By Anonymous on Tuesday, February 19, 2002 - 12:52 pm:
Hello!
I am trying to separate the components of a complex mixture containing nucleosides, nucleotides and nitrogen bases, may be some peptides also. I have made some attempts in reversed-phase C18, but it is not easy since these
are small and hydrophilic compounds, they are difficult to retain. I will appreciate any idea to increase retention or another separation principle I could use.
Additionally, if you have the article below, please, let me know the protocol used in it. Thank you very much.
Isocratic separation of some purine nucleotide, nucleoside, and base metabolites from biological extracts by high-performance liquid chromatography.
Anderson FS, Murphy RC
J Chromatogr 1976 JUN 23 121:2 251-62
By Anonymous on Wednesday, February 20, 2002 - 04:58 am:
We had good luck with Zorbax SB-Aq with perfluorobutyric acid as IP in separating several nucleotides. At low pH they have strong fluorescence.
By Anonymous on Wednesday, February 20, 2002 - 06:37 am:
Are these compounds stable in acidic pH? Do you have any reference of nucleotides with these kind of ion-pairing reagents?
K.T.
Thanks
By Anonymous on Thursday, February 21, 2002 - 04:36 am:
Guanosine and its analogues ARE stable at pH 1.5 and they have strong fluorescence at this pH. Two methods have been extensively validated under GLP and the results are fantastic. We work on drugs in human plasma with LOQ of 10 ng/mL with runtime of less than 10 min (a requirement in our lab). The key for human plasma samples is to find a specific SPE procedure. Some nucleotides are retained on Zorbax SB aq with about 1% ACN with 0.5% TFA. Some do require IP. In any case did we add some TFA to retain low pH to get strong fluorescence.Unfortunately, company policy does not allow me to disclose more.
Good luck.
By Anonymous on Wednesday, May 8, 2002 - 04:38 am:
I am having good results using a Genesis aq column from Jones Chromatography using 30mMol
TEAA against methanol 0-15%
for DNA RNA mixed phosphate groups
good luck
By Einar Pontén - SeQuant AB on Tuesday, June 4, 2002 - 02:17 pm:
The ZIC™-HILIC column may be a versatile tool as well.
We use some nucleotides for column testing and performance validation to certify column plate number.
http://www.sequant.com
Hydrophilic Interaction Chromatography (HILIC); Elution by an initial high content (>70%) of acetonitrile or low alcohols on a hydrophilic stationary phase. Volatile buffers may also be used so HILIC is indeed suitable for MS detection.
By Uwe Neue on Sunday, June 16, 2002 - 01:33 pm:
We have done separations of these compounds with our Polarity d-C18 packing, which has been optimized for maximum retention of polar compounds. Either check the Waters webside or contact me for further information......
By Anonymous on Monday, October 13, 2003 - 08:01 am:
Sorry to dredge up this one and a half year old thread, but, quoting above ...
>> Guanosine and its analogues ARE stable at
>> pH 1.5 and they have strong fluorescence at this pH.
If anyone can point me to a literature reference, I would appreciate it. I might have an application coming up soon.
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