Applications of UV Spectroscopy: Applications of UV Spectroscopy A seminar on Presented By Ms. Komal K. Suthar 1 st semister (2010-2011) M.Pharm. [Pharmaceutics] Roll No. 12 Guided By Mr. Shushant Thakur M.Pharm. [Pharmaceutics] Assistant Professor Department of Pharmaceutics Contents: Contents Applications of UV 1.) Qualitative analysis Pharmacopoeial identification of drug Structural analysis 2.) Quantitative analysis By using beer’s law Single compound analysis Photometric titrations Multicomponent analysis 3.) Determination of composition of complex 4.) Study of kinetics QUALITATIVE ANALYSIS: QUALITATIVE ANALYSIS ( a) Pharmacopoeial identification of drug : (1) By using absorbance & wavelength (2) By taking absorption ratio (3) Limit test (b)Structural analysis 2. Quantitative analysis: 2. Quantitative analysis A)By using beer’s law Using absorptivity value By using reference standard Multiple standard method B)Single compound analysis Direct analysis Using separation method After extraction After chromatographic separation Using column chromatography Using HPLC Slide 5: 3. Indirect analysis a)Single compound without chromophore b) Drugs with chromophoric reagent 1.For analyte which absorb weakly in UV region 2.For avoiding interference 3.Improve selectivity of assay 4. Q-techniques for chemical derivatisation C) Photometric titrations D)Multicomponent analysis Assay using absorbance Differential spectroscopy Simultaneous equation method Absorption ratio method Slide 6: 3. Determination of composition of complex Mole ratio method Continuous variation method ( job curve method ) 4 . Study of kinetics (1)QUALITATIVE ANALYSIS A).Pharmacopoeial identification of drug: (1)QUALITATIVE ANALYSIS A). Pharmacopoeial identification of drug (1)by using wavelength &absorbance e .g . morphine sulphate Take the spectrum in range of 230-360nm of 0.015 % w/v 0.1 N HCL solution which exhibits max only at about 285nm it give absorbance approx. o.65 In alkaline pH 0.005%w/v solution of NaOH it gives 0.34 So by different wavelength we can identify different compounds. Slide 8: (2)by taking absorption ratio Absorption ratio of any drug at only 2 wavelengths is constant. Aspirin at 266nm giving absorbance A1 & at 300nm it gives A2 Here absorption ratio = A1/A2 is constant For L-DOPA ratio should not be more than 0.05 p- Amino salicylic acid having absorption ratio 1.5 to 1.56 (3)Limit test: (3)Limit test limit of light absorbing impurities in oxytetracycline Prepare 0.2%w/v solution & measure the absorbance at 430nm absorbance should not more than 0.5 Slide 10: B. limit of p- chlorophenol in clofibrate Prepare test & standard solution of clofibrate & measure the absorbance at 455nm. Compare the absorbance of test & std. Absorbance of test must not exceed standard. Slide 11: C. Salicylic Acid In Aspirin In IP & BP visual comparison method is employed in which violet colour is compared which is developed by addition of neutral FeCl 3 solution. In USP instead of visual comparison method absorbance is measured at 305nm. Slide 12: D . Limit of p-amino phenol & paracetamol Sample is treated with alkaline ferricyanide reagent it produces colour with p-aminophenol & measurement is done at 710nm. B). Structural analysis: B). Structural analysis Chromophoric part is required for absorbance in UV. Change in chromophoric part produce predictable. Change in absorption property. E.g. Vit A exist in 2 isomeric form like vit-A 1 & A 2 Because of extra double bonds vit A2 shows bathochromic shift of 30nm. But here 25nm which is near by thus from wavelength we can identify structure of Vit –A (2)Quantitative Analysis : (2)Quantitative Analysis . Single compound analysis Direct analysis Here ,the compound to be analysed does not require any chemical treatment. Compound is dissolved in suitable solvent diluted and assayed or measured. - e.g Compound λ max Riboflavin powder 267nm Chloramphenicol capsule 278nm Chloroquine HCL injection 343nm Dexamethasone 241nm Slide 15: using separation method a) After extraction :- This technique applied to formation in which we have to extract that is from dosage form with the help of suitable solvent. Dilution is done with same Solvent and measure. Absorbance drug in extracting media. Slide 16: Drug Extractant λ max Chlorpropramide Chloroform 232 Chloroquin phosphate tab. Chloroform 343 Chlorpheneramine Maleate syrup n- hexane 264 Chlorpromazine HCI tab Ether 277 b)After Chromatographic Separation: b) After Chromatographic Separation Ion exchange chromatography used to separate desire component which is then measured for absorbance. E.g . Clofibrate capsules are separated by ion exchange chromatography. It is measured by 275nm. C)Using column chromatography: C) Using column chromatography It is used to separate desired compound which is then analysed by spectroscopy . E.g . Aspirin tablets are subjected to column chromatography. When salicylic acid is removed and aspirin measured at 280nm . d)Using HPLC: d) Using HPLC HPLC is used here for the separation of desired compound that to be estimated . E.g. Sulphamethoxazole + trimethoprim cotrimoxazole It is subjected to HPLC where both drugs are separated. Sulfamethoxazole and trimethoprim are separately measured. It is measured at 257nm and 287nm respectively . Folic acid tablets are also analysed by this