How to clean spectrophotometer cuvettes

Contaminated cells are the greatest single source of error in spectrophotometry. Cells should never be handled by the optical polished faces, and analysts should rinse off residual or spilled solution.

If cells are properly cared for, rigorous cleaning methods rarely will be needed, especially if the cells are cleaned immediately after use.

 Although acid solutions (viz. sulfochromic acid) will simply contaminate surfaces, alkaline solutions(Viz.. potassium or sodium hydroxide) can etch all types of polished glass surfaces, and the degree of attack depends on the pH and contact time.

Therefore, a few of the common cleaning methods are described below. These methods can be roughly categorized into few groups. One group uses water as the solvent and the other uses organic solvent.

1) Using Water as a Solvent - After cleaning with purified water, clean with ethanol and store dry. However, for more severe contamination, soak cells in a commercial cleaning solution made specifically for cleaning cells (for about 10 minutes at 30 to 50 °C). Then clean the cell with distilled water and soak them in a dilute solution of nitric acid and a small amount of hydrogen peroxide (for about 30 minutes). Finally, rinse the cell with distilled water and store cells dry.

2) Using Organic Solvents - After cleaning with the organic solvent being used, clean with ethanol or acetone and then clean using the same method as described for the aqueous solution above.

3) For stubborn contamination - the cell may be scrubbed lightly with a cotton swab. Avoid using alkaline cleaning solutions that can dissolve glass or ultrasonic cleaning devices that can damage the cell.

Rinse the clean cuvette with acetone or ethanol, than blow dry thoroughly with compressed air or nitrogen. Make sure that you don't build up pressure in the cuvette. Do not vacuum dry as this may introduce stresses resulting in birefringence in the cuvette.

 Gently clean the outside of the cuvette with acetone or ethanol using a soft lint free tissue. Check that the cuvette is clean by holding it up to the light. There should be no visible contamination or smears.

If the cells are stored for an extended period, then they should be dried quickly after cleaning by blowing dry with a compressed gas stream in a clean, dust-free environment

Biomolecules

Method Using Nitric acid

I . After use, take the cuvette from the holder and remove as much of the sample as possible using a fine-tipped disposable transfer pipette or a flat gel loading tip for 0.5 mm cuvettes. Rinse four or five times with either buffer or purified water (a buffer rinse is used for samples such as some proteins that precipitate in purified water). For each rinse, introduce and remove the rinsing agent with the pipette.

II. If a buffer rinse is used, it should be followed by further rinsing with purified water.

III. Rinse several times with acetone or ethanol, then blow dry thoroughly with clean dry compressed air or nitrogen. It is particularly important that the cuvette is completely dry before the next step. If it is not, heat from mixing acid with water or ethanol can damage the cuvette! Make sure that you do not build up pressure in the cuvette. Don't use house air without having a filter installed.

IV. Immerse in concentrated nitric acid, making sure that the air in the cuvette is fully displaced with the acid.

V. Allow to soak for 10 minutes to 1 hour (or overnight with 2 M nitric acid) if the cuvette has been used at high temperatures (with Piranha/Nano-Strip 30 min should be enough).

 

VI. Over a drip tray, carefully remove the cuvette from the acid using stainless steel tweezers, gripping the cuvette lightly by the neck, not by the optical surfaces.

VII. Using a fine-tipped disposable transfer pipette or flat gel loading tips, carefully remove as much of the acid as possible from the cuvette. (Again Piranha or Nano-strip is not compatible with plastic pipettes)

VIII. Carefully rinse the cuvette with purified water several times. Introduce and remove the water with a fine-tipped pipette for each rinse.

IX. After at least 7 rinses, measure a CD spectrum (180 nm to 260 nm for 1 and 0.5 mm cuvettes, 200 nm to 260 nm for longer path lengths, go to longer wavelengths if your signal was at longer wavelengths) of the water filled cuvette to prove that it is clean. Save the spectrum as "water_after" to document that the cuvettes were left clean. If you used multiple cuvettes save a water_after spectrum for each!

X. Rinse the clean cuvette with acetone or ethanol, than blow dry thoroughly with compressed air or nitrogen. Make sure that you don't build up pressure in the cuvette. Do not vacuum dry as this may introduce stresses resulting in birefringence in the cuvette.

XI. Gently clean the outside of the cuvette with acetone or ethanol using a soft lint free tissue. Check that the cuvette is clean by holding it up to the light. There should be no visible contamination or smears.

XII. If it is not needed immediately, store the cuvette in the box they were in (if not available, store in a box lined with a soft material) a clean and dry environment.