fluorescein + PVP

[Robert Brightman]

I'm having problems developing an LC method for a mixture of fluorescein and its impurities/degradations and proxymetacaine+ degradations in presence of 10-15% Povidone (PVP). The polymer gives rise to an early eluting large tailing peak which interferes with the analysis of the fluorescein impurities ( phtalic acid and resorcinol). In addition the analysis is normally carried out at 220nm where interfernce is amplified. In isocratic conditions fluorescein is very lately eluted and the 2 impurities mentioned very early. I'm using a C8 counter ion with TEA and methanol/acetontrile as a mobile phase and an Uptispher ODB 5 micron 250*4.6mm column. Gradient elution gives rise to a PVP peak which elutes later and seems to be variable from one column to another. Does anyone have any suggestions for either eliminating the polymer or reducing its interference?
Any help would be appreciated

bob in Montpellier, France

[Mattias]

Just last week at the HPLC2005 conference, I presented a method to remove gelatine from samples before the analysis. I have tested it on samples containing polyvidone, and it works perfect for that application as well.

The method is a coupled column method where the sample is injected onto a SEC column. After the undesired matrix has eluted, the flow from the SEC is switched to a normal LC precolumn and the analytes are trapped. Then the flow is switched again and the analytes are eluted with a gradient to a analytical column and detected with a UV detector.

Two downsides with the approach: Each injection is prolonged c:a 25 minutes due to the SEC step. Also, you need two LC pumps. The beauty is that everything can be automated, and that you can dissolve and inject the samples without (almost) any sample prep.

If you are interested I can send you a PDF file of the poster.

BR/Mattias

[shirish]

Hello

Can you send a paper to me at shirishpatel1977@yahoo.com

refer my question of removal of plasidone-s-630. i am encountering simillar kind of probleam.

some people suggesting a filters . i am just in process of procuring those .

do you have any experiences with this kind of filters .

[Mattias]

I have never worked with the Plasidone polymer, so I have no idea if this approach will work. If the size difference between the polymer and the analyte is in the right range, it is not impossible.

Ultrafiltration never worked for my application, but that is an easier approach of course.

BR/Mattias

[HW Mueller]

Mattias,
it would be instructive to know why the ultrafiltration didn´t work,..... lack of the right cutoff filter for your media, polymer adhesion, polymer too small?
Incidentally, for those that want/have to do things automatically: Was/is it Gilson that has a device which allows ultrafiltration and LC in line?

[Yury Zelechonok]

To Robert
In response to your direct inquiry.
I tried to send you e-mail but it was rejected by your server.
There are two aspects in your question if I understand it correctly: PVP interference with polar components and conversion of gradient method to isocratic one. PVP is not charged, so if the analytes participate in ion-exchange interaction you may have a good chance to avoid PVP interference. You asked about recommended column.
It is difficult to make accurate prediction, but from previous experience I would suggest for your first mixture of fluorescein with phtalic acid and resorcinol to use Primesep D column with a buffer at pH 6 - 7 and high concentration of organic modifier 60-70%. In this condition the interaction in big part will be a result of acidic properties of all three molecules while the difference in hydrophobicity will be significantly eliminated by strong mobile phase allowing isocratic elution. Phosphate buffer 10-25 mM with pH 6.5 can be used for UV detection, or ammonium acetate salt 10-25 mM without pH adjustment for MS-detection. At this condition I don't expect PVP interference, but I can not guarantee it.
In case of fluorescein + lidocaine I would choose a Primesep 200 column with 5-30% MeCN and 10-25 mM buffer pH 2-3.5. In this system the lidocaine will retain by combination of ion-exchange/RP mechanism and fluorescein will be very much neutral and retain by hydrophobic mechanism only. In both cases very different compounds can be analysed by isocratic method with independently adjustable retention. The buffer system can be phosphoric acid, sulfuric acid or TFA for UV application or ammonium formate for MS. If you want us to develop a method, pls. send us a small sample of each compound and we will be glad to demonstrate the efficiency of mixed-mode technology in these situations.