Applications:

1. Structural Analysis 3. Limit Test 4. Quantitative Analysis (Assay) 5. Study of Kinetics 6. Determination of pKa of Indicators 2. Identification of Drugs (Pharmacopoeial)

7. Determination of Complex Composition 8. Determination of Molecular Weight of Compound 9. As HPLC Detector

10. Forensic Application

11. Applications in Preformulation and

Formulation Partition Coefficient Solubility Release of Drug from Formulation

1. Structural Analysis:
Chromophoric part is required for absorbance. Change in chromophoric part produces predictable change

in absorption property. Eg.: 1. Detection of Conjugation: It helps to show the relationship between different groups particularly with respect to the conjugation. a) Between two or more carbon-carbon double (or triple) bonds b) Between carbon-carbon and carbon-oxygen double bonds c) Between double bonds and an aromatic ring.

 2. Detection of Geometrical Isomers:

Trans isomers exhibit max at slightly longer wavelength than the cis isomer For e.g., Of the two stilbenes the trans isomers show max = 294 nm while the cis isomer show max = 278 nm.

 3. Detection of Functional groups:

That is possible to detect the functional groups with the help of UV Spectrum. For e.g., Absence of absorption above 200 nm says that there is absence of conjugation, carbonyl group and benzene ring in the compound.

2. Identification of Drugs:
Some of the drugs which are official in

pharmacopoeia have specific max value at which they give higher value of absorbance. So from that drug can be easily identified. For e.g.,
1. PCM should have max = 249 nm and

absorbance 0.44
2. Morphine sulphate should have max =

298 nm.

 It is one of the best methods for

3. Limit Test:

detecting impurities in inorganic compounds. The band due to impurities are very intense. For e.g., Impurity having amount 0.05% has an value of 2000 so impurity can be identified in a transparent major component.

4. Quantitative Analysis:
The method can easily detect the quantity with the sensitivity of g/ml. By applying the mathematical equation the concentration can be detected from the absorbance value. For e.g.,

A = abc
Where, A = Absorbance

a = Specific absoptivity b = Pathlength c = Concentration

5. Study of Kinetics:
It can be used to study the kinetics of

reaction. In it, the change in concentration either of a reactant or product with time is measured. The method is based upon the fact that the one of the reactants or products exhibiting suitable absorption in the UV region is not overlapped by other species. It can be applicable to such rate which must be relatively slow.

6. Determination of pKa of Indicators:

Acid-Base indicators are weakly acidic or

basic compounds. To the reference of pH equation, the pKa can be calculated if the ratio of [HA]/[A-] is known at particular pH. The ratio can be determined spectrophotometrically from the graph which is plotted between absorbance and wavelength at different pH value.

7. Determination of Complex Composition:

Transition metal complexes are measured by

U.V. Visible absorption spectroscopy as they form colored complexes. For e.g., 1. Fe+2 forms violet color with salicylic acid. 2. Ni+2 forms green color with dimethyl glyoxime.

8. Determination of Molecular Weight of Compound:

It is limited to special compounds like

C=O, R-NH2, etc.

E.g.,
If of chromophore is 50,000 and

concentration of solution of solution of sulpha drug is 5 g/ml. Find out molecular weight of drug. Absorbance = 0.4

 We require concentration in moles/litre. 5 g/ml = 5 10-6 103 gm/L = 5 10-3 gm/L

Molarity = 5 10-3 moles/L. M= mol. wt.

M

A = bc

A = 0.4, b = 1, c = 5 10-3 , = 50000

M .. 0.4 = 50000 1 5 10-3 M .. M = 50000 0.005 0.4 .. M = 625

9. As HPLC Detector:
HPLC gives most efficient separation.
In modern analysis, HPLC is combined with

detectors HPLC and U.V. visible is commonly used in combination for specific drug subs. E.g. :- Cotrimoxazole
1. Sulfamethoxazole max = 257 nm
2. Trimethoprime max = 267 nm

10. Forensic Applications:

UV/Vis molecular absorption is routinely used in the

analysis of narcotics and for drug testing. One interesting forensic application is the determination of blood alcohol using the Breathalyzer test. In this test a 52.5-ml breath sample is bubbled through an acidified solution of K2Cr2O7. Any ethanol present in the breath sample is oxidized by the dichromate, producing acetic acid and Cr3+ as products. The concentration of ethanol in the breath sample is determined from the decrease in absorbance at 440 nm where the dichromate ion absorbs. A blood alcohol content of 0.10%, which is the legal limit in most states, corresponds to 0.025 mg of ethanol in the breath sample.

11. Applications of UV/Vis. In Preformulation and Formulation:

U.V./Visible spectrophotometry is a standard

method for determining the physico-chemical properties of drug molecules prior to formulation and for measuring their release from formulation. The type of properties which can be usually determined by the UV method are listed here: Partition Coefficient Solubility Release of a Drug from a Formulation.

 Partition Coefficient:
The partition coefficient of a drug between

water and an organic solvent may be determined by shaking the organic solvent and the water layer together and determining the amount of drug in either the aqueous or organic layer by UV spectrophotometry. If buffers of different pH values are used, the variation of partition coefficient with pH may be determined and this provides another means of determining the pKa value of a drug.

 Solubility
The solubility of a drug in, for instance,

water may be simple determined by shaking the excess of the drug in water or buffer until equilibrium is reached and then using UV spectrophotometry to determine the concentration of the drug that has gone into solution.

 Another method for determining solubility, where an ionisable group is present in the drug, is to dissolve varying concentrations of the salt of the drug in water and then excess acid to a solution of the salt of an acidic drug or excess base to a solution of the salt of the basic drug, thus

converting the drugs into their un-ionised forms. When the solubility of the un-ionised drug in water is exceeded, a cloudy solution will result and UV spectrophotometry can be used to determine its degree of turbidity by light scattering, which can be measured at almost any wavelength, e.g. 250 nm.

 Release of a drug from a formulation:

UV spectrophotometry is used routinely to monitor in

vitro release of active ingredients from formulations. For simple formulations the drug is simply monitored at its max. In the example shown in figure, the rate release of pseudoephedrine from a controlled release formulation was monitored. The release of the drug was followed by monitoring its release into distilled water using a UV spectrophotometer set at 206 nm.

 In the example given in figure, the particle size of the

ethylcellulose used in the formulation affected the rate of release.

 If UV-absorbing excipients were present in

such a formulation, the UV wavelength used for monitoring release would need to be selected carefully or HPLC coupled to UV detection might be used. For such studies, the sampling of the dissolution medium may be fully automated so that the medium is filtered and pumped through the UV spectrophotometer at set time intervals in order to take a reading.