Chromatography Forum

what is the role of an analytical column in an isocratic separation system? If the analyte of interest does not bind to the column, then why use one?
I am attempting to use a vitamin A separation from fortified corn meal blends. The method I'm experimenting uses isocratic hexane:ethyl ether:plamitate and detects at 325nm. Is it necessary to use an analytical column, at present I'm just usuing a guard column and in-line filter. Please advise.

The reason for using a column (and doing chromatography) is to separate things. If you have a solution of a pure compound, or a technique that can detect a single compound without interference from the rest of the stuff, you don't need chromatography. You can still use the same instrument and do flow injection analysis.

I think that Jumpshooter does not realize that in isocratic chromatography you do not have just bind or elute (like in SPE)... You also have (not indefinite) retention and under that mode you can still separate 2-20 compounds...

In chromatography, the binding of a compound is measured by the ratio of the time stuck to the stationary phase versus the time carried along by the mobile phase. It is neither infinite nor zero, but some intermediate value. Each substance has its own ratio that is a function of many different factors. Some factors are under the control of the chromatographer. So if you need to separate a mixture and quantify its components, you manipulate things so that the ratios (called capacity factor or k') are convenient and different enough.

Corn meal contains zeaxanthin which elutes in the same general vicinity as retinol and retinol acetate. How much signal do you get for unfortified corn meal? If it is more than a very small percentage of the expected vitamin A content, you need more column.

Hey I am new to this world of HPLC and please forgive me if my question is a bit "juvenille". But my experience is largely in real-time PCR for nucleic acids and have done some GC/MS on therapeutic drug metabolites; however, a new project requires me to learn and use an HPLC Model 1090 for Vit A (retinyl palmitate metab.). My learning curve is therefore = / <--very steep. You writers to this forum are really smart and or experienced; please excuse my juvinility--but I need to learn this material. Thanks for helping if possible?

I would suggest an afternoon or two with a good book (and I don't mean Harry Potter). You might also look at http://www.chromatography-online.org since it is free. You will have questions after your reading, and we will be happy to clarify/debate things. Best of luck with the new assignment.

There are links to a number of free on-line tutorials at:
http://www.lcresources.com/resources/reslinks.html