method. Indirect Analysis: Indirect Analysis Single component without chromophore :- Here the compound that is devoid of a chromophore is treated chemically so, that it is converted to a chromophore. e.g. phenolic compound Fecl 3 solution is added to phenolic compound to develop violet color which is then measured using an instrument. This technique is used for large number of phenolic drug. E.g. catechol amines , paracetamol ,hormones, oestrogens , morphine and its derivatives etc. Slide 21: b. Drug + chromophoric reagent Drug + chromophoric reagent which give chromogen and here the absorbance of chromogen are measured in visible range. For analyte which absorb which absorb weakly in UV region: For analyte which absorb which absorb weakly in UV region Chemical Derivatization Indirect spectrophotometric assays are based on the conversion of the analytical by a chemical reagent to a derivative that has different spectral properties. The majority of indirect spectrophotometric procedures involve the conversion of the analyte to a derivative that has a longer λmax and/or a higher absorptivity. Chemical derivatisation procedures may be adopted for any of several reason. Slide 23: If the analyte absorbs weakly in the ultraviolet region, more sensitive method of assay is obtained by converting the substance to a derivative with a more intensely absorbing by converting chromophore. e.g. Sugars Which do not absorb significantly above 220nm can be determined. spectrophotometrically by heating with enthrone in concentrated sulphuric acid and measured the absorbance of the colored derivatives at 625nm e.g.: e.g. Diazotization and Coupling of Primary aromatic Amines Condensation reactions Oxidation methods For avoiding interference: For avoiding interference E.g . Methyl testosterone tablet Max 240nm - drug excipients are interfering. Treat with hydrazide reagent. Oxidation of alpha ketone group of methyl testosterone with tetrazoliam salt. Measured without interference. Improve selectivity of assay: Improve selectivity of assay E.g. Procaine adrenaline injection assay. Adrenaline (20 µg/ml) injection. Adrenaline measured at 270nm. Procaine at higher conc. Interference at this λ max Adrenaline reacts with fe 2+ and gives purple colour measured by colorimetric. Q techniques by chemical derivatization : Q techniques by chemical derivatization Phenolic drug + FeCl 3 give violet colour. Vitamin D + antimony trichloride gives red colour and its absorbance is 500nm. By treating with primary aromatic amine + HNO 2 (HCL + NaNO 2 ) which gives diazo salt. react BMR reagent. c) Photometric titrations: c) Photometric titrations T itration done by photometer is called photometric titrations sample when titrated with titrant produce change in reaction in reaction mixture which is measured in terms of absorbance change in absorbance of solution may be used to follow the change in concentration of light absorbing constituent during titration this is end point detection method Slide 29: principle of this technique is that formation of sample titrant complex or titration product when sample & titrant react with each other S + T = ST Where, S = sample T= Titrant Derivative Spectroscopy: Derivative Spectroscopy Derivative spectroscopy involves the conversion of a normal spectrum to its first, second or higher derivative spectrum. First derivative spectrum (D1) is plot of the rate of change of absorbance with wavelength against wavelength or plot of dA/ d λ The second derivative spectra (D2) is a plot of the curvature of the first spectra against wavelength or plot of dA 2 /d λ 2 . Slide 31: In summery the first derivative spectrum of an absorption band is characterised by a maximum & cross over point at λ max of the absorption band. The second derivative spectra is characterised by two satellite maxima & inverted band of which the minimum corresponds to the λ max of the fundamental band Advantages: Advantages Firstly an even order spectrum is of narrower spectral band width than its fundamental spectrum A derivative spectrum there fore shows better resolution of overlapping bands than the fundamental spectrum & may permit accurate determination of λ max of individual bands Derivative spectroscopy discriminates in favour of substances of narrow spectral band widths against broad band thus help to identify smallest interference Differential Spectroscopy : D ifferential Spectroscopy The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing intereferents may be markedly improved by the technique of different spectrophotometry. Different spectrophotometric assay is that measured value is the difference absorbance (∆A ) between two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics. Slide 36: The criteria for applying difference spectrophotometry to the assay of a substance in the presence of other absorbing substance are that: (a) reproducible changes may be induced in the spectrum of the analyte by the addition of one or more reagents. (b) the absorbance of the interfering substance is not altered by the reagents. Slide 38: The simplest and most commonly employed technique for altering the spectral properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. The ultra violet-visible absorption spectra of many substances containing ionisable functional groups. E.g. phenols , aromatic