INDIRECT SPECTROPHOTOMETRIC

METHODS

TO BE PRESENTED BY: N.T.ARUN,

FIRST M.PHARM, DEPARTMENT OF PHARMACEUTICAL ANALYSIS.

EVALUATORS: 1. Mrs. Suganthi .A - M.Pharm (Ph.D.,) Lecturer-Dept. of Pharma. Analysis, 2. Mrs. Manjuladevi.A.S.- M.Pharm (Ph.D.,) Asst.Professor-Dept.of Pharmacy Practice, 3. Mrs. M.Dhanamani - M.Pharm (Ph.D.,) Lecturer-Dept. of Pharmacognosy.

Spectrophotometric methods

The spectrophotometric methods to be discussed (methods of molecular absorption spectrometry) are based on the measurement of absorption of radiation, in the visible and near ultraviolet regions, owing to coloured compounds formed, before the determination, by the elements to be determined. Only seldom is use made of the intrinsic colour of the element i t sel f , in i ts ionic form. In cases where an element neither forms coloured compounds nor occurs in a coloured form, indirect spectrophotometric methods are applied. Spectrophotometric methods are characterized by high versatility, sensitivity, and precision. They may be used for the determination of almost all chemical elements over a wide range of concentrations, from macro quantities (by means of differential spectrophotometry) to traces ranging from 10-6-10-8% (after suitable preconcentration).Spectrophotometric methods are among the most precise instrumental methods of chemical analysis. The advantages mentioned of the spectrophotometric methods are made greater by their availability. A spectrophotometer, which is the basic instrument in this field, is cheaper than most other fundamental instruments used in chemical analysis.

Indirect spectrophotometry This is also known as chemical derivatisation method or colorimetric method. Indirect spectrophotometric assays are based on the conversion of the analyte by a chemical reagent to a derivative that has different spectral properties, thereby shifting λmax towards longer λ. Chemical derivatisation is adopted because

1. If the analyte absorbs weakly in the UV region, a more sensitive method of assay is obtained by converting the substance to a derivative with a more intensely absorbing chromophore.

Ex: sugars don’t absorb above 220nm. This can be determined spectrophotometrically by heating with anthrone in conc.sulphuric acid and measuring absorbance of

coloured derivatives at 625nm. 2. The interference from irrelevant absorption may be avoided by

converting the analyte to a derivative which absorbs in the visible region, where irrelevant absorption is negligible. Ex: The condensation of ketosteroids (methyl testosterone in methyl testosterone tablets with hydrazide reagents produces derivative that absorb in the visible region free of interference from irrelevant absorption.

3. It is used to improve the selectivity of the assay of an UV absorbing substance in a sample.

Ex: In the assay of adrenaline in adrenaline injection by direct measurement, the maximum absorption is obtained at 279nm. It’s subject to gross interference due to bactericidal chlorocresol. Only adrenalin forms purple derivative in the presence of iron (II) is measured colorimetrically.

4. The adsorption of a indirect spectrophotometric procedure, instead of an UV procedure, may be based on cost consideration. Single beam manually adjusted visible spectrophotometers are much cheaper than UV spectrophotometer.

5. Apart from these, we have to evaluate some drugs by this method like Dicyclomine.Hcl, pempidine, piperidine ring structure drugs, ephedrine, amphetamine , atropine, metals like iron(using 1,10-phenanthroline), lead (using dithiazone), etc.

In the usual direct spectrophotometric methods of analysis, the absorbance of substance or derivative being determined is measured. The color-producing reaction may be illustrated: Sample (a1) + chromogenic reagent (a2) -product (a3) Where a represents the absorptivity of the species in question. Generally, a1 and a, are zero at the wave length employed in the analysis, and a, is fairly large in order that the method be sensitive. In the usual spectrophotometric method, a3 >> a1 or a2.Because the absorbance is directly proportional to the concentration of the colored sample or its coloured derivative, the analysis is simple and straightforward. In indirect spectrophotometry, the sample is first allowed to react with a definite concentration of a highly colored chromogenic reagent, present in excess. The product of this reaction is of less intense color than the original chromogenic reagent (a3< a2) and consequently, a fading of the chromogenic reagent solution occurs. The extent of fading is a direct measure of the quantity of sample present. Several modes of photometric measurement for

the extent of fading are applicable, each method having its own advantages and disadvantages.

CLASSIFICATION OF INDIRECT SPECTROPHOTOMETRIC METHODS Photoelectric measurements have in common the preliminary adjustment of the instrument to read first O%T and then 100%T, under specified conditions. The photometric methods described here differ essentially in the types of reference solutions used to establish the 100%1 setting. Figure 1 summarizes four methods described below.

