BIOCH 455: Spring 2006

Absorption Spectra of Different Cuvettes Study conducted by: Olexandr Sasha Kokhan

UV
4 3

Vis

near-IR
cuvettes quartz regular UV

Absorption

0 200 400

Wavelength, nm

600

800

1000

The purpose of this study was to illustrate the absorptivity of the cuvettes themselves at various wavelengths. When we are conducting absorbance measurements, we want to ensure that we are measuring the absorbance of the given compound and not that of the cuvette itself. The cuvette listed as quartz on the legend above is a very expensive ($300 each) cuvette for reading in the UV range. Besides their prohibitive cost, they are difficult to clean and are clearly NOT disposable. Using this type of cuvette was not a viable option for a large teaching lab ($300 x 18 groups = $5,400). The cuvette listed as regular is the standard disposable cuvette we used in all of our colorimetric assays (-gal, BioRad, Folin). Notice that this type of cuvette does NOT absorb in the visible range, so it works well for our purposes. Also, it is very inexpensive (10 cents each). Please note, however, that this type of cuvette DOES absorb in the UV. This is why you must use the disposable UV cuvettes (50 cents each) when reading at wavelengths below ~340 nm. Please look at the graph on the following page to see more detail in the UV range.

4.0 3.5 3.0 2.5

quartz regular UV

Absorption

2.0 1.5 1.0 0.5 0.0 200 220 240 260 280 300 320 340

Wavelength, nm

This graph is an expanded view in the 200-340 nm range. Notice how strongly the regular cuvette absorbs in this range. Now look at the spectrum for the disposable UV cuvette (green line). It DOES absorb fairly strongly below ~230 nm. Use of this cuvette is acceptable, then, so long as we read above that wavelength. (Remember, for GOT assays we read at 256 nm and at 280 nm for total protein.) The expensive quartz cuvette does NOT absorb strongly at any wavelength examined in this study. If we wanted to read below 230 nm, we would HAVE to use this type of cuvette for accurate readings. Now that you understand better the difference between these cuvettes, perhaps you can see why it is so important to make sure you are using the correct cuvette for the assay you are performing.

Study of Cuvette Staining Study conducted by: Olexandr Sasha Kokhan

0.030

cuvette stained with BioRad reagent after ~ 30 assays

0.025

0.020

Absorption

0.015

0.010

0.005

0.000 500 550 600 650 700

Wavelength, nm

This study was conducted in order to assess staining of the standard disposable cuvettes after re-use in multiple assays. The same cuvette was used for 30 BioRad assays and then examined for staining by performing a spectral analysis of the cuvette itself. After ~30 assays the cuvette did absorb some of the color of the BioRad reagent, as evidenced by an A595 reading of ~0.025. Although this is not a high absorption peak, staining as a result of cuvette re-use could influence the results of an assay. In other words, after multiple assays the A595 reading would give an exaggerated measure of the amount of protein present. This illustrates why we ask you to use multiple cuvettes when performing multiple assays.