carboxylic acids and amines are dependent on the state of ionisation of the functional groups and on the PH of the solution. Slide 39: The absorption spectra of equimolar solutions of Phenylephrine, a phenolic sympathomimetic agent in both 0.1 M HCl (pH – 1 )and 0.1 M NaOH ( PH – 13 ). The ionisation of the phenolic group in alkaline solution generates an additional n (non bonded ) electron that interacts with the ring πelectrons to produce a bathochromic shift of the λmax from 271nm in acidic solution to 291nm and an increase in absorbance at the λmax ( hyperchromic effect ). Slide 40: The different absorption spectrum is a plot of the difference in absorbance in between the solution at pH 13 and that at pH 1 against wavelength. It may be generated automatically using a double –beam recording spectrophotometer with solution at p H 13 in the sample cell and the solution at pH 1 in the reference ( blank ) cell . At 257 and 278 nm both solutions have identical absorbance and exhibit zero difference absorbance. Such wavelength of equal absorptivity of the two species are called isosbestic or isoabsorptive points. Above 278nm the alkaline solution absorbs more intensely than the acidic solution and ∆A is therefore positive . Slide 41: Between 257 and 278nm it has a negative value. The measured value in a quantitative difference spectrophotometric assay is the ∆A at any suitable wavelength measured to the baseline , e.g. ∆ A 1 at λ 1 or amplitude between an adjacent maximum and minimum, eg ∆A 1 at λ 2 and λ 1 . At λ 1 ∆A = A alkli – A Acid Slide 42: Where A alk and A acid are the individual absorbance at λ1 in 0.1M sodium hydroxide and 0.1M hydrochloric solution respectively. If the individual absorbances A alk and A acid , are proportional to the concentration of the analyte and pathlength , the ∆A also obeys the Beer – Lambert Law and a modified equation may be derived. ∆A = ∆ abc Slide 43: Where ∆a is the difference absorptivity of the substance at the wavelength of measurement. If one or more other absorbing substances is present is present in the sample which at the analytical wavelength has identical absorbance in the alkaline and acidic solutions, its interference in the spectrophotometric measurement is eliminated. ∆A = ( A alk + A x ) – ( A acid + A x ) = A alk – A acid Determination of Composition of Complexes: Determination of Composition of Complexes Transition metal complexes are measured by UV visible absorption spectroscopy as they form colored complexes. E.g., Fe ++ forms violet colors with Salicylic acid. Metal (M) & ligand (L) form complex but they are of different possibilities. M + L → (M-L) 2M + L → (M-L) M + 2L → (M-L) By UV visible spectroscopy we can find out correct composition of complexes. Fundamental Principle of Measurement: Fundamental Principle of Measurement Complexes are colored and are easy to measure for absorbance. Color intensity is directly proportional to complex formation. Go on changing proportion of M & L & measure absorbance & thus concentration combination having highest absorbance is the correct composition of complexes. Mole Ratio method: Mole Ratio method In mole ratio method concentration of either ligand or metal is kept constant. Moles of 1 component is changing & mole ratio also changes . Measure the absorbance of each mixture and plot the graph of absorbance X v s mole ratio of Y. Continuous variation method: Continuous variation method It is called Job’s curve discovered by Job. Here we change proportion of both the components. X 1 +Y 1 = Z X 2 +Y 2 = Z X 3 + Y 3 = Z Slide 48: Proportion of both the component is changed but the sum is kept constant Measure absorbance at λ max Y is plotted as Y is changing in increasing manner. At a particular ratio there is max absorbance. 4. Study of kinetics: 4. Study of kinetics Study of reaction rate of drugs can be done using UV visible absorption spectroscopy. Absorbance of drug is measured at different time intervals & the concentration is calculated. E.g. paracetamol is degraded to 4-amino phenol. Slide 50: Here we can set the instruments at 257nm & go on measuring absorbance. The rate of decrease in absorbance is equal to the rate of degradation of drugs. Also we can set the instrument at 300nm & measure the absorbance. The rate of increase in absorbance is equal to rate of degradation of drugs. Drugs measured by UV absorption spectroscopy as per IP2007: Drugs measured by UV absorption spectroscopy as per IP2007 SR NO DRUGS ABSORPTIVITY(a ) λ max ( nm ) 1 Chloramphenicol (anti biotic) IP 297 278 2 Caffeine (diuretic, CNS stimulant) IP 504 273 3 Atenolol tab ( β blockers) IP 53.7 275 4 Neostigmine inj (sympatholytic) IP 14.35 260 5 Promethazine tab (anti emetic) IP 910 249 6 Riboflavin ( Vit- b2) IP 328 444 7 Tinidazole ( anti amoebic) IP 356 310 8 Tamoxifen tab ( anti cancer) IP 325 275 9 Verapamil ( calcium channel blocker) IP 118 278 10 Warfarin(anti coagulant) IP 431 308 References: References Sharma B. K.; Instrumental Method of Chemical Analysis; Krishna Prakashan media ltd; 24 th edition; 2005; S-135,164 Beckett A.H., Stenlake J.B.; Practical Pharmaceutical Chemistry; CBS publishers and distributers; 4 th edition; 2005; 292-303 Indian Pharmacopoeia; Ministry of Health & Family welfare; Published by Indian Pharmacopoeial commission, Ghaziabad; 2007; volume I, II .