Method A; The instrument is set to read O%T with the photocell in darkness and 100%T when exposed to light which has passed through the pure solvent. After these preliminary adjustments, the faded sample-chromogenic product is read, this reading falling between O%T and 100%T. The resulting plot of absorbance vs. concentration is actually a photometric titration, and a typical curve is shown in Figure 2. In this figure the solid lines represent the situation obtained when the sample and chromogenic reagent react completely. In practice, however, the dotted line is more often obtained because of incomplete reaction between the sample and the chromogenic reagent.

At low concentrations of sample the absorbance reading is rather high, with a concomitant larger error in reading the absorbance scale, as it is crowded at high values. Consequently, better results are obtained at the low absorbance readings corresponding to higher sample concentration values. When the sample and chromogenic reagent react almost completely-i.e., a case of favor- able equilibrium-the lowest relative error occurs at low absorbance readings. In fact, the point of minimum error actually occurs at the intercept. When the concentration of sample becomes sufficiently great and rounding of the curve commences because of equilibrium considerations, error in the analysis will increase in this region because the change in absorbance for a given change in concentration decreases considerably. The point of minimum error will occur at an intermediate concentration removed from the two areas of high error, the region near zero con- centration and the region where the absorbance per unit change in concentration becomes small.

Method B; This case is a modification of Method A. The instrument is set to read O%T with the photocell in darkness and 100%T when exposed to light which has passed through the solution of chromogenic reagent that has been partially bleached by the introduction of a standard quantity of the sample material. After these two initial settings the unknown sample is read, the value of the reading falling between O%T and 100%T. The amount of standard

sample introduced into the reference solution used to set 100%T on the instrument must be somewhat higher than the sample to be read.

A typical calibration curve for this mode of analysis is shown in Figure 3. Although no readings can be actually obtained below zero, these off-scale readings are included to illustrate that the response now obtained is simply an expansion of the scale illustrated in Figure 2. This effective expansion of the absorbance scale diminishes the error in the analysis.

Method C; This method, presumably heretofore unmentioned, employs a very unusual method for setting the instrument. The instrument is set to read O%T with the photocell in darkness and IOO%T when exposed to light which has passed through the sample-chromogenic reagent solution. After these settings, the pure chromo- genic reagent absorbance is read, the reading falling between O%T and100%T. Operation in this way will yield data of the type represented in Figure 4. If the reaction between the chromogenic reagent and the sample species is complete, then a sharp break at the equivalence point, E.P., will be obtained. Where equilibrium is not so favorable, rounding Occurs, illustrated by the dotted line in Figure 4.In practice, only the straight-line portion prior to E.P. is employed. The range of the straight line can be extended simply by

increasing the concentration of chromogenic reagent employed, causing the E.P. to occur at higher concentrations of sample This particular method of operation is particularly useful for low concentrations of sample, because the absorbance readings are obtained in that section of the absorbance scale where the scale readings are well separated and read with higher accuracy. However, each sample requires a new slit width setting, making Method C somewhat more in-convenient and time-consuming than Method A. In addition, it may cause deviations from Beer's law, but this will not affect the analytical results because a calibration curve is usually employed. The absorbance of the pure chromogenic reagent solution referred to water (or to pure solvent) is given by: A1=a1bC1………………………………… (1) Similarly the absorbance of the unknown sample referred to water is given by: A1=a1b (C1-nC2) +a2bn’C2………………… (2) where C2 denotes the unknown concentration of the sample species, n the moles of chromogenic reagent that react with each mole of sample species, and n’ the moles of product formed from each mole of sample species. When measuring the absorbance of the pure chromogenic reagent solution against the sample, the absorbance is given by: A = A1 - A2 = (aln – a2n‘) bC2 =a‘bC2 …………… (3) A formula analogous to Beer’s law i.e., absorbance directly proportional to concentration-is obtained. The a’ value is determined by the difference between the absorptivity of the chromogenic reagent and that of the sample chromogenic reagent product, each multiplied by the number of moles of chromogenic reagent or product consumed or formed per mole of sample species. Because of the formal similarity between the Beer’s law equation and Equation 3, the usual treatment of photometric errors can be applied (3, 5,9,12,13). For example, if a straight line is obtained as predicted by Equation 3, one would expect lowest relative error at sample concentrations where the absorbance reading is 0.43. If the calibration line is curved, one

