Cleaning Cuvettes Kay Finn last updated: 6/05/2003 After each use, cuvettes should be washed well with H20 and rinsed with MeOH, then dried on the cuvette washer until they appear dry. Store in cuvette box. Using the cuvette washer: Always check to see that the wash spray is getting to the bottom of the inverted cuvette. Watch the spray and adjust the placement of cuvette, or press gently down on the cuvette to get the spray all the way to the top (bottom of the cuvette). When cuvettes develop a build up on the interior walls, they can be cleaned with acid solutions: * Try a dilute acetic acid solution first: soak overnight in 3M acetic acid, rinse well. * If this does not work (i.e., you can still see something on the wall of the cuvette, or the water blank has an unusually high absorbance or fluorescence), try 50% H2SO4: * Use gloves and goggles, and do this in the hood: Put cuvette in a glass beaker and add 1/2 vol H2O by eye with wash bottle. Add H2SO4 with dropper to the top, cover with parafilm, and invert to mix. The cuvette will feel warm. Cover the beaker with parafilm. Let sit for at least 1 hour. Rinse out into running water at back of hood. Rinse several times, then put on cuvette washer to rinse more. Sulfuric acid is thick and sticky, so rinse lots and check with pH paper to be sure acid is gone. You can use stronger acids if the cuvette is still dirty; see recommendations from Hellma. DO NOT USE BASE FOR CLEANING! This is a copy of email from Hellma tech help: Kay: Do not use NaOH. It will remove the outer polished layer. Use conc. H2SO4, HNO3 or Chromic Sulfuric Acid. DO NOT USE STRONG BASES! Best regards, Ed Roth Hellma Cells, Inc. - http://www3.nd.edu/~clarklab/protocols/spectroscopy/cuvettes.html  http://www.researchgate.net/post/Cleaning_polymerized_material_from_a_quartz_cuvette Amy E Keirstead University of New England (USA) Cleaning polymerized material from a quartz cuvette I (accidentally) photopolymerized an organic compound inside a quartz cuvette and now have the material stuck on the inside of the cuvette. Regular organic solvents (acetone, ethanol, hexane) did not remove the material, nor did soaking overnight in a conc. H2SO4 bath. Can anyone suggest a way to remove the polymerized material? I do not want to sonicate, as I think it will compromise the fused quartz, and am considering aqua regia. TOPICS Marcel Brautzsch · Martin Luther University of Halle-Wittenberg Maybe Methylene Chloride could solve your problem (as it usually solves polymers relatively good). A more rigorous way is to use a piranha solution (you can look this up at wikipedia) over night but you have to be REALLY careful when using it. Any residuals of the previously used organic solvents have to be removed or it can lead to an explosion. \Kevin Huvaere · EcoSynth Fill cuvette with 10% aqueous hydrogen peroxide and expose (1h or more) to the emission of a medium pressure mercury lamp; we often have such problems in our photochemical reactions and this approach makes our vessels and tubes look brand new. Jorge Gutierrez · Universidad Nacional de RÃo Cuarto Try with aqua regia, first wash three times and then just pour it into cell for a 5-10 min. Airton Luna · Instituto Nacional da Propriedade Industrial Handling this solution seem to be very dangerous. Why do not you try a photoi-rradiated Fenton treatment? In a beaker of 100 mL you put 0,03-0,05 g of FeSO4 and 50 mL of water; put the cuvette inside of it; with a seringe you insert slowly hydrogen peroxide concentrated. To be more efficient you should expose it to the solar light. Sudhir Das · The National Institute of Science Education and Research Just try with toluene. may be your problem can solved Amy E Keirstead · University of New England (USA) Thank you all for your suggestions. I think I will try the aqua regia first. Kevin, I do not have a Hg lamp, do you think 254 nm would work? Jorge, I have friends Miguel Gervaldo and Rodrigo Palacios st UNRC and was there for a visit last May. Jorge Gutierrez · Universidad Nacional de RÃo Cuarto Cool, =) they are great people! James E Hanson · Seton Hall University You can also use Nochromix - I think it is potassium persulfate and sulfuric acid - oxidizes most organics. Sikiru Akinyeye Ahmed · University of Agriculture, Abeokuta soak the cuvette inside a 250ml beaker filled with