RP-HPLC, unretained and unknown compound

[Butter]

Please, I am seeking to isolate a bioactive compound that co-elutes with about 4 other compounds at about 2-3 mins,with 3% organic at 6 mins. Mobile phase-MeOH/H2O, gradient.

Column: C18, 15cm, 4.6mm,5um.

Any opinion on how to isolate this compound will be appreciated.
Thanks in advance.

[danko]

Butter,
Do you mean 3% MeOH increase in 6 min. (i.e. 0.5 % per min)?
When does the gradient start (from time 0)?
What is the initial composition (before gradient start)?
What type of compounds are you dealing with (ionizable)?

Best Regards

[cyanogen78]

Sorry for the off-topic, but I can't send a PM to Danko. Danko, regarding with your sign I think that we are from the same nationality, please, lets make a conversation, I have some things to duscuss with you. Thank you in advance. Sorry to Butter for the off-topic again.

[Butter]

Danko
Sorry I was off for a while. Thanks for your reply.

Gradient conditions

0-6mins 3% organic. Gradient starts from time 0.

Initial composition 100% aqeous phase at T0 and 0% organic increasing to 3% in 6 mins.
compounds are unknown. I am dealing a bioactive fraction yet to be characterised.
Thank you

[Uwe Neue]

Well, there are two possibilities:

Either your C18 does not work in 100% water (dewetting), or your analyte is too polar to be retained by a RP column.

Case 1: use a C18 that works in water (Atlantis T3, or a EPG packing such as SymmetryShield RP18).
Case 2: use HILIC to retain your analyte.

[Butter]

@Uwe,
Thank you.

I think the analyte is very polar. Increasing the concentration of the organic phase only decreases the retention time accross boardand elutes all components before 6 minutes including those that elute later. The column is not any of those mentioned. I have considered buying Atlantis T3. I will not want to use HILIC because i still have other compounds of interest that elute at a later time with a higher percentage of the organic.
Thank you once again.

[YMC-Kune]

Dear Butter,

What kind of molecules are you analyzing?

You say they are bioactive, these molecules are often ionizable. Therefore, maybe changing the pH will change the ionisation of the molecules making them less polar. Do you know anything about the pH stability of the compounds?

Do you know if the compounds are retained at all or do they elute with the dead volume? If you are working with larger biomolecules (e.g. proteins) it is also worth considering the poresize of the stationary phase in order to get retention.

Have you checked that the column has reached equilibrium at low organic medifyer content after the previous run? Else you have the chance that you are injecting into a column which has a higher content in organic modifyer and analytes are, thus, eluted before they should.

So, there are many possibilities, therefore it would be the best to see the whole method (incl. gradient program, mobile phases and flow)

[Butter]

YMC
Thank you very much.

I have changed the pH between 2.8 to 7.5. It is the same.

There are actually some peaks that elute before the dead volume which I think are high molecular weight protein. I do not care about them for now because they are not of interest.

Yes the compounds are retained a bit. The dead volume elutes at about 1.5 while analytes elute at about 2.01.

Concerning equilibration, I made that mistake initially but corrected it. I actually allow enough time for equilibration before the next run and the runs are reproducible.
Gradient programme
time(mins) A B(% organic)
0 100 0
6 97 3
10 60 40
25 40 60
30 0 100
40 100 0

I get up 100% organic because there is a peak that does not elute until it clocks 100 except I run for a longer time(solvent waste)
I allow at least 5 after eact run before re-injection.

Thank you once again.

[Bryan Evans]

Some suggestions:

- Since you are running at 100% aqueous at initial run, you should probably
flush the system with (at least) 10 column volumes. For 150x4.6mm,
that means 15 mL prior to the next injection.

- Reduce the extra column volume in your LC

- Use an ODS phase that can handle 100% aqueous eluent

- Reduce particle size to 3um

- use guard cartridge (since your samples seem to be pretty dirty)

[YMC-Kune]

Dear Butter,

thanks for providing the method. It seems O.K. I, personally, would add the 5 minutes flush time to the end of the method, so that the detector signal is also recorded during equilibration (I suppose you are using 5 min "wait-time" between runs). Often you can follow UV absorption at 210 nm to get an indication whether or not your column is equilibrated at the end of the flush time.
I found 5 minutes equilibration to be enough to get reproducible chromatograms with most modern equipment, but it is a simple test to increase the flush time to 15 minutes for 2/3 injections. Probably you can run them overnight to save time?

Since you do not want to try HILIC, I agree with the others here, usage of a column that works with 100% aqueous phases would be the first bet. Apart from the already mentioned phases, YMC-ODS-AQ or Hydrosphere are two alternatives. I don't want this post to become a YMC-advertisement, so if you need any info on the columns, please do not hesitate to contact me.

Of course increasing column length will also increase retention (unless you have analytes that are following an adsorption/desorption mechanism, e.g. proteins).

Please keep us updated on your progress.
Regards,
Daniel.

[Butter]

Thank you guys. I appreciate all your in-puts.
@Bryan,

I actually use guard column even though I use SPE to clean up the sample before injection.

@YMC,
I will have to build the 5 minutes into the method as you suggested. I did that before though but just eliminated recently.

I have a phenomenex hydro-RP at the moment 4um particle size. it can tolerate 100 % and will try that soon. I want to collect the hydrophilic fractions with a semiprep-column and then use the hydro-RP for futher analysis.
Thank you very much guys. Any further in-put is welcome.