Chromatography Forum

That looks like it's eluting in the solvent front. Are you sure about that mobile phase A composition, 95% ACN seems very unlikely.

@ yes i prepared my mobile phase usi

That looks like it's eluting in the solvent front. Are you sure about that mobile phase A composition, 95% ACN seems very unlikely.

yes, i prepared my mobile phase A using 950 ml acn in 1000 ml dilute it to 1000 ml with water.,

do you think me acn concentration is a little less or more than 95% that's why my peak is like this?

No, I think that the mobile phase A should not be 95% ACN, it is far more likely to be 5% ACN as in your original pharmacopaeal method. Generally speaking BP methods and EP methods tend to be quite similar if not the same, so I suspect you have a communication issue and that the A solvent should still be 5% ACN.

Also, it is very strange for the A solvent to be stronger than the B solvent. A gradient is generally used to increase solvent strength, not decrease it so going from 95% ACN to 20% ACN sounds like it's wrong.

oh i see, i understand it wrongly

i will check my reference again., that's what i think before i run my standards using 95% ACN, but im losing my patience with my previous run before using 5%as mobile phase a.,

i will try again using 5% ACN as MPA,

thanks..

I'd also suggest that you change the flow rate back to 1ml/min for your initial work. Once you have your peaks properly separated and identified, then start optimising for speed.

I also think your friend was mistaken about the A mobile phase - 95% ACN as the starting mobile phase is not correct for reversed-phase HPLC.

Try this (I used Thermo's HPLC Method Translator at http://www.hplctransfer.com/GradientMethod.aspx, and assumed a system volume of 1.2mL):

MPA: 95% H2O, 5% ACN
MPB: 80% H2O, 20% ACN

Time MPA MPB
0.00 100 0
6.00 100 0
6.13 0 100
40.00 0 100
40.13 100 0
53.33 100 0

1.74 mL/min
Injection volume: 50uL

OR, if you want to use 1mL/min:

Time MPA MPB
0.00 100 0
10.50 100 0
10.67 0 100
69.56 0 100
69.79 100 0
92.75 100 0

thanks for the suggestion i will try again when i got back to work

do you think i should wash the column with 100% ACN first before running my gradient? would this help me eliminate those unwanted peak?

thanks

If you can't deviate from the BP, then I wouldn't. And if you're only running tablets, you shouldn't need to, unless there's something in your mobile phase water or the tablets that will build up on the column and not be washed off with 20% ACN, as per your method.

That being said, most reversed-phase HPLC column manufacturers recommend storing columns in 100% acetonitrile, so maybe you can do a wash of 100% ACN after your run, then start with a blank when you need to run the method again? It all depends on what the BP says you can do. I don't work in the pharmaceutical arena, so hopefully someone here can give you generic advice on what you can and can't do.

@ bisnettrj2:

i will try this one.

thank you for all the suggestions,

i will keep on trying until i got the correct one..

thanks so much guys

i will let you know if i got the right one

As for your unknown peak, my guess is it will still be there. Which leads me back to my earlier question - how pure is your carbimazole? How do you know it is pure (was it supplied with a certificate of analysis with a guaranteed purity and a chromatogram)?

You should run a couple of blanks before you run anything else to equilibrate your column.

As for your unknown peak, my guess is it will still be there. Which leads me back to my earlier question - how pure is your carbimazole? How do you know it is pure (was it supplied with a certificate of analysis with a guaranteed purity and a chromatogram)?

our reference standard is only a secondary standard not the one that comes from BP or USP. it has the certificate of analysis coming from the supplier but i haven't seen their chromatogram.

do you think my standard is not that pure?

all i know is that if my standard have the thiamazole peak then thats the time i can say that my standard already degraded.. because the carbimazole has been converted to thiamazole.,

>but i think it is possible that my standard in not that 100% pure.

maybe i have to request another batch of standard for confirmation

> but how come they have the same unknown peak with the thiamazole standard? do they have the same impurity?

thanks

You should run a couple of blanks before you run anything else to equilibrate your column.

> i have run blanks in my previous run, but the baseline is not that pretty, it looks like there is a lot of actives that is present in my chromatogram..

Let's see a re-run of your individual stock standards, dissolved in 95:5 H2O:ACN, with this method, before we go there. However, we've seen at least one chromatogram that shows that the peak is not in a blank run, so it leads me to believe that it is either in your standard, or it is a contamination from the glassware you're using to prepare the standards, or a contamination introduced by a filter (if you are filtering your samples, that is).

i will do the assay again on monday and post again the results

>i will make sure my glasswares are clean, but with the filters im using a new 0.22 um filter each standard separately.