Re: [ccp4bb]: How to test for PEG contamination?

[Arthur Glasfeld]

Hi MIscha, We've had problems in the past with PEG solutions, and two quick tests have proven useful in detecting solutions that have gone off: 1.  Merck sells a dip stick (not unlike pH paper) that gives a reasonably quantitative measure of peroxides.  It's called the EM Quant Peroxide Test (Cat No 10011-1).  Fast and easy to use. 2.  An obvious problem to test for is pH.  Some of our unbuffered solutions have gotten down to pH 2.   6.  On the additional thoughts category, I'm attaching an email we got from Bob Cudney at Hampton Research.  I hope he doesn't mind me sending this information around - it was useful to us. Cheers, Arthur Arthur Glasfeld Reed College Portland, OR 97202 USA ****************** Polyethylene glycols, in the presence of oxygen undergo a chemical change whereby they produce aldehydes and peroxides which in turn alter the ionic strength and pH of the solution.  Most PEG solutions, made from fresh flake PEG are in the range of 7 to 9.  Over time, unbuffered and in the presence of oxygen and light the pH and conductivity of the solution changes with a resultant acidic pH and higher ionic strength. Bill Ray and later Fran Jurnak both published papers on the oxidation of polyethylene glycols and Fran even compared different batches of PEGs. This work was done before Hampton Research existed, circa 1987-1991. Refrigerating PEG solutions slows the process some but is not completely effective.  Same for freezing.  Hampton Research purges the PEG Optimize solutions with dry argon before filling into sterile PETG bottles that have low oxygen permeability and stores them at -20 degrees Celsius while in inventory waiting to be shipped to a customer's lab.  Some grades of polypropylene bottles might be better yet (possible lower oxygen permeability), but is not transparent and people want to see their PEG so we use PETG bottles. One can add antioxidants but researchers in pharma and people studying certain enzymes and ligands do not like small molecules added to their PEG.  Actually, some PEG producers do add antioxidants to the PEG flake. It varies by producer and molecular weight of the PEG. The problem you describe is commonly known by the over 45 crystallography crowd or those who have been in the field for 15 plus years, as it would be us/them who remembers the Ray and Jurnak papers. To maintain the pH of the PEG solution it is advised to include a buffer at the desired pH.  You could for example buffer them PEG with 10 mM Hepes pH 7.  That would keep the pH at 7 and not interfere with varying pH in a screen or during optimization.  That would control the pH but you still have aldehydes and peroxides forming over time.  And then there are the minute amounts of extractables in the plastics used in tubes, bottles and plates, and... well, the list goes on. In the end it is not clear as to how significant the PEG/pH matter is to the crystallization community as we only receive a call or two a year where someone cannot reproduce a PEG related condition.  And this is often related to someone using a 5 to 10 year old PEG reagent that has not only changed pH and ionic strength but also like seen some evaporation and likely contamination from multiple accesses. Maybe you simply will want to add a buffer to unbuffered PEG screen solutions as you know for your sample you do not wish to screen pH values below 4. Kind Regards, Bob Cudney - Hampton Research On Jul 5, 2006, at 2:15 PM, Mischa Machius wrote: 1. Does anyone actually test their chemicals for peroxides? 2. What tests would be useful for PEGs, dioxane, glycerol, and the likes? 3. Has anyone bothered to define a threshold for 'good' vs. 'bad'? 4. Is peroxide formation a problem only for liquid PEGs, or does it also happen to a significant degree in solid PEGs? 5. What is the best storage method for (liquid) PEGs? Does storage at 4°C or -20°C prolong the lifetime? 6. Any other thoughts?