should plot 1-T vs. log C (Ringbom plot) in order to calculate the photometric error expected at any particular concentration value and to determine the useful concentration range (1,3, 11) Method D; This method is essentially a modification of Method C and resembles the transmittance-ratio method. The instrument is set to read O%T with the photocell in darkness and l00%T when exposed to light which has passed through a solution containing the chromogenic reagent and a certain amount of unknown sample. The absorbance of a solution containing the reagent and a fixed amount of standard sample is then read. The concentration of standard sample must be somewhat less than the concentration of the sample to be determined. The calibration curves are illustrated as lines C and D (Figure 4) where the reference solution contains known concentrations, C3 and C3‘, respectively, of the sample species. The equation for this method may also be derived. The absorbance of the fixed amount of standard, C3, referred to water is given by: A3 = a1b (C1 – nC3) + a2bn‘C3……………………… (4) Combining this equation with Equation 2, one obtains the absorbance of the fixed amount of reference standard referred to the unknown sample: A = A3 – A2 = (a1n – a2n’) b (C2 – C3)……………… (5)

EXPERIMENTAL Determination of Magnesium. In the determination of magnesium, Method C is used. The chromogenic reagent is Calcon [1-(2-hydroxy-lnaphthylazo) 2 -naphthol - 4 – sulfonic acid; CI 202], which forms a stable chelate with magnesium (4). This chelate has a different absorbance spectrum from that of the unchelated Calcon (Figure 5) and at 620 mµ, where the free Calcon absorbs strongly, the addition of magnesium decreases the concentration of free Calcon causing a bleaching effect. From this bleaching effect a calibration curve can be constructed using Method C. and a colorimetric method for magnesium is thereby obtained. The color of the chromogenic reagent depends upon pH as shown by the following equilibria: pk2=4 pk3=13.5 H2In- HIn-2 In-3 + H+ Pink Blue Pink The effective stability of the magnesium-Calcon chelate is also pH dependent e.g., at pH 10 the reaction is essentially: Mg+2 + HIn-2 MgIn- + H+ Blue Pink

Consequently buffer must be added to maintain a constant pH or reproducible results are not obtained. The ammonium chloride-ammonium hydroxide buffer system at pH 10 is satisfactory. At pH values below 10 the stability of the magnesium-Calcon chelate decreases appreciably. At pH values above 10 the colour stability of the chelate decreases, attributed to hydrolytic attack and air oxidation of the Calcon. The quantity of buffer added also affects the calibration curve. The amount used is a compromise

between the sensitivity- of the method and the buffer capacity of the solution. The results are given in Table I. Determination of Calcium: Calcium cannot be determined in the same way as magnesium because the Stability of the calcium-Calcon chelate is much less than that of the magnesium-Calcon chelate. At pH 10 the reaction Ca+2 + HIn- CaIn- + H+

does not proceed sufficiently far to the right to create the bleaching effect required for a successful colorimetric determination. This difficulty can be readily surmounted by adding an excess of magnesium-EDTA to the buffer solution. The calcium then replaces the magnesium from the magnesium-EDTA buffer solution: Ca+2 + MgEDTA-2 Mg+2 + CaEDTA-2 The replaced magnesium ions then chelate with Calcon, giving rise to the same bleaching effect that occurs in the magnesium determination. A similar principle has been proposed by Menon and Das, but using Erio-T as chromogenic reagent and Method A for photometric measurement. The results in Table are obtained using Method C for the spectrophotometric settings. These results were obtained after the solutions had cooled to room temperature, approximately 1 hour, as the heat of reaction and heat of dilution of the reagents cause a rise in temperature. Longer standing time should be avoided, because after 3 hours the absorbance decreases. Reagents and solutions: Standard calcium solution, EDTA solution, Magnesium stock solution, Calcium stock solution, Buffer solution A (NH4-NH4cl), Magnesium-EDTA solution, Buffer solution B (NH4cl- NH4OH), Calcon. Procedure for Magnesium: Prepare a calibration curve by delivering 5-, l0-, 15-, and 20-ml. aliquots of stock magnesium solution containing 2.4 γ of magnesium per ml. into 50-ml. volumetric flasks. Into each flask pipette 5 ml. of buffer solution A, 10 ml. of color reagent and dilute contents to volume with distilled water. Also prepare a blank, omitting the magnesium. Make the sample solutions in

