Chromatography Forum

I have a Chiral AGP column 150X4mm. Void time is 1.6 when 0.8ml/min.
The longest retention I got for Levetiracetom (C8H14N2O2 with two amide groups) is 2.7min.
I have one or combinations of the following conditions: pure water, KH2PO4 from 1mM, 10, 20 mM , 100mM, at ph 2.5, 4.5, 7, 9.5; with or without octanesulfonic acid of 5mM, 10 mM and 100 mM with 0%, 2%, 10%, and 20% of ACN, MeOH or IPA.
Do you have any suggestions?
OR how much you do not like the idea that I am going to live with retention of 2.7 min.

What conditions gave you the 2.7 minutes? and can you move further in that direction?

It looks to me like you've pretty much exhausted the all the possibilities except temperature (sub ambient?).

Tom,
Thank you for asking. It was pH7 at 100mM KH2PO4.
Unfortunately, there was interference from the Blank/diluent. To get a good method I may have to try other column.
By the way, why a C18 column can hold my API well but this chiral column failed to? Is it supposed to be that the chiral column is more hydrophilic than the C18 and should be able to hold my hydrophilic API better than C18? Where is my mistake?

Levetiracetam


Piracetam

I have a method for piracetam using pH 7.0 0.025M phosphate buffer - methanol (95:5). The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 0.5 mL per minute. Oven is set on 25C. Using this method I could separate the degradation product: 2-oxo-pyrrolidine acetic acid.

syx, thank you for your input though I am trying separate the R and S-isomer(Levetiracetam). Do you have a method for this chiral separation?

Ups... sorry. I do not think that my method could separate the isomers.
Perhaps you should try other methods, chiral or maybe normal phase chromatography.

By the way, why a C18 column can hold my API well but this chiral column failed to? Is it supposed to be that the chiral column is more hydrophilic than the C18 and should be able to hold my hydrophilic API better than C18? Where is my mistake?

I'm sure others nore knowledgeable than I will provide better information, but my understanding of the AGP column is that pH will be main determinant of retention, because the iso-electric point of the AGP is about 2.7. Because the ionic nature of the AGP is likely to be most significant factor, as the pH increases, the negative charge increases, and the retention of amines etc. also increases. C18 columns use different parameters to work their separation magic.

With your sample and column, I suspect you've tried the best permutations, however I would contact the column manuafcturer to ask for suggestions. Chiral columns are very diverse, and retention behaviour can be difficult to predict. I've found the technical support from most manufacturers ( Daicel excepted ) to be very helpful.

There is at least one chiral method listed in Pubmed using a different chiral column ( normal phase ). I'd be concerned that your retention time is currently too short to separate the enantiomers.

Please keep having fun,

Bruce Hamilton

By the way, why a C18 column can hold my API well but this chiral column failed to? Is it supposed to be that the chiral column is more hydrophilic than the C18 and should be able to hold my hydrophilic API better than C18? Where is my mistake?

I don't think you made a mistake (from what you have explored so far, quite the opposite).

I actually don't know whether the "bulk" retention mechanism for the AGP column (as opposed to it's enantioselectivity) is closer to reversed-phase or to normal-phase. The answer lies in what happens to retention as you increase the organic solvent percentage; if retention increases, it behaves like normal-phase; if retention decreases, it behaves like reversed-phase.

In any case, the surface of a protein is much more complex and convoluted than a C18 silica. The good news is that there are many different active sites with which analytes can interact. The bad news is that there are relatively few of any one kind of active site.

The protein columns tend to be generally useful for chiral separations because the geometry of the active sites can to some extent be modified via mobile phase changes (forcing subtle conformational changes in the protein). However, if you can't get retention, then you need to look at a different column. If this were my problem, I'd follow Bruce Hamilton's advice and check with the column manufacturers for suggestions.

Bruce and Tom,
Thank you for your detailed explanation. But I do not get this yet:

as the pH increases, the negative charge increases, and the retention of amines etc. also increases.

If the stationary is negtively charged and the analyte is negtively charged amine at high pH, the analyte should be pushed away by static electric pushing, right?
I will contact the manufacture again. Thanks again,

Hi,

Try this :
Enantiomeric separation on a chiralpak AD-H column using a mobile phase consisting of hexane and isopropanol in the ratio (90:10, v/v) at a flow rate of 1.0 ml/min. The resolution between the enantiomers was found to be not less than 7 in the optimized method. Interestingly, unwanted enantiomer, namely R-alpha-ethyl-2-oxo-pyrrolidine acetamide ((R)-enantiomer), was eluted prior to its mirror image.

Will work for you.

JM

JM, Thank you so much for the detailed method.

But I do not get this yet:

as the pH increases, the negative charge increases, and the retention of amines etc. also increases.

If the stationary is negtively charged and the analyte is negtively charged amine at high pH, the analyte should be pushed away by static electric pushing, right?

A quick search of the Internet found:-
http://www.chromtech.com/ct-ab/Bulletin/2002/pH.pdf
which confirmed the pKa as 2.7, and the effect of pH on retention.

" When a hydrophobic amine (in this example propranolol)
is chromatographed at pH 7, the retention is very
high (k´ almost 50). The reason is that AGP has a strong
negative charge and the amine is positively charged,
leading to strong attraction. If pH is decreased to 4,
retention is much shorter, due to the decrease in negative
charge of AGP. "

At the same site, there is also a very impressive pdf file called "Chromtech Chiral Users Guide " that has plenty of information on the effect of pH on AGP method development, and applications as well.
http://www.chromtech.com/chir01.htm

With regard to the normal phase method with Chiralpak AD-H provided above, which was extracted, without attribution, from the abstract, the Pubmed citation is below.

A validated chiral LC method for the enantioselective analysis of Levetiracetam and its enantiomer R-alpha-ethyl-2-oxo-pyrrolidine acetamide on amylose-based stationary phase.
Rao BM, Ravi R, Shyam Sundar Reddy B, Sivakumar S, Gopi Chand I, Praveen Kumar K, Acharyulu PV, Om Reddy G, Srinivasu MK.
J Pharm Biomed Anal. 2004 Sep 3;35(5):1017-26

Bruce Hamilton

Bruce,
you are not only intelligent but also working diligently. Thank you so much for the informations, which is more than a clarification for my question. Have nice weekend,

Hi, does any one here know the order information for the Chiralpak AD-H column? Google indicates it is a UK company. But I did not find the telephone number, fax, address, or email yet. Please let me know if you have those handy.

I already find the 1800 number, Thanks.