Quinine by HPLC-UV.

[WK]

I have very poor peak shape for this analyte at low pH and C18BDS type column - obviously.
I have had a good look at literature and the general opinion is high pH and zirconia or GC-MS with SPE clean up.
Does anyone know a better way - my samples are soft drinks?
Any comments would be appreciated.

[Victor]

This analysis will not work on any type of BDS C18 column. However, you should get good results using one of the following columns and acetonitrile-phosphate buffer ( pH 2.5-3.0)

Symmetry C18 (Waters)
Discovery C18 (Supelco/Sigma)
Luna C18 2 (Phenomenex)
Inertsil ODS-3
(GL Science)

I wouldn't do it by GC because it's more difficult (though not impossible!) to inject aqueous samples.

[SIELC_Tech]

WK,

What are you trying to achieve-retention of quinine (quinidine) or separation of it from your other products before analysis. If you provide us with your email I will send you a method for quinine with peak symmetry of 0.97-1.03.
my email is : mail@sielc.com

[Nalizer]

Are you analizing pure quinine? I am observing two peaks on my chromatograms (besides compounds in my reaction mixture) and peaks are not pretty, symmetry is over 2 (huge tailing). I don't care too much about peak shape because I am monitoring my conversion, but wondering if one of the peaks is my side product.

TIA,
Mark

[WK]

Thanks for your posts - try looking at this:

http://www.sigmaaldrich.com/Brands/Supe ... &id=930543

For quinine with dipotassium hydrogen orthophosphate

[Victor]

This is one of the columns, and the same conditions as I recommended on 28 September. But this analysis will not work on every C18 column, even if it is claimed as a very inert Type B by its manufacturer.

[WK]

Thanks again Victor:
I had found the application in the Supelco catalogue where it refers to Potassium Phosphate 25mM - so I initially assumed KH2PO4. It didn't work.
I have recently discovered later in the catalogue under applications that it means K2HPO4 so I will try this now.
You're post on 28th September only referred to phosphate buffer too. Until a recent extremely large posting on buffer preparation I hadn't come across K2HPO4.
This is all very interesting to me! I guess a lot of basic compounds can be done this way. I had been trying mobile phase additives and all sorts...

I have 2 types of Amide column here (Discovery & Zorbax) so I will try it on both.

[Victor]

No WK, I think you are confused.

If you make up a 25mM solution of KH2PO4 it will have a pH of around 4.5. It is not a buffer at this pH. To get a pH 3 buffer you must titrate it with phosphoric acid. If you read the buffer prep correspondence, you will realise how many different ways there are of doing this. If you start with 25mM KH2PO4 and titrate with a fairly strong H3PO4 solution such that you only need a small volume, you will get a buffer with 25mM [K+] but a larger concentration of phosphate.

You can start with K2HPO4 if you like but I recommend you adjust it to pH3 with H3PO4. As long as you do this adjustment the same way each time, it's not really important which initial salt or means of adjustment you use.

[WK]

Sorry Victor,
I should have said I am adjusting the pH to pH3.0 in all cases.
I tried this with KH2PO4 and its no good.
The crux is that K2HPO4 should work - I am testing this now.

[SIELC_Tech]

WK,

If at some point you decide that pH adjustment is too much, check this out : quinine and quinidine analized with AC?/water?sulfuric acid with perfect symmetry. With more efforts you can even separate dyhydro impurities in quinine and quinidine

http://allsep.com/makeChr.php?chr=Chr_073

[WK]

Hello Victor,
I have now settled with 25mM K2HPO4 (to pH3.0 with phosphoric acid) and acetonitrile. I have investigated the range 15 to 30% acetonitrile and 15% is good. This is with a Discovery RP-Amide 150mm x 4.6mm x 5um.

Thanks for everybody who helped - this is a very useful method.
I wonder why K2HPO4 is so different to KH2PO4, since its only function is to buffer at ph3.0.

[tom jupille]

Your actual phosphate concentration is now much higher than 25 mM.

[HW Mueller]

If there is a difference in the buffer due to using K2HPO4 or KH2PO4 you used the wrong technique.

[Victor]

Tom is right-the phosphate concentration is higher if you adjust it this way.
However, the analysis on the column you are using is not very sensitive at all to phosphate concentration (or to K+ concentration for that matter). It should work therefore whichever you start with KH2PO4 or K2HPO4.
I suspect some other glitch in your procedure but I am glad you have overcome it and all is working ok

[WK]

Thanks for the information.
I will try to repeat with KH2PO4 to see if its some sort of glitch - I'm having fun with buffer crystallization right now. I need to develop a robust procedure for changing between 100% acetonitrile to K2HPO4/acetonitrile and back etc. The detector cell gets the full force of the changeover at the moment - even with water as an intermediary.
I think K2HPO4 gives more crystal problems than KH2PO4.
WK