a similar manner, replacing the stock magnesium solution with the sample aliquot. In cases where the sample contains excess acid or base, preneutralize the sample before adding the buffer solution and color reagent. Allow the solutions to stand to cool for approximately 1 hour and again dilute to volume. Set the spectrophotometer at 620 mµ. Place the blank solution and 5-ml. stock magnesium solution in I-cm. cells. Set the dark current in the usual manner with the shutter closed. Introduce the 5ml. stock magnesium solution into the light path and set the instrument to read 100%, T by adjusting the slit width. Put the blank solution in the light path and record the absorbance. Repeat using lo-, 15, and 20-ml. stock solutions. From the absorbance of the stock magnesium solutions. Construct a calibration curve. The concentration of the sample is taken directly from the curve. Procedure for Calcium: Prepare a calibration curve by delivering 5-, l0-, 15-, and 20-ml. aliquots of stork calcium solution into separate 50-ml. volumetric flasks. To each flask pipette 5 ml. of buffer solution B and 10 ml. of color reagent, and dilute to volume with distilled water. Also prepare a blank solution, omitting the calcium. Prepare sample solutions in a similar manner, replacing the, stock calcium solution with a sample aliquot. In cases where the sample contains excess acid or base, neutralize the sample before adding the buffer solution and color reagent. Allow the solutions to stand for 1 hour and again dilute to volume. Read the stock calcium and sample solutions using the procedure described for magnesium. From the recorded data prepare a calibration curve and determine the concentration of the sample directly from the calibration curve. A Beckman Model B spectrophotometer was used.

CHEMICAL DERIVATISATION: In the early days of quantitative pharmaceutical analysis, chemical reactions were involved in almost all the methods; titrimetry, gravimetry, and colorimetry. Later, however, the role of chemical reactions decreased considerably. The reason for this was the spread of UV spectrometry and other spectroscopic techniques, as well as various chromatographic methods. Using these techniques, chemical reactions can be completely omitted (e.g. in spectroscopic methods, HPLC with UV or RI monitoring, TLC densitometry based on natural absorption or

fluorescence), or the reaction takes place in the measuring cell or detector only (e.g. in voltammetry methods, HPLC with electrochemical detectors, gas chromatography with a flame ionization detector). The role of derivatisation reactions in stationary or flowing systems is again increasing. The reasons for this seem to be the following: (a) Using preliminary chemical reactions the field of application of spectroscopic and chromatographic methods can be greatly expanded: spectrometrically inactive materials can be determined by UV-visible spectrophotometry and by HPLC with UV detection (b) The application of derivatisation reactions is sometimes a useful tool in the spectroscopic or chromatographic identification of unknown components. (c) The main reason for the increasing use of derivatisation reactions is certainly the continually increasing demand on pharmaceutical analysts for more selective and/or sensitive methods Even an outline summary of the vast number of papers in this field is beyond the scope of this review: only the most important monographs dealing with derivatisation reactions in chromatography and spectroscopy are listed. DIFFERENT METHODS:

Diazotisation & Coupling. Condensation Reaction. Reduction of tetrazolium salts. Acid-dye method. Oxidation method. Metal-ligand complexation.

Derivatization of Carboxyl Groups for the Spectrometric Assays The majority of carboxylic acids (among them drugs and biologically important derivatives) are spectrometrically inactive or absorb only in the UV region. Several derivatization methods are available for their UV-visible spectrometric determination or for HPLC analysis with spectrophotometric or fluorimetric detection. The aim of this study has been to develop a general method for the transformation of carboxyl groups to carboxamide derivatives with chromophoric or fluorophoric properties suitable for spectrometric or HPLC determination. The key reaction of the method is between the carboxylic acid and diethyl

chlorophosphite leading to a mixed anhydride R-COOH + P (OC2H5)2Cl = R-CO-O-P (OC2H5)2 + HCl. (1) This reaction takes place very rapidly at room temperature using acetonitrile or a mixture of acetonitrile and tetrahydrofuran as solvent. Diethyl chlorophosphite should be used in large excess to avoid interference from water in the solvent (which should be under 0.05%) and to ensure complete reaction. After completion of the reaction, any reactive and spectrophotometrically active primary or secondary amine or hydrazine can be added to form the corresponding carboxamide or hydrazide derivative: R-C0-0-P (OC2H5)2 + R'-NH2 = R-CO-NH-R' + P (OH)(OC2H5)2 (2) Reaction (2) is somewhat slower than reaction (1), but it can be completed at moderately elevated temperatures within 1-2 h, even with the least reactive derivatives. At first aniline was used as the amine component in a slow, single-step process, the carboxanilide derivatives having a strong absorption band at 243 nm (e-14,500). Here the use of 2-nitrophenylhydrazine as the amine component is described. This reagent was for the detection of carboxylic acids and anhydrides and for the quantitative determination of carboxylic anhydrides and chlorides. The use of 2-nitrophenylhydrazine as a colorimetric reagent for the determination of some water-soluble carboxylic acids was described. The carboxylic acids were coupled witi1 the reagent with the aid of water-soluble carbodiimides. The advantage of 2-nitrophenylhydrazine over aniline is that in alkaline medium (pH>12) the acyl derivatives are highly conjugated with absorption maxima around 550 nm, greatly increasing the selectivity of the method.

