Gradient Help---ASAP

[LC_Trouble]

Hi Everyone,

When i run my samples some of the peaks are co eluting and i tried using the gradient but i coudn't see a single peak.what would be the cause of this problem.

Thanks.

[tom jupille]

Come on, we're not mind readers!

What are you trying to analyze?
What instrument are you using ?
What column (chemistry and dimensions)?
What kind of detector?
What mobile phase (composition, pH, flow rate)?
How much are you injecting?
Is this a method which you have run successfully in the past?
Or is it a published method?
Or are you developing this ab initio?

[Peggsy]

I totally agree with Tom, which most likely explains the lack of replies.

BTW, is there a column connected........

[LC_Trouble]

Sorry for not giving the details;

1. Mobile Phase-100% Meoh+1% Formic acid
2.i m just running the standards,in that there are 6 co eluting peaks at diffrent mins for eg at 45mins(2 peaks),65mins(4 peaks).
3.reverse phase HPLC-
4.intially i didnt run with gradient,but once i found there are coeluting peaks i tried using the gradient.unfortunately no peaks turned out!
5.can you please give me an eg of how is a gradient normally used.

Thanks.

[JA]

We need to repeat the questions:

What are you trying to analyze?
?
What instrument are you using ?
?
What column (chemistry and dimensions)?
?
What kind of detector?
?
What mobile phase (composition, pH, flow rate)?
100% Meoh+1% Formic acid
How much are you injecting?
?
Is this a method which you have run successfully in the past?
?
Or is it a published method?
?
Or are you developing this ab initio?
?

With respect to the first question, you don't have to give away any proprietary details. The class of compound or functionalities can sometimes be enough. Is there a sample matrix involved (biological?) or is the sample a simple synthetic product. Given the mobile phase you're using and the retention times observed I'd say it's pretty important to also know the column type (be specific) and perhaps it's history of use (see question 7).

[LC_Trouble]

We need to repeat the questions:

What are you trying to analyze?
i am a learner,not analyzing anything particular.when i ran some 8 std's together at 40ppm i found only 2 peaks.but during my individual run i see all the peaks.
What instrument are you using ?
agilent 1100 series
What column (chemistry and dimensions)?
i dont rember
What kind of detector?
DAD
What mobile phase (composition, pH, flow rate)?
100% Meoh+1% Formic acid
How much are you injecting?
0.2 micro lt /ml
Is this a method which you have run successfully in the past?
no
Or is it a published method?
no..i am learning
Or are you developing this ab initio?
no


thanks for responding me. i just wanted to know how to separate the coeluting peaks using the gradient system.

thanks

[skunked_once]

Just a wild guess but I think that your compounds are all eluting at or shortly after the column void volume. If you are using a reverse-phase column with 100% methanol, almost everything is going to elute right away and you won't get any separation. You need to learn more about HPLC operation using the information avaiolable here and elsewhere. The important parameters to learn about are type of column, mobile phases, and principles of gradient separation. Operation of an HPLC system is too complex to explain in one post. We admire your desire to learn so do some reading and don't be afraid to post again with more detailed questions.

[Kostas Petritis]

In gradient elution you should start with solvents of low elution strength and going to higher elution strength during your gradient.

So if you see all your peaks eluted with 100% methanol, I would suggest that you start with 100% water 0.1% formic acid or TFA and then go up to 100% methanol with the same amount of acid added into it in 20 min. According the results you get out of it, you can fine tune your method accordingly...

[JA]

Hi,
Appreciated that you are new to chromatography; we all were once, as they say, however there are some things you really can't miss: The column manufacturer and type (pretty much everything is labelled these days), the identity or chemical name of your "std's". Without these, people may have to start taking wilder guesses at the problem and in some cases people won't reply at all. Especially when we are considering things which (on face value) appear to go against the normal lines of thought: compounds eluting at 45 and 65 minutes with 100% methanol mobile phase.

We have ascertained that your "std's" are at a concentration of 40 ppm, or ~ 40 ug/ml and you're injecting 0.2 ul. Depending on the nature of the compounds, the DAD range chosen and possibly the presence of secondary interactions, I don't feel it's outside the realms of possibility that some of your "std's" are below the limits of detection in a mixture.

Presumably from the separate injection of the 8 "std's" you were able to make a note of 8 distinct retention times and magnitude of response from each?

[danko]

Hi LC_Trouble,

I can imagine you had trouble running gradient. I’m just curious: What was the other eluent (besides the 99% methanol + 1% formic acid)?
If you insist on Reversed Phase separation, you might be better of with the conditions Kostas suggested previously (I’m sure he meant 99.9 % water and 0.1% formic acid as the weak eluent and 99.9 methanol plus 0.1% formic acid as the strong one)


Best Regards