Chromatography Forum

Hello,
I am having problems and I hope one of you can please help me.
I have to develop a RP isocratic method for simultaneous evalutaion of two compounds. One of them is extremely polar and the other extremely non polar.
I am using c18 column and cannot change it.
the problem is that at high concentration of organic phase the polar drug is eluted right at the beginning at same time as solvent peaks. And at low concentration of organic phase the non polar compound does not elute at all.

Also, I have noticed, that when I change my mobile phase, or the percentage of organic solvent in the mobile phase, the ;location of the solvent peak changes. Is this normal?
Shouldn't the solvent peaks appear at same time irrespective of the solvent composition as long as the flow rate is the same?

the second question is bothering me alot. Can someone please help.

(1) You need to use a gradient method instead of an isocratic method.

(2) The "solvent peak" can shift a bit (you did not say how much relative to the flow rate and time), but should always be near the T zero point. If the injection solvent volume is large (or the injection solvent is different than the mobile phase) it can cause the solvent peak to shift a bit more than normal.

Develop a good method and you should have a nice separation, good retention and resolution of both compounds without any "shifting" solvent peaks.

thankyou very much for your reply,
I think I understood my problem of peak shifteing.

But I HAVE to develop an isocratic method only. Any suggetsions would be helpful. Ion pairing agent (TFA) is not helping to retain the polar compound (It carries a positive quaternary ammonium charge)

octane sulphonate 2-10mM should pull the quaternary amine away from the solvent front and is available in high purity from sigma aldrich (ion pair grade).

Is the hydrophobic compound ionizable? If so, is it an acid or base? You could reduce its retention by fully ionizing it using a buffer of suitable pH. Since you wont change the ionization of the quat you could potentially solve this without using ion pairing.

Do you have a logP value for the quat? If its too low, i.e. <~-1, you'll likely have to use an ion pairng agent.

In cases like this one shouldn´t forget that two methods, one for the polar the other for the nonpolar compound, could be THE way to go.

(1) You need to use a gradient method instead of an isocratic method.

(2) The "solvent peak" can shift a bit (you did not say how much relative to the flow rate and time), but should always be near the T zero point. If the injection solvent volume is large (or the injection solvent is different than the mobile phase) it can cause the solvent peak to shift a bit more than normal.

Develop a good method and you should have a nice separation, good retention and resolution of both compounds without any "shifting" solvent peaks.

hi thankyou for your reply.
I am using 100% ACN in blank.
When i use 50% ACN in mobile phase solvent peak is at 1.9
on reducing ACN concentration in mobile phase to 10%, solvent peak is shifting to 2.5-2.9
dont know if this is normal.
Flow rate is same.

t0 should not shift that much. The "solvent peak" you are looking at is slightly retained.

t0 should not shift that much. The "solvent peak" you are looking at is slightly retained.

thankyou very much for the reply.
could you suggest how can i correct this problem.. my column is only 150 mm long

Without seeing your chromatograms, it's very hard to make specific suggestions. Depending on your wavelength and detector sensitivity, you may not be seeing a "solvent peak" at all; as I posted earlier, my guess is you may be looking at some trace impurity which is actually slightly retained with a very low % organic mobile phase. It happens, and unless you're doing a PhD thesis on t0 determination, I'm not sure that it's worth worrying about.

Without seeing your chromatograms, it's very hard to make specific suggestions. Depending on your wavelength and detector sensitivity, you may not be seeing a "solvent peak" at all; as I posted earlier, my guess is you may be looking at some trace impurity which is actually slightly retained with a very low % organic mobile phase. It happens, and unless you're doing a PhD thesis on t0 determination, I'm not sure that it's worth worrying about.

Well It is mys studentship thesis.. yeah!

octane sulphonate 2-10mM should pull the quaternary amine away from the solvent front and is available in high purity from sigma aldrich (ion pair grade).

Is the hydrophobic compound ionizable? If so, is it an acid or base? You could reduce its retention by fully ionizing it using a buffer of suitable pH. Since you wont change the ionization of the quat you could potentially solve this without using ion pairing.

Do you have a logP value for the quat? If its too low, i.e. <~-1, you'll likely have to use an ion pairng agent.

hi thankyou very much for your reply.
Can I use octane sulphate instead of sulphonate?

Can I use octane sulphate instead of sulphonate?

Yes. In fact a wide range of anionic surfactants can be used. The sulfonates were popularized by Waters in the 1970s and have become the de facto standard, but there's nothing magic about them.