P (OC2H5)2Cl 2

NO2

NH NH2

NO2

NH NH3+Cl-

NO2

NH NH P (OC2H5)2

N

N

N

OH

OH-

R

C

O O

P(OC2H5)2

NO2

NH NH C

O

R

N N C

O-

R

N-O2

PO(OC2H5)2-

As is seen in reaction (3) the excess of diethyl chlorophosphite reacts with the 2- nitrophenylhydrazine reagent and the product decomposes upon addition of sodium hydroxide to form the colorless 1-hydroxy-1,2,3-benztriazol. Both reactions are almost instantaneous and as a result of this the reagent blank is negligibly small at 550 nm. By contrast isobutyl chloroformate, which is more often used to form mixed anhydrides, cannot be applied in this work because its reaction product with 2-nitrophenylhydrazine does not decompose in alkali: this would cause extremely high reagent blank absorption. Several carboxylic acids have been determined by the proposed method. The general procedure is the following. The sample containing up to 5µl of carboxylic acid, is dissolved in 250 µl of anhydrous acetonitrile or (in the case of bile acids) in a 7:3 v/v mixture of acetonitrile and tetrahydrofuran. A 50µl portion of diethyl chlorophosphite is added and the mixture is allowed to stand at room temperature in a well-closed vial for 15 min. A 250 µl portion of 2-nitrophenylhydrazine reagent is

then added (0.04 Min acetonitrile) and the mixture is heated at 700c for 30 min. The contents of the vial are then transferred to a 25 ml calibrated flask with the aid of about 15 ml of ethanol. A 5 ml portion of 0.5 M aqueous sodium hydroxide is added and the flask is made up to volume with ethanol. The absorbance is read against a reagent blank at the absorption maximum (ca. 550 nm). The absorbance is stable for several hours.

Table 1

Spectral data of carboxylic acids after derivatization with 2-nitrophenylhydrazine

Acid Amax (nm) Molar absorptivity• Regression datat a b

Acetic acid 550 6520±1.4% 0.008 1.072 Palmitic acid 550 6850±0.8% 0.004 0.270 Cholicacid 550 7280±1.3% -0.001 0.187 Benzoic acid 560 8030±1.2% 0.006 0.795

• Mean of eight determinations ± r.s.d.

T Regression equation: absorbance = a + b x concentration (mg/100 ml):r >0.9995.

Diazotisation & Coupling:

The amine is first diazotized with an aqueous solution of nitrous acid (generated in situ by the reaction of hydrochloric: add and sodium nitrite) at 0-50C

Ar-NH2 + HN02

H+

Ar-N+≡N + 2H2O

The colourless diazonium salt is very reactive and when treated with a suitable coupling reagent (Ar'-H), e.g. a phenol or aromatic amine, undergoes an electrophilic substitution reaction to produce an azo derivative.

Ar-N+≡N + Ar’-H Ar-N=N-Ar’ + H+

The azo derivatives are coloured and consequently have an absorption maximum in the visible region. The λmax and € max depend on the Ar and Ar’

groups. Among the most widely used coupling reagents are 1-naphthol, 2-naphthol and N-(1-naphthyl)-ethane-1, 2-diammonium.dichloride (the Bratton -Marshall Reagent) which give high absorptivities. For example, the azo derivative of diazotized sulphadiazine coupled with the Bratton-Marshall reagent is

which absorbs intensely around 545 nm owing to its extensive conjugation. Sulphamic acid or ammonium sulphamate is added to the solution of the diazotised amine before the coupling stage to destroy the excess nitrous acid, which inhibits the coupling reaction.

HN02 + NH2S03H N2+ H2S04 + H2O

The sensitivity and selectivity of the procedure permit the assay of low concentrations of impurities that contain a primary aromatic amine group in the presence of the parent substance lacking the amine function. British Pharmacopoeia tests for free amine impurities in Frusemide, Iothalmic acid and Iodpamide Meglumine Injection are based upon diazotisation and coupling. Substances that can be hydrolysed (e.g. Bendrofluazide; Chlorothiazide) or reduced (e.g. Chloramphenicol) to a derivative containing a primary aromatic amine group can also be assayed spectrophotometrically by diazotisation and coupling of the amine derivative.

