Chromatography Forum

my chromatography condition is 20mMkH2PO4(PH=3):acetonitrile=96:4,which is 0-13min A:B=96:4；13-13.5 A-60,B-40;13.5-15.5 A：B=96:4,15.5-16 A-96,B-4;16-21 A：B=96:4.my column is C18,250*4.6,5.I have used 6 columns to analysis my sample and internal standard.my trouble is that the retention of my sample becomes longer and longer,while my internal standard stay stable.when the two meet,i have to change another cloumn.the cloumn cannot go back by flushing.my sample is Tazobactam,which have several N and one -COOH,the internal standard is Metronidazole.I use this condition to analysis serum sample using acetonitrile and inject just the water phase by N2. thanks for your reply.

Tazobactam is very basic compound. Please let us know which C18 column you are using, and why you have choosen pH3. Increase in retention time can be linked to the fact that more and more active silanol groups are on the surface.
Maybe a more basic pH and or a very well base deactivated column would give more stable retention times.

dear,guoguo,i have no idea also ....but, i 'll always support you!!

Tazobactam is very basic compound. Please let us know which C18 column you are using, and why you have choosen pH3. Increase in retention time can be linked to the fact that more and more active silanol groups are on the surface.
Maybe a more basic pH and or a very well base deactivated column would give more stable retention times.

Excuse me ,you have said that the compound is" very basic",however ,it has a -COOH??

I have used inertex c18,GL C18,herdra ods-3,and now thermo c18.i have no idea that why you said that more and more sianol will be active.why this happened?

inerstil ods-3 ,not gl c18

You are right Penny, there is a -COOH, but also N=N and it is difficult to solve in Water.
Most published HPLC methods are using an ion-pair reagent and pH6,5.
If retention time shifts many parameters can cause that problem. If retention time increases the compount is longer retarded, what can happen when the column is used for a long time.

Well

First, the molecule has a bunch of Nitrogene atoms apart from the -COOH branch. So silanols may be a factor.

Secondly although a bit old experiance from similar compunds. Usually you are stuck with a pH around 3-5 due to stability reasons. Also the "classic" mobile phase usually contained a counter ion such as a hexylammonium or tetrabutylammonoin salt when using traditional C18.
The mobile phase in general apart from the counter ion seems to be in line with published and pharmacopiea methods on the same/related compunds.

Perhaps a simple google search can help you with mobilephase composition?!

Rangtaiyang,

Do you see peak shape changes – especially tailing? Try and calculate the tailing of the peak from the beginning (on the new column) and later on. Representative chromatograms would be helpful here.

Guys,
Free silanol following few injections on a modern column and at pH 3 ???????

Best Regards

It is interesting.

if it were me, I'd check:
1) check the LC system to make sure it works well;
2) check the sample prepare to ensure no garbage was retained by the columns;

A Q, why you have three sections of 96 A and 4 B in the end of the gradient? What are you trying to do?

Gerhard,

I could not find pka for this but found aminoazobenzene has pka 2.82. Do you have good literature showing it is very basic?

At physiological pH, tazobactam is a highly hydrophilic drug.
The pKa is 2.1; i.e., at pH 7.4, the drug is almost completely
ionized (15). The pKa of piperacillin is 4.14. The degree of
ionization at pH 7.4, therefore, is slightly lower. Since on re-
versed-phase HPLC columns its retention time is longer than
that of tazobactam, piperacillin appears to be somewhat less
hydrophilic than tazobactam.
After deproteinization with acetonitrile, drug concentrations in serum and
CSF were measured by high-performance liquid chromatography (HPLC). Piperacillin was separated by using a potassium dihydrogen phosphate-acetonitrile mobile phase and a LiChrosphere-C18,5-mm column and detected at 220 nm.
Tazobactam was separated on a dual-column HPLC system (precolumn, RP 2, 10 mm; analytical column, Spherisorb ODS II, 5 mm; mobile phase, 100 mM sodium dihydrogen phosphate, 5 mM tetrabutylammonium hydrogen sulfate, 5% (pre-column) or 10% (analytical column) acetonitrile, pH 6.5) and detected at 210
nm. The serum and CSF samples were measured against a calibration series
That was published from:
Kinetics of Piperacillin and Tazobactam in Ventricular
Cerebrospinal Fluid of Hydrocephalic Patients†
R. NAU,1* M. KINZIG-SCHIPPERS,2 F. SO ¨ RGEL,2 S. SCHINSCHKE,1 R. RO ¨ SSING,1 C. MU ¨ LLER,2 H. KOLENDA,3 AND H. W. PRANGE1
Departments of Neurology1 and Neurosurgery,3 University of Go¨ttingen, D-37075 Go¨ttingen, and Institute of Biomedical and Pharmaceutical Research,2 D-90562 Nu¨rnberg-Heroldsberg, Germany
Received 14 June 1996/Returned for modification 11 December 1996/Accepted 19 February 1997

Dear Gerhard,

Thanks for the literature.

If I understand it correctly, pka=2.1 it is a mild acid(it is an acid stronger than acetic acid). I still did not get how it is "very basic".

Sorry guys: the only relevant pKa of the tazobactam is that of the carboxylic acid. The triazole ring on the side is such a weak base that it is irrelevant: its pKa is 1.17. With other words, the triazole does not even think of becoming protonated until a pH lower than 2.1 or so.

The rest of the molecule is completely innocuous.

If Uwe is right, pH should be adjusted to pH2.0. I had similar compounds and everybody told me what Uwe said.
It would be helpful to get some more information about Retentiontime shift, for example what is the RT with a new column and what is the RT after X injections. Another important question: what`s about the tailing of the peak, what is the asymmetry factor and is it increasing when RT is increasing?

the peak will become more asymmetry after 48hrs running while RT is increasing.