I,
I am looking at ATP and Adenosine Levels in cell cultures, I have a standard RP C18 column. Can i use it to separate and identify them? if so , what are the LC parameters?
Lots of examples for you to use on the web. Just do a keyword search and you will find plenty. Here are some examples (but not necessarily ones that are best. It will be worth your time to review many and find the simplest ones to start with that best match the tools you have available).
"A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures" http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299834/
"Analysis of adenosine by RP-HPLC method and its application to the study of adenosine kinase kinetics" http://www.ncbi.nlm.nih.gov/pubmed/17763527
I used RP-HPLC with inverse phase C18 columns to separate nucleosides and deoxyribonucleosides. It can be used also for nucleotides. I found on Merck index the values of absorbance that you need for detection. You will have to buy standard single nucleotides you want to separate and run them one at time to register the type of peak, retention time and concentration. I think if you search on Google about the technique for ATP and adenosine you find papers.
Dear Bill Letter, Thanks for your reply, I have searched the web but found that many of the procedures use Modified RP-C18 Columns (like end capping...), how ever i have only RP-ODS column.
Of the Two references that you have given, the first one involves derivatization and fluorescence detection, Unfortunately I do not have a fluorescence detector. The second reference was of great help to me, but the only problem being that the methd was optimised only for ADO under fast eluting conditions, but my analysis requires me to separate and identify many nucleotides and nucleosides simultaneously, I am not sure this will work in an isocratic system.
analytical biochemistry 147, 180-185 (1985), will be help full for you
I do not understand your comment about columns? All RP C!8 columns are fully endcapped. That is what "ODS" column stands for. It means C18.
Yes, some of the methods use derivitization, some HILIC, some regular C18 columns with simple mobile phases and some analyze the compounds in their native form too. All kinds to pick from. *You provided us with no information about what equipment you have available to use and only requested info on possible methods by HPLC using a C18 column. So I provided you with a few examples (of hundreds that I found on the web in a few minutes) to get you started. The web is great source of information. Take advantage of the info and learn how to become proficient at searches. This is an important skill to learn. Search the web and review the many methods for one that is applicable to what you have (in terms of tools, experience and equipment). There are many options so putting in some time to research it will pay off for you later on. Also, keep in mind that many methods in the literature are of poor quality, so you should think about how to improve the methods when you are working with them. Good luck on your project!
Dear Bill,
It was my mistake for not giving you the full picture. Any way I digged into the literature and after 2 days, was able to standardise the seperations. Thanks for the info.
My suggestion is the attached publication, a fast and simple method for nucleotides determination by C18 HPLC.
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