Condensation Reaction:

Many colorimetric procedures are based on the rapid reaction that occurs under suitable conditions between amines and carbonyl compounds. The reactions involve the nucleophilic attack by the amine on the carbonyl carbon with the elimination of water. Substances containing a carbonyl group react with a variety of reagents containing an amino group:

R'C O

R''

NH2R'''R'

C NR'''R''

H2O

When R"' = Alkyl or aryl, the product is a Schiff's base

= NH2 (hydrazine), the product is a hydrazone

= NHCONH2 (semicarbazide), the product is a semicarbazone

= OH (hydroxylamine), the product is an oxime

A number of hydrazine and hydrazide reagents have been described for the colorimetric assay of ketosteroids. The selectivity of the reactions for steroids with the keto group in different positions depends on the reagent. Isoniazid (isonicotinic acid hydrazide) reacts with 4-en-3-oue and 1, 4-dien-3-one steroids in acidic solution to form yellow derivatives with λ max around 400 nm.

N CONHNH3O

A N CONHN

A

The reagent is used in the assays of Nandralone D e c a n o a t e Injection and Betamethasone Sodium Phosphate Injection. A similar reagent 2, 4-dinitrophenylhydrazine, is used i n the a s s a y of M e t h y l testosterone Tablets. The hydrazone formed on condensation of the 3-, 17- or 20-Ketosteroid with (carboxymethyl) trimethylammonium chloride hydrazide (Girard's reagent T) is water-soluble owing to the quaternary ammonium group, and lipid-soluble impurities may b e removed by extraction into chloroform. Alternatively, the hydrazone itself may be extracted i n t o dichloroethane as the ion-pair with bromothymol blue. Amino compounds can be assayed sp e c t r op h ot o m et r i c a l l y using a

suitable carbonyl reagent. One of the most frequently employed re- agents is 4-dimethylaminobenzaldehyde (Ehrlich's reagent) used, for example, in the assay of procaine hydrochloride in Lymecycline and Procaine Injection. The condensation product which absorbs at 454 nm is

CHN N COOCH2CH2N(C2H5)2H3C

H3C

In the presence of a steroid with an α-ketol (21-hydroxy-20-keto) side-chain group, tetrazolium salts are reduced to their coloured formazan derivatives. Several formulations containing corticosteroids are assayed using triphenyltetrazolium chloride. The reaction is carried out in an alkaline medium (tetramethylammonium hydroxide) at 30-35° for 1-2 h and the absorbance of the red product is measured around 485 nm. The oxidation of the α-ketol group and the reduction of triphenyltetrazolium chloride to triphenylformazan are shown:

Steroids esterified in the 21-position, e.g. Hydrocortisone Acetate, hydrolyse in the alkaline solution to yield the free 21-hydroxysteroids and are also determined by this procedure. Precautions are taken throughout the assay against the effects of light and oxygen.

Acid-dye method: The addition of an amine in its ionised form to an ionised acidic dye, i.e.-methyl orange or bromocresol purple, yields a salt (ion-pair) that may be extracted into an organic solvent such as chloroform or dichloromethane. The indicator dye is added in excess and the pH of the aqueous solution is adjusted if necessary to a value where both the amine and dye are in the ionised forms. The ion-pair is separated from the excess indicator by extraction into the organic solvent, and the absorbance is measured at the λmax of the indicator in the solvent. Usually, the most intensely absorbing form of the indicator is measured, with the addition, if required, of acidified or basified ethanol. Alternatively, the absorbance of the indicator may be measured in aqueous solution after back extraction from the organic solvent. The molar absorptivities of the ion-pairs formed between quaternary ammonium compounds and methyl orange or bromothymol blue are typically 2 x 10 4 - 4 X 104. The acid-dye method therefore provides a more sensitive technique for certain amines and quaternary ammonium compounds that absorb weakly in the ultraviolet region, e.g. Hyoscine butyl bromide (€ 257 = 202). The correct choice of pH may permit the selective assay of a mixture of an amine and a quaternary ammonium salt. For example, in the assay of a tertiary base and a quaternary ammonium salt both substances are ionic at pH 3, and the resultant absorbance of the ion-pair extracted into the organic solvent measures the total concentration, whereas at pH9 only the quaternary compound is ionised and forms the extractable ion--pair. The acid-dye technique is used for the assay of formulations containing certain quaternary ammonium salts or amines, i.e... Biperidine Lactate Injection, Clonidine Hydrochloride Injection and Tablets, Neostigmine Methyl sulphate injection and Benzhexol Hydrochloride tablets. Oxidation method: Oxidation of the side chain of weakly absorbing compounds containing a simple phenyl group produces a carbonyl derivative that has a much

