Chromatography Forum

I was told that TLC should be performed prior to HPLC as the Rf factor can provide useful information regarding the choice of the solvent for preparation of the stock solution- the one giving a higher Rf value being used as the solvent for stock solution. Is this true and if so, if we do not use the solvent giving the highest Rf value how can it affect our process?

It makes sense to do a TLC prior to HPLC but these days it is not necessary. Again it depends on your application and type of compounds that you are trying to analyse by HPLC. If you know the chemical structure of your molecules or at least the type of the molecules, you can use an appropriate HPLC method without doing any TLC because there sould be a closely related HPLC method and relavant information should be available on line. in journals etc. To find a TLC solvent system also takes time .

Good luck!

Ananda

What does stock solution mean here? If it means mobile phase then the highest Rf would be 1 ("theoretically"), which means no retention, that is no chromatography. If a standard´s stock solution is implied then you may get trouble with the peak spread, often discussed (I call that "wash-in").

A TLC in conjunction with HPLC can be very useful to discern absolute (permanent) retention of part of your sample.

Sonal wrote:

>I was told that TLC should be performed prior to HPLC as the Rf factor >can provide useful information regarding the choice of the solvent for >preparation of the stock solution- the one giving a higher Rf value being >used as the solvent for stock solution.

This is true, not necessary but useful. A solvent solution producing a higher retardation factor value, ie less retention of the analytes on the plate (using reversed phase TLC) will give you a solution which is more likely to elute all compounds from the analysis mixture ('spots' lower on the plate).

>Is this true and if so, if we do not use the solvent giving the highest Rf >value how can it affect our process?

The solution/solvent you do choose may not elute the compounds from the HPLC column, or will take a lot more solvent and/or time for the analytes to elute from the HPLC column.

No one likes to inject analytes onto a column if it is not known they can be removed from the column by passing a solvent through it.

As a general practise, it is easier to use a solvent that is too strong initially and weaken it for maximum separation of analytes than to use one too weak and then TRY to find a solvent that will elute the analytes from the column.

All this discussion has assumptions which may or may not be applicable to your analytical situation, but are beyond the limits of this discussion.

First of all I would like to clarify that I performed TLC(normal phase), with pure solvents only-no mixtures, and am working on reverse phase HPLC. I checked my drug solubility and it was soluble in DMSO, DMF and ACN, DMSO and DMF giving a higher Rf value than ACN.

I would like to know in which solvent should I prepare my standard stock solution(preparation in the mobile phase is not feasible), my mobile phase being methanol-buffer.

What I did was prepared my stock solution and dilutions in ACN and was getting the drug peak also, but later came to know that it should be prepared in the solvent which gave a higher Rf value. This is what I wanted to confirm whether this statement is true or not.

In HPLC, the standard rule is to prepare your sample in the mobile phase, or in a solvent composition that has a lower elution strength than the mobile phase. Sometimes, this can create trouble, and people are trying to dissolve the sample in a more universal solvent such as DMSO. While this works for dissolving the sample, it may lead to strange peak shapes, especially if the injection volume is not negligible.

Bottom line, if you are working on a single problem, dissolve the sample in mobile phase!

OK, not being in industry I can be a bit less diplomatic than Uwe. As I indicated above, the advice on standard solvent is 180° wrong (probably someone told you to find a solvent which best dissolves your material). If not enough material dissolves in the mobile phase (are you really sure??), or in a solution of even lower elution power, than inject a few µL of a saturated standard .... solution (assuming you don´t work nano), sort as what Uwe mentioned.
And: Some participants (me included) have mentioned milliliter injections of sample dissolved in solvents of lower elution strength than the mobile phase.

Sonal

Doing normal phase TLC is not useful in developing a HPLC reversed phase method, at least most of the time.

You have now clarified your questions with details and I will try to comment.

The statement you refer to is FALSE.

Your drug Rf being higher using DMF and DMSO for TLC dissolution solvent to the Rf of using AcN is probably an effect of the dissolution solvent not being evaporated from the plate before insertion into the moble phase. This 'modifies' the mobile phase - surface interactions and affects the final Rf value. The DMF or DMSO migrates with the drug up the plate.

