Helpful hints re method development for analyzing moisture content in phosphoramidites and nucleic acids? ALternative is indirect KF and does not have adequate sensitivity.
By Anonymous on Wednesday, May 28, 2003 - 08:17 am:
Dow in Canada did a presentation on water in solvents using GC a couple of years ago. If my memory is correct the presentation dealt primarily with the Valco PDID detector, but there was also TCD info. If you can find info on past EAS programs you might be able to contact the author and find out just what they did.
By Ralph on Wednesday, May 28, 2003 - 12:04 pm:
Without knowing how much sample is available and in what physical form it is in, a possible way is to extract with the minimum amount of a suitable solvent e.g acetone (dried with mol sieve) and use a Porapak Q column or equivalent. Always run a blank of the solvent alone to get the background level.
If you have an FID there is a way to get at low level water content by packing some calcium carbide into your injection liner. On injection acetylene is released which is easily detected.
By Anonymous on Thursday, May 29, 2003 - 11:51 am:
Thanks for your comments. I will take at look at EAS notes. Sample quantitites are not limited, visual appearance is fine crystalline material. Normally, I prepare 0.3-10 mg/ml in ACN for LCMS work.
Deoxynucleosides are manufactured to meet a specification of <0.5% water in the vacuum dried product. Water is bad and these compounds are extremely hygroscopic. It is desired to lower the spec but we must have methods that can be reliably used at these levels.
As I stated earlier, the indirect coulometric method can be employed for analyzing the compound dissolved in ACN. Of course, low water ACN is employed. I was told that GC TCD can be done using the polar DB624 column, ramping 50-200 C at about 10 C/min. Water should elute about midway during the run. I know this column is used for general volatile analyses but short finding a ref method, I am looking at some development time.
By Anonymous on Thursday, May 29, 2003 - 12:02 pm:
If you use column 2-4ft FSOT lined SS 1/8 inch OD 2mm ID packed with Hayesep R 100/120, use 120 to 150°C oven, 10 to 40 cc/min. carrier and injector you should be able to quantitate water at extrememly low levels. Inject 0.2 to 0.5µL, preferably with an internal standard of methanol or ethanol, use linear regression to determine amount of water in sample via std addition.
Use 6in of replaceable column at inlet as your disposable injection port liner to avoid sample buildup.
By Ralph on Thursday, May 29, 2003 - 12:57 pm:
I don't have any experience of water in acetonitrile on this column but my initial feeling is that you will get a poor peak shape with this column --- BUT -- if you have a DB624 then why not give it a quick try with some ACN spiked at 1 mg/ml. You have plenty of sample so I would use 200mg per ml of solvent. Water at 0.5% will give 1 microgram water/microlitre injected which is easily detected. As for temperature program try splitless 60-85° at 25°/min then 5°/min. You may have a lot of interfering peaks from these samples
My preference would still be for a gas solid phase though :-)
By Ralph on Thursday, May 29, 2003 - 01:01 pm:
Rod's method would good to use
By Anonymous on Thursday, May 29, 2003 - 09:28 pm:
Ralph et al
I use it to measure water in acetone. Spec is 0.3% max.
I check and find 0.25-0.27% water usually.
I have measured <100ppm in neat AcN with HP6890 µTCD.
Run one or two blanks first to demonstrate consistency of trace water response first.
By Ralph on Friday, May 30, 2003 - 11:30 am:
Just in case there was any confusion because of the order the replies came up, my concerns were in using DB624 for the analysis. I would fully agree with Rod's method which uses a gas solid phase.