A spectrophotometer is used to measure the amount of light absorbed by, or transmitted through, a solution. Obtain a sample cuvette. Use a pen to mark the top of one side of the cuvette. This mark will be used to insure the cuvette is oriented the same way each time in the sample holder. Rinse the cuvette several times with the reference. The reference is typically the solvent used to make up your sample solutions. Then fill the cuvette approximately 3/4 full. Wipe the outside of the cuvette to remove solution and fingerprints. Place the reference solution in the sample holder. Note the position of the pen mark on the cuvette; the mark must face this direction each time the cuvette is placed in the holder. Measure the reference solution. The signal obtained from this measurement will be subtracted from all sample measurements. Remove the cuvette from the sample holder. Take the cuvette used to measure the reference. Rinse the cuvette several times with the sample solution. Then fill the cuvette 3/4 full. Wipe the outside of the cuvette, and place it in the sample holder. Measure the sample solution. The same reference signal can be used to analyze solutions that differ in concentration. Once the reference signal is obtained, measure each of the sample solutions in the order from least concentrated to most concentrated.
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