Major components of a singlebeam spectrophotometer Spectral Isolation

Major components of a single-beam spectrophotometer

• Spectral Isolation – Excluding that of other wavelength • Use of filters, prisms, and diffraction gratings • Combinations of lenses and slits – To render light rays parallel – To isolate narrow portions of the light beam – Highly coherent beam of light that has minimum scatter

• Monochromator – Isolates the desired region of the spectrum to be used for measurements • Light of a desired wavelength • Narrow wavelength range of the spectrum • Slits – Isolate a narrow beam of the light and improve its chromatic purity

• Solutions • Dilute enough – In which the change in absorbance varies significantly with small changes in concentration • Absorption – Depends on the nature and concentration of the substance in the solution

• Cuvets – Glass , silica (quartz), or plastic – I. 0 -cm light path • Borosilicate glass cuvets – suitable for measurements in the visible portion of the spectrum

• Quartz cells – For readings below 340 nm • Plastic cells – Visible and UV range – Cleaning, etching by solvents, and temperature deformations – For disposable, single-use

• Cuvets may be cleaned – By copious rinsing with tap water and distilled water – Mild detergent – Soaked in a mixture of concentrated HCl: water: ethanol (I: 3: 4) • Cuvets used for measurements in the UV region – Handle with special care – Scratches, fingerprints, residual traces of previously measured substances

• Spectral bandwidth – The spectral purity of a filter or other monochromator – Wide-bandpass filters – Narrow-bandpass • for resolving and identifying sharp absorption peaks that are closely adjacent

• Measuring at the peak absorbance & at narrower bandwidths of light – Increase in absorbance – Improved linearity with concentration • Agreement with Beer's law – Achieve maximum sensitivity • for peak absorbance readings to be within 99. 5% of true values, the spectral bandwidth should not exceed 10% of the natural bandwidth

Effect of spectral bandwidth (SBW) on the absorption spectrum of coproporphyrin I. • Curve a, I nm • Curve b, 10 nm • Curve c, 20 nm

• Peak absorbance • Natural bandwidth

• Linearity – Linearity is satisfactory as long as the d. A increments remain constant • Background interference – Inclusion of blanks – Not measuring at the peak absorbance – Reading absorbance at two or three wavelengths