greater absorptivity than the parent compound. Commonly used oxidation reagents are alkaline potassium permanganate solution, acidified potassium dichromate solution, or perchlorate solution. The product formed from simple monophenyl compounds e.g. ephedrine or propanol amine, is the corresponding benzaldhyde derivative which exhibits intense absorption, at around 240 nm owing to the interaction of the carbonyl 𝜋 electrons with the ring electrons.

The assay of Ephedrine Hydrochloride Elixir involves the extraction of the benzaldhyde into cyclohexane and measurement of the absorbance at its λmax 241nm. Compounds with a diphenylmethylidene [(C6H5)2C<] nucleus are oxidized to benzophenone which has a λmax in hexane solution at 247nm. Metal-ligand complexation: Many organic reagents form complexes with metal atoms by the formation of coordinate bonds and covalent bonds. Ligands with two or more donating groups may share more than one pair of electrons with a single metal atom by coordinating to two or more positions. The chelates formed by these multidentate ligands with metal atoms are often coloured and consequently their concentration may be determines by visible spectrophotometry. Many examples of the photometric assay of either the concentration of heavy metal ions by the addition of an excess of a chelating agent or the concentration of organic substances by the addition of an excess of a suitable complexing metal have been described.

Characteristic colours which vary in hue with change of pH are given by the reaction of iron (II) ions with phenols that contain two adjacent hydroxyl groups. Thus, on mixing a solution of adrenaline with a buffered solution containing iron(II) sulphate, a purple complex (λmax- 540 nm) is formed that has a maximum intensity at pH 8-8.5.

Assays of adrenaline in Procaine and Adrenaline injection and in Lignocaine and Adrenaline injection of Methyldopa Tablets, Methyl dopate injection and Isoprenaline Hydrochloride Injection are based upon the coloured complexes formed with iron (II) sulphate-citrate reagent, buffered with glycine buffer solution. Alternatively, the concentration of iron(III) ions in a simple may be determined using the chromogenic reagent Tiron (1,2-dihydroxy-benzene-3,5-disulphonic acid, disodium salt), which forms an intense red complex. One of the first substances to be used as a general reagent for the photometric assay of heavy metal ions such as Cd, Hg, Cu, Pb and Zn was dithizone(1,5-diphenylthiocarbazone).The coloured complex formed, for example with lead:

is soluble in organic solvents and may be extracted from aqueous solution into an immiscible organic solvent such as chloroform or carbon tetrachloride. Lead dithizonate bas a carmine red colour in carbon

tetrachloride and the high absorptivity (€ 520 = 7 x 104) permits the assay of only a few p.g., of lead. Thio-Michler's reagent [4,4'-bis- (dimethyl amino)-thiobenzophenone] is an extremely sensitive chromo­ genic reagent for Pd ( €520 = 2.1 x 105) and for Hg (€560 = l.2 x10 5 ). Determination of mimosine by a sensitive indirect spectrophotometric method (diazotisation) A simple and sensitive indirect spectrophotometric method is described for the determination of mimosine based on its reaction with diazotized sulfanilamide (DZSAM). DZSAM couples with N-(1-naphthyl) ethylenediamine (NEDA) forming a pink colored azodye, absorbing maximally at 540 nm (£max= 27 mM−1 cm−1). In the present method, mimosine was first reacted with known excess of DZSAM and the unreacted DZSAM was determined by coupling with NEDA. The reaction of mimosine with DZSAM proceeded optimally at neutral pH. The decrease in absorbance of the DZSAM–NEDA-coupled product obeyed Beer’s law in the concentration range of 0.005–0.15 µgml−1 of mimosine. The present method was applied to estimate mimosine in plant extracts containing lesser than 0.05µgml−1 with recovery at 99±0.41%. The method described is superior to other reported methods in terms of ease of adaptability and sensitivity.

Effect of pH on the reaction of mimosine with DZSAM. Mimosine reacted with DZSAM at varying pH: (1) pH 6.0, (2) pH 6.5, (3) pH 7.0, (4) pH 7.5 and (5) pH 8.0. Absorbance of the azodye formed plotted against the concentration of mimosine.

