UV/Vis Spectrophotometry

Introduction: -It is one of the most frequently applied techniques in pharmaceutical analysis. -A type of absorption spectroscopy. -Quantitative determination of inorganic, organic & biological species. -Measurement involves: UV region(190-380nm) & Vis. region(380-800nm) . Instrument which measures the ratio, or function of ratio of the intensity of two beams of light in the ultravioletvisible region are called ultraviolet visible spectrophotometer. -UV/Vis spectrophotometry Molecular spectra: -The most common transitions in UV/Vis region for electronic spectra are (n* & * ). Where n is non-bonding electron and * refers to antibonding orbital.

-A molecule possesses following internal energies : Eint = Eelect. + Evib. + Erot -Absorbs light or radiation in both UV & Vis. regions. -Absorption occurs when the energy of light matches that is required to induce electronic transition in molecule along with vibrational & rotational elements. - The energy elements are quantised. -These energy levels are intimately related to the structure of a molecule. -Vibrational & rotational transitions superimpose upon the electronic transition.

-One particular transition(Abs.Max.) is given by more molecules than any other transitions. -The related(minor) transitions are given by fewer molecules.

E = (Eelect. + Evib. + Erot.)upper - (Eelect. + Evib. + Erot.)lower -The vibrational & rotational changes introduce fine structure into the spectrum. -The resulting absorption involves a band of wavelengths rather than a single line. -The variation of the gain of energy approximates to a Gaussian distribution.

Quantitative use of UV/Vis spectrophotometry: -A relation based on Beer-Lambert law is used to solve quantitative problems. A= log I0/It = abc Where, A is absorbance, I0 the intensity of incident radiation at particular wavelength, I t the transmitted light, a the molar absorptivity, b the pathlength of sample, c the concentration of the absorbing species. Instrumentation: Two types: 1)Single beam UV/Vis spectrophotometer 2)Double beam UV/Vis spectrophotometer

Single beam UV/Vis spectrophotometer

Double beam UV/Vis spectrophotometer

1)Stable sources: -A source must generate a beam with sufficient power for each detection & measurement. -The emitted radiation should be stable for reasonable periods. -However, the problem of the source stability can sometimes be minimized by double beam design. Common energy sources for the following regions: a)Visible region. b)Ultraviolet region. a)Visible region: -Common source of radiation for visible region is a a tungsten filament lamp. -It emits radiation of 350-2,000nm. -Stray light effects from IR region is removed by the suitable filters. b)Ultraviolet region: -The convenient light source for ultraviolet region is a deuterium discharge lamp. -Continuous spectrum covers 185-380nm. Some instruments use Xenon arc lamp as a source. It produces intense radiation by the passage of current through an atmosphere of xenon. Its spectrum is continuum over the range between about 200 and 1000nm with the peak intensity at about 500nm. 2)Sample holder: -The material of the sample container ideally should be transparent at the wavelength of measurement. -In UV/Vis spectrophotometer: *Above 320nm, the cuvette(cell) should be flat fused glass/fused silica or quartz/plastic. *Below 320nm, the container should be fused silica/ quartz cell. -Pathlength is 10mm. 3) Monochromator To produce a beam of monochromatic radiation that can be selected from a wide range of wavelengths a)Filters: Glass, gelatin & interference filters b) Prism & grating: Consists of a system containing: -an entrance slit -a concave mirrors or collimating lens -a dispersive device, prism/grating -a foucing lens -an exit slit.

a)Prism: -White polychromatic light is dispersed into constituents colours(spectrum) because of refraction. -The required wavelength may be selected from it.

c)Grating: -Diffraction grating is the element in the monochromator of most modern UV/Vis.spectrophotometer. -It gives a uniform dispersion through out the entire spectrum(not possible in prism monochromator) -In this tool, the principle of diffraction & interference(productive & destructive) is used for dispersion. -Dispersion is brought about by directing a polychromatic beam through a transmission or onto the surface of a reflection grating. -The grating is a hard, optically flat, polished surface that has a large number of parallel & closely spaced grooves. -A diamond tool is used to make the grooves.

4)Detector: ( a device to convert radiant energy to an usable electric signal). -It is to: * measure & convert radiant energy/light intensities to an usable electric signal. -Precise determination of the light intensities are required for accurate determination of substance. Photon detectors: -Barrier-layer cells, Phototubes, Photomultipliers, Photodiodes etc. -Photomultiplier detector is commonly used in UV/Vis. Spectrophotometer. -It is for to measure the low power radiation. -Consists of photoemissive cathode & several dynodes(which emit several electrons for each electron striking them).

5)A signal processor & readouts: -An electronic device to amplify the electrical signal from detector or transducer. -Changes signal from DC to AC. -Change the phase of signal & filter it to remove unwanted components. -Performs mathematical operations i.e.differentation, integration & conversion to logarithm. -Finally there are several readout devices e.g, DArsonaval meter, digital meters, recorder, cathode-ray tubes, LCD panels, computer display etc. Pharmaceutical application of UV/Vis. Spectrophotometry: 1)Qualitative, 2)Quantitative, 3)Structure elucidation: 1)Qualitative -Ultraviolet absorption spectra has been extensively used in pharmaceutical chemistry/analysis as a supporting tool/evidence for identification of organic drug substances. -Comparison of absorption spectrum of the authentic sample, wavelength of maximum absorption( max ) , absorptivity, ratio of intensities at two wavelength, pH induced spectral change, absorption spectrum of derivative of the sample . -Above are the useful criteria of identity & purity. -For complete identification of a compound, spectrophotometric measurements must be supplemented by chemical tests & other physical measurements. 2)Quantitative: -Selection of wavelength: At max. -Concentration: Abs.0.9(0.3-1.5). -Selection of solvent: Solubility, Spectral profile, Ultraviolet cut-off of some common organic solvents

Examples: Drug

Solvent

Wavelength(nm) 265 278

Acetazolamide 0.1M HCl Cyanocobalamin Water

Bisacodyl tabletsChloroform Frusemide tablet Riboflavin Paracetamol Alcohol Acetate buffer 0.01M NaOH

264 252 444 257

Procedure to calculate the concentration of the absorbing substances: i) Use of a standard absorptivity. ii)Use of a calibration graph. iii))Single or double point standardisation. 3)Structure elucidation: i)Presence and absence of unstauration(multple bond) , heteroatoms like S,O, N(nitro, nitroso, carbonyl, thionyl) or halogens. ii)Effect of conjugation(alternating double bond & auxochromes:NH 2 ,OH, NO2 ,CH3) is shift of max . Hypsochromic shift(max to longer wavelength) Bathochromic shift (max to shorter wavelength) iii)Effect of geometric isomerism e.g. Calcitriol (Cis isomer) max at 265nm Isovit D2 (all trans) max at 287nm iv)Effect of cross conjugation: Characteristic absorption is due to the main chromophoric e.g in progesterone, prednisolone, prednisone. v)Effect alkyl substitution( increasing the no. of substitution increases the max in crotonaldehyde, alfa- ionone, progesterone). vi)Number of rings in the structure: i. Benzene (1) 261nm ii. Napthalene (2) 312nm iii. Anthracene (3) 375nm vii)Determine the keto-enol tautomerism (max gets shorten in enol form) viii)Spectrophotometric titration (sharp change in absorbance as indicator )