Chromatography Forum

Hello!

Forgive my ignorance but I would like to know if it is possible for a guard column (C18) to completely retain highly nonpolar compounds? I am asking this because I was previously using TLC with RP18 silicagel plates for my analytes and some compounds did not move from the starting line even when hexane was used as developing solvent system. I don't know if this occurs due to highly nonepolar compound or maybe somekind of reaction between the stationary phase and analyte takes place. If highly nonpolar compounds are the case, I am worried that I may be damaging my C18 column even when guard column is used (compounds do not elude from the column if the gueard column doesn't completely retain them?). However, until now I haven't notice any rise in the column backpressure.

Any thoughts?

This has been descreibed many times. The record in our lab is stripping leukotrienes from about 8 mL eluent of a column via a C-18, 2 x 20mm (or 4 x 5mm, I can´t look it up here) guard. If you don´t have the right mobile phase you can´t get any chromatography. This hexane on a RP-TLC . . . I wouldn´t take that as representative of RP-HPLC. Remember that there has to be an equilibrium between the stationary and mobile phases to move something along a chromatography "bed".

There is always the possibility that analytes/matrix compounds will be "completely retained" in a C18 column. Another problem can be precipitation of your compounds when you use such organic solvents (did you check their solubility in hexane)?

a guard column will not block right away even of the compound is completly retain with every injection because the amount could be small.
i would take 3 decisions:
1. leave it as it is. after all the role of the guard is to catch the bad stuff and clogg in the end. far cheaper then changing the column entirely
2. you could look into your sample preparation procedure and see if you can get the problematic compound out before injecting it. SPE would be a good solution either on-line of off-line
3. maybe you can look at an alternative stationnary phase. one that would not retain this compound that bad.

the decision will mainly depend on what you are doing, what you need to check, how much it costs you, how much time you can waste/spend, can you change anything to begin with or is it "The Method", final.

in general hexane is not the solvent of choice for RP chromatography.

Thank you for your answers. I am not using hexane as a solvent in HPLC, I just wanted to point it out what efect it had regarding TLC. In HPLC I use THF:MeCN:H2O mixture

Dear aalbre,

it is certainly possible to retain non polar compounds on a C18 column. This is sometimes done on purpose when using a column as a trap column.

As in your case this retention is unwanted you will have to see, if a different column would work better. Some column with a shorter carbon chain might be better suited. Maybe try a C8 and or a C4. Are there any specific reasons you chose a C18 column, or is it because you did yout TLC experiments on C18 material?

Why do you expect the sample compound to be trapped on the guard column? Are you sure you can detect the moelcule with your detection system?
Are you looking at big molecules? In that case you would also need to think about pore size of the stationary phase.

Regards,
Daniel.

thanks for your reply YMC-Kune.
I chose this C18 column because it was the only one that drew a distinction between two structural isomers. Otherwise my analyte molecules and its derivatives aren't big at all (range from 288-380 Da).
I have a PDA detector set at 518 nm (specific absorbtion max) but setting lambda to lower wavelenghts does not show any new peaks in the chromatogram, so I believed that the unwanted compounds are completely retained either on the guard column or worse, on the C18 column...

Dear Aalbre,

How did you detect the spots on TLC? Are you sure it is detectable? (Nothing worse then searching for a non detectable peak)

You can wash the whole system with methylene chloride and record the signal (make sure you are going via THF to MeCl3 and back via THF because of miscibility).

Else, I am affraid it will be quite difficult to analyse both party in one method. You could try a column with small pores, mybe you are lucky and that larger molecules are not retained (how large is large?) If you need the content of the high molecular weight molecule as well, you most likely have to develop two methods. The one on the C18 will give you the proportion of isomers, one on a C4 or C8 will give you the ratio between the high and low molecular fraction.

Hope this helps,
Daniel.