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APPLICATIONS OF VISIBLE AND UV SPECTROMETRY

V. Suhasini M.PHARM

(PHARMACEUTICS)

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APPLICATION OF VISIBLE SPECTROMETRY

Substance that are having intrinsic color (berberine , clofazimine ,cyanocobalamin , riboflavin and rifampicin) can be determined directly by colorimetric method whereas colorless substance can be estimated only after converting it into colored derivative by treating them with appropriate chromogenic reagent. Colorimetry is used in the quantitative estimation of

1. Inorganic compound 2. Biochemical specimens 3. Organic and pharmaceutical compounds

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INORGANIC COMPOUND Inorganic field it is used in the estimation of

both cation and anion . The cations estimated by colorimetry

cations Chromogenic agent

color λmax

ammonium

Nessler’s reagent

Orange 425nm

antimony Potassium iodide

yellow 425nm

arsenic Ammonium molybdate

blue 840nm

Magnesium

Titan yellow red 535nm

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BIOCHEMICAL SPECIMENS In biochemical field it is used to determine

the amount of glucose , ketone bodies , blood and proteins

anions Chromoenic Agent

colour λmax

fluoride Thorium chloranilate

purple 540nm

phosphate molybdenum

blue 820nm

silicate molybdate yellow 400nm

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Organic and pharmaceutical application In assay of glyceryl trinitrate tablets first the

nitric acid is obtained by quantitative hydrolysis of the ester. The nitric acid thus obtained by hydrolysis of nitrates reacts with phenol 2,4 disulphonic acid to form a yellow colored product in ammonical solution . The intansity of the yellow color for potassium nitrate at 450 nm to determine the contents in tablets

S OO

OH

S

O

O

OHOH

O

N+

O-

OS

O

O

OH

S OO

OH

HNO 3

NH3

O

N+

O-

OS

-O

O

S OO

O

OH

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colorless analgesic drug paracetamol , chemically called as p- acetamido phenol is assayed by uv spectrophotometer . A colorimetric method is also reported for its estimation at 450nm after converting it into a yellow colored chromogen by reacting with NaNo2 in presence of Hcl at 5o c after preliminary hydrolysis

OH

NH

O

CH3

OH2+

OH

NH2

+ CH3

O

OH

OH

NH2

+ Na N+

O-

O

+ ClH

OH

N+

N+

Cl

NaCl+ + OH2

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Ergometrine maleate and ergot alkaloids from a blue colored compound on reaction with p-dimethyl amino benzaldehyde and a trace of ferric chloride in sulphuric acid. The blue colored compound then can be measured at 578nm

The amount of carbachol in the injection is determined by colorimetric method by reacting it with reineckatte solution in acetone to from a pink red color which is measured at 526nm

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Sulpha methoxazole react sodium nitrite and Hcl at 5oc. The diazotised sulphamethoxazole can be readily coupled with N (1- naphthyl) ethylene diamine dihydrochloride to form a purple colored complex which can be measured at 538nm

NH2 S

O

ONH

ON CH3

Na N+

O-

O

++ ClH0 - 5 OC

S

O

ONH

ON CH3

N

NCl

+

NH

NH2NH

NH2

S

O

ONH

ON CH3

NN

Purple co lured complex

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Vitamin A and vitaminD can be estimated

colorimetrically by by reacting them with antimony trichloride in chloroform , where the intense blue and green color obtained respectively is measured between 520-590nm. the color formed is stable for few secounds only hence the reagent is added directly to the cuvette during measurement

calciferol tablet and solution are determined by colorimetric method after extracting it by light petroleuam from the formulation .the residue of light petroleum extract in ethanol free chloroform solution , on reaction with antimony trichloride yield a brown orange color which can be measured at 500nm

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Compounds containing amino group can be analysed colorimetrically by using suitable carbonyl reagent. The most frequently employed reagent is p- dimethyl amino benzaldehyde (ehrlich’s reagent) .the drug procaine injection is assayed based on the condensation reaction of procaine and p-dimethylaminobenzaldehyde to yield yellow color measured at 454nm

NH2

O

O N

CH3

CH3

+ NCH3

CH3

O

N

O

O N

N

CH3

CH3

CH3

CH3

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Determination of phenolic compound Phenols react with ferric ion and forms violet

color which can be used for its quantitative estimation .this can be used for quantitative estimation in colorimetry . The formation of color is attributed to the existence of keto-enol tautomerism in phenols

Phenol is mostly in enol form it forms colored complex with ferric ion

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Phenol react with diazonium salts to yield colored dyes

N+

N+Cl + OH N N

OH

+ ClH

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Advantages of colorimetry method This method is not time consuming and

hence it is ideally suited for routine work The operations involved in the estimation are

simple, particularly when the photoelectric colorimetric is used

Determination of minute quantities of substance (<1 to 2%) is possible

The instrument employed for the work is inexpensive

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APPLICATIONS OF ULTRAVIOLET SPECTROSCOPY The methods based on the absorption of

radiation are extremely important from the viewpoint of an analytical chemist.

