

                                                by citrate              

λ_max=600 nm                                                                         λ_max=750 nm

range: 1-125 mM                                                                 range: 1-35 Mm

 

Determination of Reducing Sugar in Black Liquor:⁷⁸

The lignin will drag interference in the system of the sugar determination. Dual wavelength spectrophotometry can remove the interference when making use of the DNS to detect the reducing sugar content in the black liquor.

 

The dual-wavelength spectrophotometric method has advantages of good accuracy and precision, high sensitivity and simultaneous determination of multi-component, so Use DNS dual-wavelength spectrophotometric determination of reducing sugar content of black liquor, select the 520 and 570nm test wavelength can exclude the interference of lignin.

 

From the relation curves between the absorbency of the lignin and its concentration at 520nm and 570nm, the magnification coefficient of the differential signal was defined as K₂=5.338, K₁=3. 288. The correction index k=1.2. Then the concentration C(g/L) of the glucose solution can be figured out through formula C=(S/k-0.0165) × M/0.821.Then a group of glucose solution with same glucose addition but different lignin addition had been measured.

 

Separation and purification of amylose and amylopectin from cassava starch and content determination:⁷⁹

The amylose and amylopectin in cassava starch were separated by n-butanol crystallization method. The purity of amylose and amylopectin was characterized by blue value of starch-iodine complex. According to principle of dual wavelength spectrophotometric method, the contents of amylose and amylopectin in cassava starch were determined at selected wavelengths of 624 nm and 538nm with respective reference wavelengths of 440nm and 750nm.

 

SOIL ANALYSIS:

Determination of nitrate in soil extract⁸⁰:

A rapid method is described for determining nitrate concentration in a soil extract solution based on its UV absorbance at 210nm. The interference of non-nitrate species is accounted for by subtracting an empirically-determined multiple of the absorbance of the extract solution at 270nm from its absorbance at 210nm. The value of the multiplication factor, R, is calculated from the 210: 270nm absorbance ratio of the extract solution after it has been treated with Raney-Nickel catalyst to remove nitrate. The method was tested using 10 Illinois soils. The composite value of R for these soils was 3.05 ±0.22. This value, as well as each individual value of R for the respective soils, was used to calculate the nitrate concentration. The results were then compared to one another and to results obtained by the steam distillation method.

 

INORGANIC ANALYSIS:⁸¹

Spectrophotometry usually involves the formation of a complex of the trace element with a selective ligand reagent that, as a metal-ligand complex, absorbs light in the visible and UV regions. Spectrophotometry is still used for the rapid determination of copper, iron, and of other metals such as Cr (VI) in environmental samples because Spectrophotometry is a conventional and inexpensive technique.

 

Analytical characteristics of dual wavelength procedure for individual determination

Table 4: Analytical characteristics of dual wavelength procedure for individual determination

S. No	Compound	Reagents	Remarks	Ref
1	Copper in waste water	Erichrome blue black R (EBBR)	550 and 660 nm(λ)  PH 12	82
2	Sulphide in domestic water	Coloured complex solution (Ag+ + triton-x-100 + cadion 2B + sodium tertraborate solution	446 and 556 nm (λ) PH 9.2	83
3	Arsenic (III) in waste water	Ethyl violet	560 and 630 nm (λ) PH 6-7	84
4	Mercury (II) in water	4-(2-thiazolylazo) resorcinol	442 and 547 nm	85
5	Iron (III) in rocks	Diantipyrinylmethane (DAPM)	388 and 470 nm Produced red-brown complex in acidic media	86
6	Titanium (IV) in rocks	Diantipyrinylmethane (DAPM)	388 and 514.9 nm Produced yellow complex in acidic media	86
7	Chromium (VI)in waste water	Rhodamine B and phenol red	430 and 550 nm	87
8	Germanium in ashes	Salycyl fluorone	466 and 504 nm	88
9	Methylmercury in fish tissue	Dithizone	475 and 628 nm	89
10	hydrogen peroxide in the wood pulp bleaching streams	Molybdate solution	297 and 350 nm	90

 

Determination of nickel in the presence of cobalt⁹¹:

When nickel is to be determined, λ₁ is set to the absorbance maximum for the nickel ion, 397nm and λ₂ is changed several times until a constant level is obtained, when solutions containing suitable amounts of cobalt are used. The preliminary testing for the determination of nickel in the presence of cobalt as perchlorate is shown in Fig. From these results, the most suitable combination of λ₁ and λ₂ is seen to be 397 and 620nm; with this combination, ∆A for cobalt is zero or negligible, so that the interference of cobalt can be eliminated completely.