dilute HNO3 for about 2 hours. try it, it works like magic. remember all nitrates are soluble. Karthick Muthusamy · Department of Chemistry Soak the cuvette in Chromic acid i will solve ur problem. Try it Rajesh Jagannath Tayade · Central Salt and Marine Chemicals Research Institute Aqua regia is good for quartz use it for cleaning Eurico Melo · New University of Lisbon Be careful. If you are speaking about fused quartz cuvettes (one single square section quartz tube) all the treatments above, including sonication, are harmless for the cuvette. It is not the case if they are glued optical cuvettes. I know that chromic acid overnight (but not much longer) followed by a couple of hours in basic edta is ok. Another more drastic (and dangerous) method is the ethanol-nitric acid reaction that I have used in extreme situations. I agree that sonication may open the glued corners. At least it is my experience despite the recommendation of some brands. Boopathy Ramasamy · Council of Scientific and Research Institute-Central Leather Research Institute, Chennai, India Use hydrogen peroxide as suggested by kevin or use dilute chromic acid, it is also a acidic nature of strong oxidizing agent. This can be prepared by taking 2-4 % of potassium dichromate in conc. sulphuric acid. settle and take clear orange colored liquid phase, which is chromic acid. Aug 16, 2012 Cuvette Cleaning When making circular dichroism measurements, it is essential that we use good quality cuvettes that are clean and free from damage. Good quality and free from damage are easy enough. Cuvettes that are well suited to CD measurements can be purchased from manufacturers such as Hellma or Starna, and any that show defects on visual inspection can be discarded. But what about clean? There is a lot of information on the internet on cleaning laboratory glassware, including cuvettes, but not all of it is useful and some of the procedures described are very hazardous.  In this blog post, we will summarise our ideas about cuvette cleaning, and describe how we ourselves clean the cuvettes that we use for CD spectroscopy. In doing this our objective is to obtain cells that are both clean and free from birefringence caused by stressing of the cell during the cleaning process. Figure 1: Example of Hellma Cuvette Cell Caution: please take care. The proprietary cleaning agents discussed in this article should always be used and disposed of in accordance with the supplierâs instructions. All other materials should also be handled with care. Wear the appropriate protective clothing and ensure that you have read the safety information on any materials that you use. Use the procedures described here only for quartz cuvettes removed from their holders or cartridges. Do not use them for the flow cells in the Automated Circular Dichroism spectrometer (ACD) while they are mounted in their cartridges. It is not always easy to know when a cuvette is contaminated. The simplest way is to run reference CD and absorbance spectra of purified water in the clean cuvette before starting the experiment. These spectra can then be compared with water spectra run in the same cuvette after use. There may be slight differences due to baseline drift, but there should be no trace on either the CD or absorbance spectrum of a change due to the sample. But even this is not perfect. There may be contaminants present that are not CD active, and either do not absorb in the wavelength region we are scanning over, or are present at very low concentrations. For low molecular weight water soluble samples, simply rinsing with purified water followed by rinsing with acetone or ethanol and blow drying with clean, dry, compressed air or nitrogen, is often sufficient to clean the interior of the cuvette, with the exterior of the cuvette cleaned by wiping gently with acetone or ethanol on soft lint free tissue. Biomacromolecules, particularly denatured proteins, can be quite sticky and their removal from quartz surfaces often needs more aggressive cleaning methods. Starna and Hellma supply their own brands of cuvette cleaning agents, usually solutions of anionic and non-ionic detergents in the pH range 9.5 to 12. Their websites also give guidance on cuvette cleaning and their recommended protocols are fine as far as they go. However, they are not always enough to