[J_Holland]

Hi, I'm a graduation student from the Netherlands. I've got a few questions about RP-HPLC(in my case with DAD) in general, which I can't answer by googling

-Why can't I measure ethanol or methanol in HPLC? (Or any simple alcohols)
-I think I can't measure simple acids like: glycolic acid or methanal? But I'm not sure why.
(I guess in both cases its because they don't absorb UV light. But I'd like to know why they don't, or if there is a rule they follow)

-My mentor said its obvious that by chancing my isocratic mobile phase (MeOH/MilliQ/H3PO4) from 50/50/0,1 to 5/95/0.1 I got more separation in my analysis. And that I probably got the most separation out of it (with those solvents). I just can't seem to grasp why that's the case.

I've been searching alot but I cant seem to find the answers anywhere. If someone can help me I'd be really thankfull

[Arkcon]

-Why can't I measure ethanol or methanol in HPLC? (Or any simple alcohols)
-I think I can't measure simple acids like: glycolic acid or methanal? But I'm not sure why.
(I guess in both cases its because they don't absorb UV light. But I'd like to know why they don't, or if there is a rule they follow)

You've guessed correct, the biggest problem is the lack of a UV chromaphore.  The molecules you mention are small, so there's limited atomic interactions to absorb light.  This is the "rule" that molecules follow.

-My mentor said its obvious that by chancing my isocratic mobile phase (MeOH/MilliQ/H3PO4) from 50/50/0,1 to 5/95/0.1 I got more separation in my analysis. And that I probably got the most separation out of it (with those solvents). I just can't seem to grasp why that's the case.

Can't you?  What has changed?  And yes, I see the percentages -- but, what do the changes do?  What are your particular analytes, what is the nature of your column media, and how did this change help?

You can consider reverse phase chromatography as a tubular separation funnel -- a long tube doing a constant equilibrium of a solvent extraction.  How does the change in solvent affect the separation?

I've been searching alot but I cant seem to find the answers anywhere. If someone can help me I'd be really thankfull

This isn't a favorite topic for textbooks.  But there are online sources, and training courses (for a fee) on basic HPLC.  But often, a new person starts with a published method, and develops the advances they need from there.

[J_Holland]

Thanks a lot for your fast response. I'm glad to hear my guess was right. I've been reading online courses, but even so I couldn't find any concrete answer to those two questions.

And sorry I should have mentioned earlier, I'm researching bronopol and its degradation products(2-bromoethanol/ bromonitromethane/ nitromethane/ 2-bromo-1,3-nitroethanol/ tris(hydroxymethane)nitromethyl), on a 25cm long c18 column (HPLC-DAD).

I know that by increasing one of the two solvents, compounds in a sample will have more retention with the mobile or stationairy phase(based on polarity in my case). I guess that increasing the amount of Milli-Q in my mobile phase, means that my compounds have less affinity with my mobile phase and more affinity with the stationairy phase, and therefor stay longer on the column.

I just found this strange, since bronopol and its degration products have a lot of polar functional groups, and therefor I didn't think that by increasing the polar solvent, the affinity with a non-polar stationary phase would increase.

It could be that I'm overthinking this, It's just that my deduction is contradicting itself, which results in me doubting myself.

[J_Holland]

Could anybody explain how come I get more seperation by using more water as mobile phase?

[scwilson]

In general, separation improves as analytes are forced to interact more and more with the column, since the column is our primary means of separation. Of course, if too much column interaction is forced, all analytes indiscriminately interact with the column and separation is poor. Basically, as long as column residence time is reasonable, increased column interaction with the analytes results in better separation. Remember, these are general guidelines, NOT absolute guidelines.

You're using RP-HPLC. Reversed-phase. Does that mean your stationary phase is polar or non-polar? Compared to typical RP-HPLC solvents--acetonitrile, methanol, ethyl acetate, water, etc.--which is most polar? Would using that polar solvent in greater proportion increase interaction of your analytes with the column or decrease it? How would that affect separation?

[J_Holland]

Im guessing by using more water in my mobile phase, I force my analytes to interact more with the column. I've also read that MeOH reacts with RP column, while water doesn't, and therefor reacts with active sites on the column. So by adding less MeOH i'm also increasing the active sites in the column and therefor adding more retention and seperation to my compounds.

[Arkcon]

God.  I was hoping you'd figure that out.  I don't know about the methanol interacting with the column however, I think its more about methanol not being there to give the analyte a chance to interact with the column.