Cellular Chemistry

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Cellular Chemistry

Cellular Chemistry

Analysis Of Spectrophotometry

Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Namely, light has (Schwedt, 2003, pp. 16-17):

a particle nature which gives rise to the photoelectric effect

a wave nature which gives rise to the visible spectrum of light

A spectrophotometer measures the intensity of a light beam after it is directed through and emerges from a solution. An example with copper sulfate (CuS04) is shown on Figure 1.

The red part of the spectrum has been almost complete absorbed by CuSO4 and blue light has been transmitted. CuSO4 absorbs little blue light and therefore appears blue.

In spectrophotometry, greater sensitivity can be gained by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum. For this, red wavelengths have to be isolated.

In a spectrophotometer, a light source gives off white light which strikes a prism, separating the light into its component wavelengths (See Figure 2.). Thus, light waves can be separated by frequency.

The spectrophotometer can measure the amount of light (of certain frequency) transmitted or adsorbed by the solution. This light that has not been absorbed by the solution in the cuvette, will strike the phototube. The photons of light that strike thephototube will be converted into electrical energy. This current that is produced is very small and must be amplified before it can be efficiently detected. The signal is proportional to the amount of light which originally struck the phototube and is thus an accurate measurement of the amount of light which has passed through (been transmitted by) the sample (Schwedt, 2003, pp. 16-17).

Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra. Concentration of every component may be found from the spectrophotometer measurements and calibration curve made using the samples of known concentration.

Laboratory procedure

We will need (Schwedt, 2003, pp. 16-17):

4 calibration samples of water contaminated with Toluene and MTBE. Concentrations are 1 ppm, 10 ppm, 25 ppm and 50 ppm

Your test samples from other experiments

Safety equipment (gloves, glasses, etc.)



100-250 mL Wastewater beaker

Make sure that you read and understand the lab procedure.

Follow the safety procedure for the work in the lab.

Turn spectrophotometer on. Let it warm up for about 15 minutes

In the meantime, prepare your calibration samples

Rinse the quartz cuvette with deionized water several times. Careful with the cuvettes.

Place 1 ml of sample into cuvette using a pipette. Use new pipette tip for every sample.

Make sure you don't touch the clear sides of the cuvette. Wipe the cuvette with very fine paper to remove any water droplets or dust. Avoid scratching the cuvette.

Place cuvette into spectrometer.

Take a reading.

Remove cuvette and discard liquid into beaker with wastewater.

Rinse the cuvette with deionized water 3 times before ...
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