remove protein baked onto the cuvette surfaces at high temperatures. The same applies to other proprietary glassware cleaners such as Decon 90, which is similar in composition to the cleaning agents supplied by Hellma and Starna. So what can we do if these agents fail to clean our cuvette properly? One option is to use a solution of inorganic oxidiser in sulphuric acid. Sulfochromic acid, which is potassium dichromate in sulphuric acid, is NOT recommended. It is very hazardous, difficult to dispose of, and may leave residual Cr3+ ions absorbed on the cuvette walls. The aptly and frighteningly named piranha solution, hydrogen peroxide in sulphuric acid, does not have the same problems of contamination with heavy metal ions, but is also very hazardous and its use is forbidden in some laboratories. The proprietary cleaning agent Nochromix, in which the inorganic oxidiser is ammonium persulphate, (NH4)2S2O8¸ is an alternative, although we have never found a reason to use it ourselves. Detergents containing protease enzymes, such as Tergazyme, could be used for cuvettes contaminated with proteins, but there is always the possibility that the proteases themselves will become the contaminants. Strong bases such as potassium or sodium hydroxide are not recommended as they tend to etch the surfaces of the cuvettes, and the manufactures warn against sonication as it can crack the cuvettes. A possibility for cuvettes more heavily contaminated with protein is to use a dilute acid, nitric acid at 3% concentration, for example. An overnight soak in dilute nitric acid usually works. Failing that, concentrated nitric may be used (but remember the hazards of working with any acid, particularly oxidising acids). Here is what we do for cuvettes that have been used with biomacromolecules. 1.  Assume that the cuvette will always need soaking after it is used with biomacromolecules. 2. Have enough cuvettes available to allow for them to be properly soaked without interrupting the workflow. 3.  After use, take the cuvette from the holder and remove as much of the sample as possible using a fine-tipped disposable transfer pipette. 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 a fine-tipped pipette. 4. If a buffer rinse is used, it should be followed by further rinsing with purified water. 5. 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. 6. Immerse in concentrated nitric acid, making sure that the air in the cuvette is fully displaced with the acid and store overnight. CAUTION: make sure that the vessel containing the acid is clearly marked with its contents and is stored in a safe place. 7. Allow to soak for 10 minutes, or overnight if the cuvette has been used at high temperatures. 8. Over a sink or drip tray, carefully remove the cuvette from the acid using stainless steel tweezers, gripping the cuvette lightly by the neck, not the optical surfaces. 9. Using a fine-tipped disposable transfer pipette, carefully remove as much of the acid as possible from the cuvette, ejecting the acid slowly into a large beaker of water. 10. Carefully rinse the cuvette with purified water several times. Introduce and remove the water with a fine-tipped pipette for each rinse. 11. Repeat the above step using acetone or ethanol as the rinsing agent, then blow dry thoroughly with compressed air or nitrogen. Do not vacuum dry as this may introduce stresses resulting in birefringence in the cuvette. 12. 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. 13. If it is not needed immediately, store the cuvette in a box lined with a soft material in a clean, dry environment. 14. When it is needed, remove the cuvette from the box and blow away any dust with compressed air or nitrogen. In general, take care of your cuvettes. Do not use abrasives on them. Handle them gently at all times. Do not twist or pull them, as this may introduce stresses resulting in birefringence. Do not vacuum dry them as this can also introduce stresses. And whatever else, work safely.  Hellma® is a registered trademark of Hellma GmbH and Co. Müllheim, Germany. Starna®is a registered trademark of the Starna Group, Hainault, United Kingdom. Decon® is a trademark of Decon Laboratories Ltd, Hove, United Kingdom. Nichromix® is a registered trademark of Godax Laboratories Inc. MD, USA. Tergazyme⢠is a trademark of Alconox Inc. NY, USA Â