Now concerning HPLC drug dissolution solvent

You do not want to inject a strong solvent solution onto a reversed phase column as HW Mueller and Uwe Neue have indicated.

Several reasons, but first and foremost your chromatography will most likely suffer as the components dissolved in the strong solvent are less likely to form an initial sharp focused plug at the head of the column.

ALWAYS inject your dissolved sample in the mobile phase or in a WEAKER solvent than the mobile phase. This will insure usually a sharp sample plug at the head of the column necessary for good chromatography.

I wish you success in your endeavor.

Thankyou for your responses. As mentioned by you I should dissolve the drug in a solvent of lower elution strength than the mobile phase.

The elution strength of methanol for a C18 column is 1.0, of ACN 3.1 and of DMF 7.6. There is no solvent with a lower value than methanol. These values I got from http://home.planet.nl/~skok/techniques/ ... ended.html

My drug is sparingly soluble in methanol and when I preapred my stock solution in the mobile phase there developed a slight turbidity, so I switched to ACN in which the drug is soluble.

In reversed-phase chromatography, the solvent with the lowest elution strength is water. The advice above means that you should dissolve your sample in your mobile phase, or in something that contains a bit more water than your mobile phase. If you are running a gradient, dissolve your sample in the starting composition of the gradient, or something with a bit more water.

You may complain that your sample is not soluble in such things, but this is generally not correct. With the high detector sensitivity in LC, you usually need only a very small amount of sample to dissolve.

Rereading your post, Sonal, I may have misunderstood, your tlc mobile phase was neat DMF or DMSO perhaps? Or was it methanol and a buffer?

In any case, that is not important, as normal phase TLC is not HPLC reversed phase.

Now, you never told us in the forum the composition of your buffer.

You probably only need to dissolve your drug at 1mg/mL or less as Uwe suggested in his last post. If your drug will not dissolve in the buffer/methanol mobile phase try dissolving it at an even lower concentration and inject a larger amount of sample. Instead of 1mg/mL at 20µL then inject 200µL at 0.1mg/mL.

You should be able to find out the maximum concentration of the drug your buffer will allow. Determine that and you are close to a solution.

best wishes,

Chromatographer1

Rereading your post, Sonal, I may have misunderstood, your tlc mobile phase was neat DMF or DMSO perhaps? Or was it methanol and a buffer?

In any case, that is not important, as normal phase TLC is not HPLC reversed phase.

Now, you never told us in the forum the composition of your buffer.

You probably only need to dissolve your drug at 1mg/mL or less as Uwe suggested in his last post. If your drug will not dissolve in the buffer/methanol mobile phase try dissolving it at an even lower concentration and inject a larger amount of sample. Instead of 1mg/mL at 20µL then inject 200µL at 0.1mg/mL.

You should be able to find out the maximum concentration of the drug your buffer will allow. Determine that and you are close to a solution.

best wishes,

Chromatographer1

If the other advices won't work you can try ACN/buffer to solve your samples, but that could lead to some problems with peak shape. Try to inject as less as possible.
If your compound of interest isn't charged you can try to use even a mixture of ACN/MeOH to solve the samples.

Thankyou all of you for your responses. But I was just wondering what difference does it make if my first peak (solvent peak) is a bit distorted(with a small additional peak at 2.78 and that too not always present along with my regular peak at 2.45 ). I agree with you all that the stock solution should be made in a weaker solvent than the mobile phase (methanol-phosphate buffer), but my drug peak is coming ok. This is also confirmed by Consumer Products Guy as mentioned in my post on solvent peak.

The appearance of the "solvent peak" (more properly, the "dead time", t0) actually is of little concern, so long as your analyte peak is sufficiently strongly retained. For pharmaceutical analysis, both the US FDA and the ICH "suggest" k' values > 2., which should put your analyte well out of the way of any disturbance at t0.

However, as has been pointed out, dissolving the sample in a stronger solvent than the mobile phase can cause peak shape problems even for wekk-retained peaks.