Ultraviolet methods find extensive use in the identification of various hydrocarbons , vitamins , steroids , heterocycles ,and conjugated aliphatics

I. QUANTITATIVE ANALYSIS II. QUALITATIVE ANALYSIS

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Quantitative analysis The conc of a drug or absorbing substance in

a given sample can be easily analysed by measuring the absorbance of the solution prepared in a transparent solvent in a uv spectrophotometer. this quantitative UV method is applicable to determine the conc of a single component in the given sample and to determined the conc of different components in mixture

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The methods adopted to analyse single component sample

1. Standard adsorptivity value method 2. Calibration graph/curve method 3. Single or double point standardization

method

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standard absorptivity value method In this method the conc of the unknown compound can be

determined by using the measured absorbance and standard absorptivity value

For example if the absorbance of methyl testosterone in ethanolic solution measured in a 1cm cell at its λmax 241 nm is found to be 0.890 and the standard absorptivity value at 241 nm is 540 then the conc of methyl testosterone can be determined by the following equation

A = abc A - absorbance of the solution =0.890 a - standard absorptivity value = 540 b -pathlength of the sample cell = 1cm c – conc of the unknown to be determined 0.890 = 540 x 1 x c ; c = A/ab = 0.890/540=0.00165g/100

ml

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Calibration graph/ curve method In this method the absorbance of a number

of standard solutions of the reference substance at conc encompassing the sample concentrations are measured and calibration graph is constructed.

calibration data is highly essential if The absorbance has non linear relationship

with conc It is necessary to confirm the proportionality

of absorbance as a functions of conc The absorbance or linearity is dependent on

the assay conditions

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CALIBRATION CURVE

0.4 0.3absorbance

0.2

0.1 conc 0 5 10 15 20

Conc in µg/ml

absorbance

0 0.0005 0.05010 0.10015 0.150

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Single or double point stardardization method

The absorbance of a sample solution and a standard solution of the reference substance will be measured in single point procedure

The standard and sample solution should be prepared in the same manner, where the conc of the standard solution should be close to the sample solution

The conc of the substance in the sample is calculated from the proportion relationship that exists between absorbance and conc

C test = A test x C std A std

C test , C std Conc of test and std solution

A test , A std Absorbance of test and std solution

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Quantitative analysis of substance in multicomponent sample

The identification and conc of one or more components in the sample can be analyzed by modifying the simple spectrophotometric procedure adopted in single component sample

The interference of one component by the other component may arise due to manufacturing impurities, decomposition product , formulation excipients will cause unwanted absorption termed as irrelevant absortion , imparts systematic error in the assay of the drug

By adopting multicomponent analysis the actual amount of sample under investigation can be easily calculated after removing the irrelavant absorption

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The of all multi-component spectrophotometric analysis is that at all wavelength

The absorbance of a solution is the sum of absorbance of the individual component

The measured absorbance is the difference between the total absorbance of the solution in the sample cell and that of the solution in the reference cell

The different multi-component analytical method

I. Using absorbance corrected for interference II. After solvent extraction of the sample III. Simultaneous equation method IV. Absorption ratio method V. Geometric correction method VI. Orthogonal polynomial method

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Using absorbance corrected for interference By knowing the absorbing interferon's its conc and its

absorptivity it is possible to calculate its contribution to the absorbance of the mixture.

Then the conc of the component of our interest can be calculate from the corrected absorbance ( total absorbance – interfering absorbance )

Example: determination of conc of epidrine Hcl and chlorocresol in epidrine Hcl injection

Assay after solvent extraction of the sample If it is not possible to calculate the contribution of

absorbing interferants to the total absorbance or the interferance from other absorbing substance is large , then it may be possible to separate the absorbing interferon's from the analyte by solvent extraction procedure

This procedure is appropriate for acidic or basic drugs whose state of ionization determines their solvent partition behaviors .EX : assay of caffeine in aspirin & caffeine tablet

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Simultaneous equation method This method is based upon the fact that the

total absorbance of a solution at a given wavelength (λ1 & λ2) is equal to the sum of the absorbances of the individual components present

