0 Reading In modern instruments, the reading Ps so adjusted that the absorbance of the sample is read directly This measurement Scane a range of warlengthe and simultaneously measures the absorbance of sample and Solvent The Spoctrum is recorded absorbance against wavelength on a chart recorder Light Source of UV is H /dauterium lamp Visible light- tungsten filament lamp These two sources cover the whole range of wavelengths ie ,200. - 400 -UV. 400-780nm - -Visible The Optical system used for the generation of Monahromatic light is done eithia by a Prism which splits a Multi-wavelengle Source rudiation into components parts by regraction / grating based on diffraction of light
- Detector are commonly photomultiplier tubes, with designs based on the wavelength range to b2 detected 3 - Absorption spectra arise due to a transition in a molecule or its part with a special I 1 arrangement in the excite state That arrangement present in a part of molecule is called chromophore - N=N- - Conjugation of double bond lower the energy of e transition hence the chromophore wavelength Bathochromic longer wavelength reduced shifts / (red absorbs at Shifts town longer is called as shift If conjugation of a chromophore is by protonation or some chemical reaction, absorption maxima shift towards shorta wavelength (higher frequency), its called hypsochromic shilts (blue shifts). Colorimeter x Beer lambert's law. red shift the shift Batho only in Visible region. Hypso d Monochromatic radiation. Transmittance recorded as optial Density Steps in developing a Spectrometric Analytical method I Run the sample for Spectrum. 2 wavelangth. Obtain a monochromatic wavelength for the Max absorption
3 Calculate the wnc of your sample using Beer lambort oqn A Ect 2.0 Spectrometer Reading Abotomo 0.0 Absun bance > 200 250 300 350 400 4.00 2 0 Wavelengh (nm) transmission O 100 Spectrometric Analysis using standard Curve 1.2 Swors 0.8 to 0.4 V I 2 3 4 Happy lone of glucose (g() Avoid very high or low absorbencies when drawing a standard curve. Best result are obtained with 0.1 LACK Plot Absorbane vs. cone to get a straight line. Sample Cells. LDA UV spectrophotomete Quartz (Gystalline silica).