Chromatography Forum

Hello to all.

I received sample from our contract laboratory for HPLC analysis. After several tests I found that retention of principal peak (see picture) on standard C18 column is very low. On Supelcosil C18 250x4.6mm (with mobile phase 15% MeOH in water, flow 1ml/min.) is RT about 4 mins. I think that such chromatogram cannot be considered as trustworthy.
Do you have any ideas for more suitable column? I leer at ZIC®-HILIC column but its price is quite high with uncertain result.



Thank you for comments.
Regards

My first thoughts are to acidify the mobile phase and decrease the %methanol.

On another note, be careful posting an exact structure on the internet. If that is not a common molecule, you might get in trouble for that...one of my colleagues did.

Try adjusting the mobile phase pH to 2.3 – 2.4 with phosphoric acid.
Or better yet; prepare a 10 - 20 mM sodium phosphate buffer at the pH range mentioned above.
And use it instead of water.

Best Regards

+1 on suggestion to cut MeOH conc. - or switch to ACN, may have to go pretty low on that too.

... On another note, be careful posting an exact structure on the internet. If that is not a common molecule, you might get in trouble for that...one of my colleagues did.

OK, if you will agree, I'll change the address referencing to picture after solving

And now about your recommendations:
Decreasing of methanol content - I tried it during method development but C18 phase probably colapse because peaks became broad and separation was completelly lost. Minimum content 10 - 15% of methanol was needed.

Adjusting of the mobile phase pH - was tested but without observable effect on separation. I tested pH range 3-8 (with phosphate buffer and sodium acetate buffer for higher pH).

Switch to ACN - the separation was not so good as with methanol. Positive influence of other solvents (IPA, THF) was not observed.

The suggestions here sound good. I would also suggest that you run the sample by gradient also with a hold @90% Meoh or ACN for several minutes. Although the molecule has some polar functionality I would suspect it is still quite hydrophobic. You may be seeing an impurity coming off relatively unretained while your target molecule sits on the top of the column.

Marc

...You may be seeing an impurity coming off relatively unretained while your target molecule sits on the top of the column.
Marc

No, the target molecule really elutes at approx. 4 min. with mentioned conditions. I start with gradient elution during method development (usually 20 - 80% of ACN). There is not really other significant peak.

Maybe you could try a phenyl column?

Excuse me, I don't understand structure of this molecule (C(4)OO???). This is cyclic peroxide or ...? Are you sure that this peak is not impurity?

Sadilek,

You haven’t tried pH < 3, as suggested above. That means the stationary phase’s free silanol groups are mostly ionized and even more importantly negatively charged. The oxygen in your molecule will try to avoid these sites and consequently the molecule will elute much faster than it would if pH was in the region of 2.2 – 2.4. Another good reason for adjusting the sodium phosphate buffer to the latter pH is its pKa.
Which reminds me to mention that sodium acetate (pKa = 4.75) is far from the right choice if the aim is to achieve buffering at pH higher than 8.
And maybe, with this column, you shouldn’t go higher than pH 8 anyway. Because you know what happens with the silica support/particles at pH > 8.

Best Regards

1, it is a neutral molecule. PH does not matter.
2, It seems not to be high hydrophilic with two ester and one OH;
3, Decreasing MeOH should work. if Not, find a column which can tolerate 100% aq or high percent aq..

Good Luck,

To get more retention is easy: use a column that is compatible with 100% water. Any column with an embedded polar group will do, or a good C18 that is designed to function in 100% water. My choices: SymmetryShield RP18 for the first one or Atlantis T3 for the second one.

danko: "...Because you know what happens with the silica support/particles at pH > 8."
Yes, I know
"...sodium acetate (pKa = 4.75)"
It was used for pH 5, for pH 7 - 8 I used phosphate buffer (pKa=7.21).

ym3142: "...find a column which can tolerate 100% aq or high percent aq."
Yes, it is the way which probably will help.

Uwe Neue: Thank you for recommendation. I will try this two columns.

Thank you for your advices. Have a nice day.

it is a neutral molecule. PH does not matter

The analyte’s oxygen draws the electron cloud towards it self thus creating a permanent dipole moment (partly negative). This is how hydrogen bonding works. So negatively charged oxygen (i.e. ionized silanol) will repel this particular molecule even in its neutral state.

Otherwise I too believe that some (not all) polar embedded stationary phases might work.
Low pH buffering could be tried with the available column – takes a couple of hours.

Best Regards

It is hard to believe that this molecule has such low retention. I would suggest injecting compound without column, measure peak area and the measure peak area with the column. If this is indeed your compound then I would go with 5% ACN and 0.1%TFA to fully suppress ionization f compound and stationary phase. Repulsion effect in case of this compound and silanols are overestimated. Even benzoic acid (which is less hydrophobic) will retain with this conditions on any RP C18 column for over 20 minutes (250 mm column). As alternative you can add ionic interaction by going to mixed-mode column. You will add strong interaction and observe synergy of two mechanisms. Consider functionalities and properties of your molecule. In most cases it is not important what is exact structure of your compound, look at mechanisms of retention and how stationary and mobile phase can be tailored to your separation:
http://www.sielc.com/application_082.html
http://www.sielc.com/application_136.html (at 40% ACN not 5%)
http://www.sielc.com/application_139.html (much more polar acidic molecule then yours)
http://www.sielc.com/application_145.html
http://www.sielc.com/application_163.html

Regards,

Vlad