Chromatography Forum

Hello everyone,
I have to find a method to analyze Sodium Ascorbyl Phosphate with HPLC.
HPLC is an absolutly new thing for me. I tried it with a C18-column ( 150 mm ) and methanol/water ( 90:10 and 10:90 ). The retentiontime was too low and the peak tailed. Then I found out, that the mobile phase must have a low pH to get the neutral form of the substance. I took TFA and got a pH ~ 2,5. The retention time was too low ( with the void volume ) and the peak tailed. I took a 250 mm column ( C18 ) and tried it with ACN/water with TFA ( pH ~ 2,5 ). No change. I read a lot of books and ask me, if I have the wrong acid or if I should try it with an ion-pair-reagent. My problem is, that I have very less time to find the method ( 2 weeks ). Can anybody help me? It`s urgent!

A typical C18 column probably will not work very well.
I use the following system (the MP is acidified water only) for Citric acid (a

Column: YMC ODS-AQ S5µ (4.6mm x 250mm)
Detector: 210nm UV
Inj Vol: 25µL
Flow: 1.0 mL/min.
Mobile Phase: Adjust the pH of Water to 2.0 with 85% Phosphoric Acid. Mix well and filter

Retention Time of Citric Acid ~10 minutes w

Total Run Time 20 minutes

I hope that this helps

You can use the method for "dronic" acids. You molecule is very polar and you need to use ion-exchange properties of it:

http://www.sielc.com/application_138.html

Regards,

Vlad

I've assayed magnesium ascorbyl phosphate (MAP) successfully using an Inertsil C8 (5-micron / 100A) and a 0.1% aqueous phosphoric acid mobile phase (pH 2.2 or thereabout). MAP elutes at ca 6 min at 1 mL/min on a 250x4.6 column with acceptable peak shape. It'll separate from ascorbic acid as well as aminopropyl ascorbyl phosphate. The sodium salt should act similarly, I'd predict. Drop me a line if you have any questions or would like to see chromatogram.

Thank you for your tips. I found a method wich works. I took a mobile phase with KH2PO4 and tetrabutylammoniumhydroxide in water with 10% methanole and brought it to pH 2,5 with o-phosphoric acid. On a C18 (150 mm) the peak will come after 7 min. ( Flow 1ml/min ).