Methode Development

[vienna]

hello,
I'm trying to develop new methode for determination of Folic acid and Cyanocobalamin by simultaneous mesurement. Im Using the Paired ion Chromatography (tetrabutylammonium phasphate and Heptane sulfonic Acid) for mobile phase, and C18 Column. But the peak didnt separate well. and the retention time of that two compound are still the same, which is 1.4 min.
What can I do now? And what paired ion Chromathography that I should use? Can somebody tell me?

[Gerhard Kratz]

Please try the following chromatographic conditions:
Mobile Phase A) 10mM Ammonium formate (pH3,5)
B) Acetonitrile/10mM Ammonium formate(pH3,5) = 30/70

Time(min) B%
0 0
15 100
Flow rate: 1,0ml/min
Column temperature 40°C
UV 254nm
Injection volume 5µl
RP18 column endcapped 4,6mmID x 150mm L, new generation

Folic acid should have RT around 10,8 min, and Cyanocobalamine around 12,3min.

Good luck

[Chandresh K. Soni]

Dear Vienna,
please try on Methanol as org. Modifier or the Mixture of Acetonitrile and MEthanol with gerhard suggested mobile phase with extended run time up to 25 minute. it will also give you more separation in this method

[bisnettrj2]

http://www.chem.agilent.com/Library/app ... 682971.pdf

http://www.chem.agilent.com/Library/app ... 9313EN.pdf

Check these two references. No paired-ion chromatography, simple reversed-phase with gradient separations.

[vienna]

Please try the following chromatographic conditions:
Mobile Phase A) 10mM Ammonium formate (pH3,5)
B) Acetonitrile/10mM Ammonium formate(pH3,5) = 30/70

Time(min) B%
0 0
15 100
Flow rate: 1,0ml/min
Column temperature 40°C
UV 254nm
Injection volume 5µl
RP18 column endcapped 4,6mmID x 150mm L, new generation

Folic acid should have RT around 10,8 min, and Cyanocobalamine around 12,3min.

Good luck

Dear gerhard

Are you sure that I dont have to use the paired ion chromatography for mobile phase?
Because I've try your methode but not much change. Only RT move from 1.4 min to 2.5 min, and the peak shape is not too good.
what happened?
I've try to change the collumn length to 250mm but RT of those two compound still almost the same. The resolution of two peak only 1.1.

what should I do now?

Vienna

[Gerhard Kratz]

Dear Vienna.
If you used the same column, that is not a surprise. Your column is modificated by the ion-pair reagent and shows now a different selectivity. If you have a new column available you should get better peak shape and better resolution. You cannot clean your actual column from the ion-pair reagent. Please try it with a column that has never seen any ion-pair reagent.

[novus]

Did RP18 Column was same as C18 Column?

[vienna]

Dear Vienna.
If you used the same column, that is not a surprise. Your column is modificated by the ion-pair reagent and shows now a different selectivity. If you have a new column available you should get better peak shape and better resolution. You cannot clean your actual column from the ion-pair reagent. Please try it with a column that has never seen any ion-pair reagent.

Ok, thanks. I'll try to use another collumn.
But, as you say that we cannot clean collumn that modificated by the ion pair reagent. why?
can I just flush it with water? Because the ion pair reagent that I use is water soluble.

[vienna]

Did RP18 Column was same as C18 Column?

Yes it's the same. RP mean Reverse Phase

[Gerhard Kratz]

Dear Vienna.

You can try to clean your column, but the Heptane sulfonic acid is bonded to the surface and it is not possible to remove it 100%. Even if you inject 50°C warm water. Please use the ion pair column only for methods which use ion pair reagent, don't use it for other method developement work. I did this mistake once and developed a new method on a column were I used heptane sulfonic acid before. After the method was developed I bought a new column to do the validation work. Chromatogram was totally different and I had to start again.

[lmh]

I think there may be a misunderstanding about washing columns here. Although I'd agree it's almost impossible to get an ion-pair reagent off a C18 column once it's been used, water is usually not the best way to wash it off anyway. The ion pair reagent should (mostly) interact hydrophobically with the column, and will be washed off more effectively with the usual strong eluants.

[vienna]

ok thanks to all

[maimun.hossain]

Please try the following chromatographic conditions:
Mobile Phase A) 10mM Ammonium formate (pH3,5)
B) Acetonitrile/10mM Ammonium formate(pH3,5) = 30/70

Time(min) B%
0 0
15 100
Flow rate: 1,0ml/min
Column temperature 40°C
UV 254nm
Injection volume 5µl
RP18 column endcapped 4,6mmID x 150mm L, new generation

Folic acid should have RT around 10,8 min, and Cyanocobalamine around 12,3min.

Good luck

Is it possible to analysis at 0.0006 mg/mL conc. level?????