Q&A for How to Do Spectrophotometric Analysis

You must "zero" your spectrophotometer before using it so all of your absorbance readings can have a baseline to be compared to. For example, if your protein sample was diluted with distilled water, you would zero or "blank" the spectrophotometer using just distilled water, that way the only difference between the absorbance readings can be attributed to protein concentration in the sample.

DNA, RNA, protein isolation, protein purification, enzyme kinetics, determining optimal pH of a sample, determining concentrations of unknown samples (as described above), and determining the pKa of various samples.

This depends on what you're looking for. Here is how you would prepare a sample to determine protein content in a solution. 1) Lyse cells from experiment with lysis buffer (we use 4% SDS lysis buffer) in an eppendorf tube. 2) Sonicate the cells to help break them open, allowing proteins from inside the cell to move into the lysis buffer. 3) Boil samples at 100 degrees Celsius for 10 minutes. 4) Centrifuge your samples at 13,000 g for 2 minutes. 5) I flick the the eppendorf tube with my finger to ensure proper mixing and then centrifuge again. 6) Take 10 microliters of your sample and add it to 990 microliters of double deionized water. Vortex to mix. 7) Take your sample (1mL) and measure its absorbance in the spectrophotometer as described above.

The user's manual will describe in detail how to do maintenance and operations using SP. Because this instrument requires precision measurement, almost all modern SPs have a built-in calibration routine which must be run prior to using the instrument. A spectrophotometer (SP) is an instrument which measures the amount of transmitted light of particular wavelength. This instrument is used in chemical and biochemical analysis, but a variant SP can also be used to study the physical property of a substance. Most spectrophotometers come in two categories: prism and grated wafers.

The duration of time a sample can be left out changes with different solvents. if measuring in something like a 96 well plate, I wouldn't wait an hour simply because of the environmental factors affecting such a small sample size. While you can technically measure after this long, I would personally call into question the validity of any results obtained that way.