Chromatography Forum

Hi Guys,

I am trying to develop method for Thimerosol Assay on HPLC or UPLC. I did some litreture serach but i found methods with only atomic absorbance. If any one have any idea for column or mobile phase to start with, please i need your help.

Thanks

Thimerosal (Thimerosol) chemical structure and octanol-water partition coefficient:



Considering this info I would try HILIC with 70-80% ACN and 30-20% water and low UV detection.
Others with more information can share their point of view

Regards

I searched the WEB a little bit and find these related articles:

1.Simultaneous determination of methylparaben, propylparaben and thimerosal by high-performance liquid chromatography and electrochemical detection
Journal of Pharmaceutical and Biomedical Analysis, Volume 15, Issues 9-10, June 1997, Pages 1359-1364
Seong Ho Kang, Hasuck Kim

-in this article authors used C18 column, mobile phase= methanol : 0.02 M aqueous phosphoric acid (59:41, v/v) and electrochemical detection

2.HPLC analysis of thimerosal and its degradation products in ophthalmic solutions with electrochemical detection
Talanta, 1992, vol. 39, no.12, pp. 1619-1623
PROCOPIO J. R. ; DA SILVA M. P. ; DEL CARMEN ASENSIO M. ; SEVILLA M. T. ; HERNANDEZ L


Hope this helps.

A HILIC cloumn may do the separation work. However, due to relatively weak Hg bond, you may not able to see the molecular ion through ESI, that's why ICP-MS is a better detector for Thimersoal than MS

Thanks guys..i would like to go with UV detector...i will try HILIC columns or C18 columns with low UV detection. I will scan at 200-400 and will figure out maxima. I welcome you guys with more suggestions..

Thanks

Hi,

Found a paper (doi:10.1016/S0021-9673(00)01073-6) using a Hypersil C column (210 X 4.6 mm I.D., 5 um).

UV detection at 226 nm was performed. The mobile phase was methanol–water–orthophosphoric acid (66:35:0.9, v/v) of pH 2.5. The flow-rate was fixed at 0.6 ml/min.

Thanks,

did they mention run time with hypersil column and any column temp?

you should be able to retain this on any C18 column if you go to a lower pH (phosphoric or TFA). If you remove charge from the acid (by going to lower pH and suprressing ionization of analyte) LogP is going to be much higher. This should retain similar to benzoic acid...and UV is not a problem you can do 215 or 230 due to the presensce of aromatic ring, carbonyl and sulfur

Thnaks guys for your input. I tried with C18 column and mobile phase was methanol: water: phosphoric acid (50:50:0.1). Peak was detected at higher concentration(0.05 mg/mL) but shows too much fronting. Thimerasol in my sample is so low that i am not able to detect properly at sample level concetration. (0.01 mg/mL). Is there anyway to fix this problem? My sample is in methanol: water (50:50). What can i do for fronting problem?

Thanks in advance for your help.

Dissolve the sample in mobile phase (i.e. with the phosporic acid), and let us know if you still have fronting, or if some other strange things are happening.

I tried with sample dissolved in mobile phase, but still it shows fronting. Again this is summury what i did.

Mobile Phase is Water: methanol: Phosphoric Acid (50:50:0.1)
Wavelength: 222 nm
Column C18

I tried on UPLC too with C18 column. I got good absorbance on UPLC but here also same problem of fronting. I almost there but i am not solve the problem of fronting. Pls help...

Unfortunately, I know nothing about the hydrolytic stability of such a compound. However, fronting that does not go away with proper handling of the chromatography could be related to compound stability. With my suggestion to add phosphoric acid to your sample I had two things in mind: if the fronting is caused by incorrect sample solvent, and/or departure in pH, then the addition of the acid should make the fronting problem go away. This was not the case. Next, the presence of the phosphoric acid could cause a deterioration of your sample. You should be able to see such a thing with a few injections; if you get a declining peak area of your acidified sample with time, this would indicate instability of the analyte under acidic conditions.