Chromatography Forum

Hi

Whats is the basis of selection of compound/chemicals used for column performance (lets say in case of RP columns)???… I just got curious in this because different column manufactures mentions different sets of chemicals/compounds in their COA

Thanks

There should be two tests for your column. One is a plate count test, the other one is a selectivity test. For the plate count test, a small UV-active hydrocarbon is used for reversed-phase columns. Examples: toluene, naphthalene, acenaphthene. Such an analyte gives only one type of interaction and the determination of the plate count is uncomplicated.

For a selectivity test, much more sophisticated tests should be used in order to measure different components of the properties of the packing. You want to measure hydrophobic retention, silanol activity, hydrogen bonding, steric interactions. Several tests have been published that accomplish this. Of course, I am of the opinion that my own test (or variations on it) is the best...

Hi Neue,

My manufacturer uses the following four compounds in RP columns. Wil you please explain the reasons for using these specific test compounds.

Are the difference concentrations of these compounds at maufacturer's and my lab going to affect the test results that I get.

URACIL
TOLUENE
NAPHTHALENE
BIPHENYL

Uracil is generally used as a "void volume" (t0) marker, as it is (approximately) unretained on reversed-phase columns

toluene and naphthalene are two of the compounds Uwe mentioned for a plate count test. Although Uwe didn't mention it, biphenyl falls in the same general class.

In effect, those compounds will confirm that the column is well-packed, will provide some information on the overall consistency of the bonded phase, but provide little if any useful information about selectivity or secondary (silanol) effects.

I like to use a real-life critical pair to assess column performance. For example, in one of our products we track the resolution of the active peak from the later-eluting fragrance component.