By sonal on Thursday, April 15, 2004 - 02:37 am:

It is mentioned that the pH of the mobile phase be +/-2 of the pKa of the drug. I have to develop a method for its analysis by HPLC. I wanted to know whether +2/-2 is preferred. My drug is an ionizable molecule with a pKa of 4.5.

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By sonal on Thursday, April 15, 2004 - 02:44 am:

If a drug is converted to its lactone form is there any way that we can prevent the conversion.Can we add an antioxidant to the stock solution of the drug?

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By Uwe Neue on Thursday, April 15, 2004 - 01:45 pm:

Sonal: the retention time of a compound will vary with the pH, if it is in the range of +2/-2 around the pK of the compound. You can either stay away from this range, or you can make sure that the pH is always adjusted accurately. If you do the latter, the retention can be as reproducible inside this range as outside this range. If an acetate buffer is fine for your application, there is nothing fundamentally wrong with using this buffer.

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By sonal on Thursday, April 15, 2004 - 09:35 pm:

Uwe Neue: We would definitely not be using the range of +2 to -2 rather only one pH ,either +2 or -2. I wanted to know which pH is preferred as the compound has to be in one state only either ionized or unionized. If we use -2 whole of the compound will be in the unionized state and if we use +2 it will be in the ionized state. Also I have no separations to be done as I have only one drug whose method I have to develop. My drug is an acidic drug.

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By Chris Pohl on Friday, April 16, 2004 - 08:11 am:

Sonal

As a general rule, it's usually preferred to operate with analytes that are in their neutral form when using a reversed phase column. This generally gives better retention, better peak shape and better reproducibility. The major exception to this would be basic solutes where the pH necessary to produce the neutral form of the analyte might result in significantly shorter column life, depending upon the basicity of the analyte. Of course, the other exception to this is in cases where you need to adjust selectivity in order to improve the resolution of two closely eluting analytes. Increasing the ionization of one of the poorly resolved components can be an effective tool in adjusting resolution.

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By Anonymous on Saturday, April 17, 2004 - 02:22 am:

how do i separate Phenoxy Ethanol and Methyl paraben on reverse phase column

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By sonal on Monday, April 19, 2004 - 01:06 am:

Thankyou Chris Pohl and Uwe Neue for your reply. regarding my second question, to prevent the lactone conversion of the compound is there any other method besides maintaining the ph to a specified value for eg by adding an antioxidant so that I can manipulate my ph to control the retention characteristics.

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By Chris Pohl on Monday, April 19, 2004 - 06:13 pm:

Sonal,

Depending upon the stability of the lactone in question, you may be able to convert your compound back to the free hydroxy acid by simply elevating the pH of your sample for a sufficient time prior to injection. Generally, the rate of formation of the lactone will be too slow to occur to a significant extent during the chromatographic separation. Of course, performing the separation at a pH significantly higher than the pKa the acid will help prevent formation of the lactone during chromatography.

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By sonal on Monday, April 19, 2004 - 10:32 pm:

Chris Pohl

In one of the references it is mentioned that the pH has to be maintained at around 4 and the pKa is around 4.5. According to what you have said that the lactone formation takes time then can I take the ph around 2.5 so that the compound remains in the unionized or shall I maintain the stated ph i.e around 4 only.
Also in the stock solution which I shall prepare everyday is there any chance of conversion to the lactone?
Can adding an antioxidant be of any use?

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By Chris Pohl on Tuesday, April 20, 2004 - 08:13 am:

Sonal,

Formation of the lactone is nothing more than formation of an internal ester. Depending upon the size of the ring formed during the lactone formation reaction, there can be considerable differences in the stability and rate of formation. Without knowing just how stable your specific lactone is, I can't give you a definitive answer. However, if you're chromatographic parameters are compatible with operating at higher pH (i.e. if you have sufficient retention and suitable selectivity at higher pH) then your best option would be to operate at a pH equal to or greater than the pKa since the lactone can only be formed when the hydroxy acid is in the nonionized form. Regarding your stock solution, your best option would be to adjust the pH such that there is essentially none of your compound present in the nonionized form (i.e. at least two pH units higher than your pKa). This should prevent formation of lactone in your stock solution. Finally, an antioxidant should have no effect on this reaction since lactone formation is esterification reaction not an oxidation reaction.

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By sonal on Wednesday, April 21, 2004 - 07:30 am:

Thankyou Chris Pohl for your reply. Actually I am new to this area and have not yet started with my work. So whenever I read something new I have queries in my mind.
The reference I mentioned in my earlier message says that the pH should be maintained at 4 to prevent the conversion of the compound to lactone and you are saying to maintain the ph above the pka of the compound.
Also is TLC necessary before proceeding for the method development by HPLC. What inference can we get from it? Can the solvents optimized with this method be extended to hplc. Can we use mixed solvents for tlc like mixture of buffer and the organic solvent or just the pure organic solvent needs to be tested?

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By Chris Pohl on Wednesday, April 21, 2004 - 08:03 am:

Sonal,

I can't imagine a good reason for recommending pH 4 in order to minimize lactone formation unless it's related to the optimal conditions for retention of your analyte. As I said previously, the higher the pH the better with regard to preventing lactone formation.

Regarding the use of TLC prior to HPLC method development, I would say that generally there is relatively little benefit to this approach. While I suppose it might be conceivable that one could screen a wide variety of mobile phases more efficiently using TLC, the fact is that conditions used for TLC are not exactly analogous to conditions used for HPLC since in TLC one does not normally move analyte bands all the way across the plate during development. Since in HPLC one must use mobile phases sufficient to carry the analyte bands through the column, the conditions which are optimal for TLC will not necessarily be directly relevant to optimal HPLC conditions. I would say that a better strategy would be to use a "generic" gradient from 5% acetonitrile to 95% acetonitrile with a suitable buffer to scan for appropriate eluent conditions for a given set of analytes. From there you can identify suitable conditions for isocratic operation, assuming isocratic conditions are feasible.

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By sonal on Thursday, April 29, 2004 - 07:59 am:

Thankyou Chris Pohl for the reply