Procedure: To a slightly acidic solution containing 5mg of nickel and various amounts of cobalt, add 5ml of 70% perchloric acid, and dilute to 25ml with water. Measure the absorbance at 397 vs. 620nm in a 1-cm cell.

 

The dual-wavelength technique in inorganic spectrophotometry with highly sensitive chromogenic reagents:

Many highly sensitive chromogenic reagents for metals with molar absorptivity’s of the order of 10⁵ are now known. the analogs of 4-( 2-pyriclylazo)- l.3 diaminobenzene (PADAB) are used for the determination of micro- amounts of cobalt.

 

In conventional method the net absorbance at maximum wavelength against water or reagent is measured. In dual-wavelength measurements λ₁ is set to the wavelength for the absorption peak of the chelate and λ₂ to the wavelength for the absorption peak of the reagent.

 

Procedure: To a 25ml volumetric flask transfer a suitable aliquot of sample solution containing up to 0.1 p.p.m of cobalt. and add 0.1-0.2 ml of ethanolic 0.05% reagent solution. Adjust to pH 5.0 with 0.2 M sodium Acetate-O.2 M acetic acid buffer solution, and mix. Then add 10 ml of hydrochloric acid. dilute to volume. and mix. Measure the absorbance for λ₁ 588 nm and λ₂ 425 nm.

The dual-wavelength method increases the apparent molar absorptivity of the cobalt-5-Cl-PADAB complex

 

DUAL WAVELENGTH SPECTROPHOTOMETRY: AS A DIAGNOSTIC TEST OF THE PULP CHAMBER CONTENT⁹²

Dual wavelength spectrophotometry is a method that has previously been considered as a diagnostic tool to determine pulp status. Pulse oximetry is a method based on dual wavelength spectrophotometry technology.

 

The purpose of this in vitro study was to determine the feasibility of using dual wavelength spectrophotometry to identify teeth with pulp chambers that are either empty, filled with fixed pulp tissue, or filled with oxygenated blood.

 

In phase I (human tooth model) of the experiment, a human third molar was prepared so that its pulp space could be filled with oxygenated blood and later emptied.

 

In phase II (dog tooth model), the lower jaw of a beagle dog was removed and placed in formalin, thereby fixing the pulps of the teeth. The pulp of the right canine was removed via an apical approach, and attachments were placed in a similar position to those on the human tooth, to allow filling and emptying of the pulp space. Cavit was placed over the exposed fixed pulp in the left canine. Ten readings, which were separated by light source and detector removal and replacement, were taken of the right canine pulp space when it was empty or filled with oxygenated blood, or the left canine pulp space when it was filled with fixed tissue. Distinct and reproducible changes were measured for pulp spaces filled with air, tissue, or oxygenated blood.

 

In phase III (stimulated pulp testing on dog teeth model), Blood was introduced into the root canal space, the chamber was rinsed with water and replaced with air, according to a predetermined code.

 

The continuous dual wavelength spectrophotometer used in this study was designed to noninvasively monitor oxygenation changes in muscle. The instrument detects the presence or absence of oxygenated blood at 760 nm and 850 nm. The identification of pulpal contents was correctly determined in all 20 of the predetermined conditions. The findings indicate that continuous wave spectrophotometry may become a useful pulp testing method.

 

CONCLUSION:

A simple rapid spectrophotometric method for the determination of two component systems by means of a dual-wavelength method is described. By proper selection of the combination of two wavelengths λ₁ and λ₂, one component can be masked instrumentally even when its concentration varies. The component to be determined is measured as follows: two light beams of different wavelengths λ₁ and λ₂ from two gratings are time-shared through a single cell by means of a rotating sector and the difference between the absorbance at wavelengths λ₁ and λ₂ is measured.

The ability of double-wavelength spectroscopy to extend the proven effectiveness of uv-vis absorption spectroscopic techniques by overcoming limitations in selectivity has been demonstrated certainly in the field of biochemistry but also in general analytical spectrophotometry. Since the fundamental advantage of double-wavelength spectroscopy is its capability of reducing interferences by one dimension, many unsolved analytical problems, both qualitative and quantitative, can potentially be solved with presently available instrumentation.

 

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Received on 11.02.2022    Modified on 29.07.2023

Accepted on 19.04.2024   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2024; 14(3):166-174.