Therefore it is possible to analyze individual components of a mixture even if there is an overlap in the spectra

M N

Absorbance

λ1 λ2

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The individual absorption spectra of substance M and N showing wavelegth for the assay of M and N in admixture by the method of simultaneous equation

The absorption of M at λ1 and λ2 is am1 and am2

The absorption of N at λ1 and λ2 is an1 and an2

The absorbance of dilute solution at λ1 and λ2 is A1 and A2 respectively

The conc M(Cm) and N (Cn) in diluted sample can be determined by equation

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λ1 A1 = am1 bCm + an1bCn (1)

λ2 A2 = am2bCm + an2bCn (2) for measure in 1 cm cell b = 1 rearranging equation (2) Cn = A2 – am2Cm an2 substitnute Cn in equation (1) and rearranging

Cm = A2an1 – A1an2 am2an1 – am1an2

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Qualitative analyis In qualitative analysis , UV spectra is mainly used

to detect the presence of different chromophores,polynuclear compound and to detect the extension conjugation

Detection of functional groups Uv technique is applied to detect the presence or

absence of the chromophore Uv wavelegth range from 200nm to 400nm,it

shows the absence of conjugation carbonyl group (aldehydes and ketones ) benzene or aromatic compound bromo or iodo atoms

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Detection of conjugation : Two or more carbon – carbon double bond or triple

bond Carbon-carbon double bond, carbon –oxygen double

bond Aromatic ring with double bond Example of various conjugated chromoporic group

chromophore example λmax

CH2=CH2 Ethylene 174nm-C=C-C=C- Butadiene 217nm-C=C-C=C-C=C-

1,3,5 Hexatriene

267nm

-C=C-C=O- Crotonaldehyde 218nm

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Distinction of conjugated and non conjugated diene

uv spectra is also useful in distinguishing a conjugated and non conjugated compound

Example : The non conjugated ethylene

having one double bond has λmax at 174 nm can be easily distinguished from butadiene which appears at longer wavelength λmax 217nm due to the presence of conjugated double bond

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Detection of impurities if a substance is contaminated with

impurity ,the impurity will also absorb light at different wavelength then the absorption spectra of impure substance will differ from characteristic absorption of pure substance thus impurities present in pharmaceuticals or organic compounds can be easily detected by measuring its absorbance at specific wavelength

Example : the presence of impurity xanthanoic acid in propantheline bromide can be determined by measuring the absorbance of 0.5%W/V in 0.1M NAOH at 281nm .if the absorbance is more than 0.31, then it indicates the presence of xanthanoic acid impurity

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Identification of unknown compound An unknown compound can be identified by

comparing its spectrum with the known spectra. If the two spectra coincide , the two compound must be identical . If the spectra do not coincide, then the expected structure is different from the known compound

Detection of polynuclear hydrocarbons Benzene and polynuclear compounds have

characteristic spectra in ultra violet and visible radition . Thus the identification of polynuclear hydrocarbons can be made by comparison with the spectra of known polynuclear compounds .

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Example : benzenes shows adsoption maximum at 256nm . The methyl group (toluene ) substitution in benzene ring in the shift of λmax from 256 to 261 nm

Polynuclear hydrocarbon

Absorption maximum

312nm

375nm

480nm

580

Naph th alen e

Anthracene

Naphthacene

Pentacene

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Structural elucidation Elucidation of structure is another important

qualitative application of uv spectroscopy. For example structure of vitamin A1 and A2 can be easily elucidated by uv spectra. Vitamin A1 absorbs at 325nm and vitamin A2 absorbs at

351 nm . The absorption maximum appears at longer wavelegth for vitamin A2 due to the presence of additional ethylene bonds CH3 CH3

CH3

CH3

OH

CH3

Vitamin A1

CH3 CH3

CH3

CH3

OH

CH3

Vitamin A2

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Detection of possible tautomers If a molecule exists in two tautomeric forms

preference of one over the can be detected by ultra violet spectroscopy

Example : 2 hydroxy pyridine which exists in equilibrium with its tautomeric from pyridone-2 the spectra of these two compounds were found to favour pyridoxine-2 which is an α,β unsaturated ketone and clearly, the equilibrium is shifted towards the right

N OH N O2- Hydroxy Pyridine Pyridine - 2

References Instrumental methods of chemical analysis by R.Chatwal Pharmaceutical analysis fourth edition by S.Ravi sankar Principle and applications of uv and visible

spectroscopy by Rajasekaran

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Thank you