Use the warm-up time to prepare your samples.
Take care with cuvettes as they can be quite expensive, particularly if they are made from glass or quartz. Quartz cuvettes are designed for use in UV-visible spectrophotometry. When handling the cuvette, avoid touching the sides the light will pass through (generally, the clear sides of the container). [3] X Research source If you accidentally touch these sides, wipe the cuvette down with a kimwipe (which are formulated to prevent scratching the glass).
If you are using a pipette to load your samples, use a new tip for each sample to prevent cross-contamination. [4] X Research source
In a classroom lab, the wavelength will likely be given to you. Because the sample will reflect all light of the same color as it appears, the experimental wavelength will always be a different color than that of the sample. Objects appear as certain colors because they reflect light of particular wavelengths and absorb all other colors. Grass is green because the chlorophyll in it reflects green light and absorbs everything else.
Digital spectrophotometers can be calibrated in the same way, they will just have a digital readout. Set the blank to 0 using the adjustment knobs. When you remove the blank, the calibration will still be in place. When measuring the rest of your samples, the absorbance from the blank will automatically be subtracted out. Be sure to use a single blank per session so that each sample is calibrated to the same blank. For instance, if you blank the spectrophotometer, then analyze only some of samples and blank it again, the remaining samples would be inaccurate. You would need to start over.
If the needle or readout is not 0, repeat the calibration steps with the blank. If you continue to have problems, seek assistance or have the machine looked at for maintenance.
The absorbance is also known as the optical density (OD). The more light that is transmitted, the less light the sample absorbs. Generally, you want to record the absorbance values which will usually be given as a decimal, for example, 0. 43. If you get an outlying result (such as 0. 900 when the rest are around 0. 400), dilute the sample and measure the absorbance again. Repeat the reading for each individual sample at least 3 times and average them together. This ensures a more accurate readout.
The transmittance (T) is found by dividing the intensity of the light that passed through the sample solution with the amount that passed through the blank. It is normally expressed as a decimal or percentage. T = I/I0 where I is the intensity of the sample and I0 is the intensity of the blank. The absorbance (A) is expressed as the negative of the base-10 logarithm (exponent) of the transmittance value: A = -log10T. [10] X Research source For a T value of 0. 1, the value of A is 1 (0. 1 is 10 to the -1 power), meaning 10% of the light is transmitted and 90% is absorbed. For a T value of 0. 01, the value of A is 2 (0. 01 is 10 to the -2 power), meaning 1% of the light is transmitted.
An absorbance spectrum usually has peaks at certain wavelengths that can allow you to identify specific compounds.
You can also use this method to identify contaminants in your sample. If you are expecting 1 clear peak at a specific wavelength and you get 2 peaks at separate wavelengths, you know something is not right in your sample.
