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A biotechnologists and molecular biologists uses spectrophotometer for accurate preparation and analysis of samples. The spectrophotometers has different application in the qualitative analysis of sample purity, DNA and protein quantitation, cell density measurements, enzyme catalysed reaction, and quantitative determination of a whole range of chemical and biological substances.
Spectrophotometer is based on the simple basis that different compounds will differentially absorb specific wavelengths of electromagnetic radiation in either the ultraviolet (UV, 200-400 nm), visible (VIS, 400-700 nm), or near-infrared (near-IR, 700-900 nm) range. Photometric assays may directly measure sample absorbance at a given wavelength or indirectly measure an enzymatic reaction product or related binding substance that absorbs light in amounts directly proportional to the absorbance of the target compound (such as assays for protein concentration). All spectrophotometers employ the same basic structural components designed to detect these variations between absorption wavelengths and densities.
The general components common to most spectrophotometer systems include a radiation (light) source, wavelength selector, fixed or adjustable slit, cuvette, photodetector, and an analog or digital readout. The light source (specific for either UV or visible ranges) will emit radiation that is passed through the wavelength selector, usually a diffraction grating, prism, or set of screening filters, where a specific wavelength of "monochromatic" light is selectively generated (defined by its maximum emission at this wavelength). This light is then directed toward a thin slit (usually adjustable) to regulate its relative intensity before it passes through a cuvette containing the sample of interest. Cuvettes differ with respect to their absorbance characteristics, depending on the material they were constructed, and should be selected so as not to interfere with the absorbance of the sample being measured. Finally, absorbance is detected by a photodetector which generates electrical current that can be amplified and measured to yield an absorbance value (Abs), or optical density (OD), for the given sample. The absorbance or OD reading is corrected against a blank, which contains all of the reagents in the experimental sample except the test analyte, to obtain a true measure of the absorbance of the sample.
The spectrophometer system commonly uses the general components like radiation (light) source, wave length selector, fixed or adjustable slit, cuvatte, photodetector, and an analog or digital readout. Mainly the light source emit the radiation that pass through the wavelength of monochromatic light is selectively generated. The light focussed towards a thin slit before light passes through cuvette which containing the sample of interest. Cuvette has different absorbance characteristics, and depending on the material they were constructed, and should be interference with the obserbance of the sample being measured. Finally the obserbance is detected by the photodetector which generates the electrical current and that can be amplified and measured to yield an absorbance value r optical density for the given sample. The absorbance or OD reading is corrected against a blank, which contains all of the reagents in the experimental sample except the test analyte, to obtain a true measure of the absorbance of the sample.
Pure substance of solution does not absorb the energy of wavelength of electromagnetic radiation equally, and a substance can be known by the unique pattern of wavelength absorbed. By measuring the absorbance of an uncharacterized solution over a variety of wavelengths and plotting the graph of the absorbance value on the Y-axis and the wavelength on the X-axis, we can determine the absorption spectrum of a sample the absorption maximum (¬max) of any pure substance in solution is the wavelength where absorption is greater and is normally utilised to
polystyrene (PS) cuvette
polymethylmethacrylate (PMMA) cuvette
Hewlett Packard 8452A diode array spectrophotometer
Take cuvettes which made by the different varying materials, placed in the cuvette holder, perform a background correction or baseline spectral subtraction in the wavelength range 190 - 820 nm.
Take the absorption spectrum for each cuvette. And determine the useful operating wavelength of each cuvette by looking at the wavelengths where the cuvette is completely transmitting. Verify the cut-off wavelength for each cuvette.
And then test eyeglasses also to determine their transmission wavelengths.
Treatment of result/ Post laboratory questions:
Experiment 2. Standard Concentration Curve and Determining the Concentration of an Absorbing Substance
Spectrophotometer is used for measuring the absolute concentration of an analtye which based solution on absorbance measurements. The basis for the quantitative measurement is Beer-Lambert's law, given by the equation:
A = ¥lC
where A = absorbance, C is the concentration of the absorbing substance (in mol/L), ¥ is a constant known as molar absorptivity which is function of wavelength and nature of absorbing substance, and l is the path length (in cm) travelled by light through the sample.
The determination is normally performed by first constructing a standard concentration curve of the analyte by plotting the absorbance (y-axis) of several solutions against concentration (x-axis) and then comparing the absorbance reading of the unknown concentration with that of the standardsolutions.Absorbance measurements are normally performed at wavelength of maximum absorption to obtain the best linearity and sensitivity in the calibration slope.
In this experiment, you will construct a standard concentration (calibration) curve for a colored analyte (ferricyanide) by measuring the absorbances of varying concentrations of ferricyanide.
aqueous potassium ferricyanide solution (5 mM), 100 ml
test tubes or vials
Prepare the different concentration of ferricyanide solution in test tubes or vials by mixing the different amounts of ferricyanide solution by changing the amount of distilled water.
Vol of stock ferricyanide soln
Vol of deionised water
Final conc of ferricyanide
In each tube calculate the final conc of ferricyanide solution.
Perform a blank measurement using Tube 0 or deionised water.
Perform a spectral scan on each of the ferricyanide solutions. Save all absorption spectra.
Cover all absorption spectra, and determine the wavelength of maximum absorption. Record the absorbance reading for each of the solution at the wavelength of maximum absorption.
Treatment of results
Plot absorbance (y-axis) against ferricyanide concentration (x-axis). Specify the wavelength of maximum absorption
Perform a linear regression analysis on the plot; calculate the correlation coefficient, slope and intercept of the calibration plot.