Spectrophotometer of nursing topic1.pptx
- 2. Spectrophotometer An instrument that measures the amount of light that passes through (is transmitted through) a sample. It is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color (or more specifically the wavelength). This means it use light in the visible and adjacent (near ultraviolet and near infrared) range.
- 4. Principle: when a beam of monochromatic light passed through a solution it may transmitted as a such or some of may be absorb. Proportional the transmitted light can be represented by intensity of the incident radiation. T=I/I₀ Absorbance(A) of light through a solution in inversely proportional to log of %T ₁₀ A=Log( ∕T) ₁ =Log(I/I ) ₀ Where I=intensity of transmitted light I =intensity of incident light ₀
- 5. The quantitative determination The quantitative determination of compounds by spectrometric technique is based on two law. Lamberts Law Beers Law Lamberts law- its state that light absorbed by solution is directly proportional to length of the light through the solution. hence A= Є.l where A= absorbance Є=molar absorptivity coefficient l= path length of the sample (usually 1cm)
- 6. Beers law- its state that the amount of light absorbed is directly proportional to concentration of absorbing solute in the solution. Thus A=Є.C where C=concentration of solution moles liter-1 combining equation A=Є.C.l Limitation of Beers Lamberts Law: 1. when different forms of the absorbing molecules are in equilibrium. 2. when fluorescent compounds are present. 3. when solute and solvent forms complex though some sort of association.
- 7. Types of instrument: Single beam single beam spectrophotometer measures the light intensity of the beam and after a test sample is inserted. Double beam- A double beam spectrophotometer compares the light intensity b/w two path.one path containing a reference sample & the other is test sample.
- 9. Types of Spectrophotometer Ultraviolet (UV)-Vis Spectrophotometers Uses ultraviolet light of wave lengths from 200 nm to 350 nm Infra Red (Ir)- covers the range of the electromagnetic spectrum between 0.78 and 1000 m, m. In the context of infra red spectroscopy wavelength is measured in "wave numbers", which have the units cm-1. The most useful I.R. region lies between 4000 - 670cm-1.
- 10. UV-visible spectroscopy This means it use light in the visible and adjacent (near UV and near Infrared)range. It uses light over the ultraviolet range (185- 400 nm) and visible range(400-700 nm) of electromagnetic radiation spectrum.