Calibration graph for the determination of mimosine. Changes in the absorbance of the azodye calculated as absorbance, A0 (when no mimosine was added) absorbance, Amin, with added mimosine was plotted against the corresponding concentration of added of mimosine. Standard deviation in absorbance <0.001 (n = 6). Colorimetric determination of steroids: (Condensation) Methods of colorimetric determination of ketosteroids which make rise of the-reaction of a carbonyl group with a hydrazine, a hydrazone or a hydrazide. Three of these methods use p-nitro- or 2,4-dinitrophenylhydrazine, thus allowing the determination of steroids bearing a carbonyl group at position 3, 17 or 20. Isoniazid gives colored hydrazones only with Δ4- or Δ1,4 -3-ketosteroids, whereas salicyloyl- hydrazide allows the fluorimetric determination of 17-ketosteroids. Under suitable conditions, phenylhydrazine gives a colored species only with 17,

21-dihydroxy-20-ketosteroids (Porter-Silber reaction). These compounds can also be determined with 2-hydrazinobenzothiazole or with 3-methylbenzothiazolin-2-one hydrazone in the presence of an oxidant.

Spectrophotometric determination of pefloxacin in pharmaceutical preparations (metal ligand complexation) It has been established that the antibiotic pefloxacin (Abaktal) methane- sulphonate reacts with Fe(III) at pH 1.00-8.00 to form a water-soluble complex with maximum absorbance at 360 nm. The composition of the complex, determined spectrophotometrically by the application of Job's, molar-ratio and Bent-French's methods, was pefloxacin: Fe(III) = 1:1 (pH= 2.50; λ= 360 nm; µ = 0.1 M). The relative stability constant, obtained by the methods of Sommer and Asmus was 105(pH = 2.50; λ= 360 nm;. µ= 0.1 M). The molar absorptivity of the complex at 360 nm was found to be 4.8 x 103 1 mol- em-\ Beer's law was followed for pefloxacin concentrations of 2.15-85.88 J.g ml- 1The lower sensitivity limit of the method was 2.15 µg ml-1 The relative standard deviation (n = 10) was 0.57-1.07%. The method can be applied to the rapid and simple determination of pefloxacin in aqueous solutions and tablets

INDIRECT SPECTROPHOTOMETRIC DETERMINATION OF PIROXICAM AND TENOXICAM THROUGH OXIDATION WITH POTASSIUM PERMANGANATE (oxidation) Three rapid, simple, accurate and selective validated spectrophotometric methods (A, B and C) for the determination of piroxicam (PX) and tenoxicam (TX) in bulk sample and in dosage forms are described. The methods are based on the oxidation of the studied drugs by a known excess of potassium permanganate in sulfuric acid medium and

subsequent determination of unreacted oxidant by reacting it with Methylene Blue (Basic Blue 9) dye (method A), Acid Red 27 (Amaranth) dye (method B) and Acid Orange 7 (orange II) dye (method C), in the same medium at a suitable λmax = 660, 520 and 485 nm, respectively. The reacted oxidant was found to be corresponding to the drug content. Regression analysis of Beer-Lambert plots showed good correlations in the concentration ranges 1.0-8.0, 1.0-9.0 and 1.0-7.2 µ g mL-1 using methods A, B and C, respectively, for PX and 0.3-7.0, 0.3-1.6 and 0.3-2.5 µ g mL-1 using methods A, B and C, respectively, for TX. The stoichiometric ratios for the cited drugs to oxidant were studied. The optimum reaction conditions and other analytical parameters were evaluated. The proposed methods were applied successfully to determine the examined drugs either in pure form or pharmaceutical formulations with good accuracy and precision. The relative standard deviations were ≤ 0.33 with recoveries 98.9-101.7% for PX and ≤ 0.49 with recoveries 99.4-102.0% for TX.

Estimation of clonidine hydrochloride in pharmaceutical preparations (Acid Dye Method):

An acid-dye complexing method with bromophenol blue, bromocresol purple and methyl-orange was used for the ion-pa.ir extraction and colorimetric determination of clonidine hydrochloride in pharmaceuticals containing 100 mg of clonidine hydrochloride was 98.9% and relative standard deviation 0.89%.

REFERENCE:

1. Practical pharmaceutical chemistry by A.H.Beckett and J.B.Stenlake; 4th edition; page no.: 300-306.

2. Elementary organic spectroscopy by Y.R.Sharma; page no.: 18-20. 3. www.sciencedirect.com 4. www.pubs.ac.in