RPHPLC/separation of small peak from large peak

[rajeev]

Hello to all,

I am encountering the peak separation problem in my HPLC experiment. I have provided the link to view the chromatogram .

Experimental conditions:
Column - 25 cm X 4.6mm, 5u particle size
Solvent A - 100% water with 0.05% HFBA
Solvent B - 60% acetonitrile +40% water with 0.05% HFBA

Elution condition- Binary gradient
5min-0% solvent B
6min- 10% solvent B
30min - 22% solvent B
31min - 100% solvent B
41min - 100% solvent B
42 min - 0% solvent B (100% solvent A)
52 min - 0% solvent B (100% solvent A)

(I hope this kind of gradient elution is correct for this column)

As you see the chromatogram, the separation is not good between large peak with 8.9min retention time and small peak with 10.08 min ( .
I am interested to quantify these peaks including peaks at 17min and 19 min . Where as the peak at 14.9 min is ref.std

In an recent experiment i tried with increased con. of HFBA (0.1%) in solvent B but with no success (if it is required i can also attach the chromatogram).

Is there any one got better idea to trouble shoot this problem , I will appreciate the future comments.

Thanks
Rajeev

[philippem]

Hi Rajeev

You could extend the time for 100 % A , i nitial condition f.i.

7 min 0% solvent B
8 min 10 % solvent B
32 min 22 % solvent B
33 min 100 % solvent B

probably you will increase retention time for first 2 peaks peak , but be aware that the smaller peak, now eluting at 10.08 min, may "disappear" in the noise of your baseline.

by the way your internal standard has a great peakarea in comparisment to the other peaks to be quantified ! your calibration line is OK ?

regards

philippe

[rajeev]

Hi Philippem,

Thanks for your suggestion.

The peak from reference standard is in the limit, I zoomed this chromatogram to show the small peak.

In the calibration experiment, i tried from 2nm to 10 nm range and it was linear up to 10nm. But, in the next experiment i will decrease the reference standard con. to 2.5 nm. is this make sense?

Rajeev

[tom jupille]

Assuming reasonable retention, there are only two ways to improve resolution between a pair of peaks:
- more plates
- better selectivity

For more plates:
- higher temperature
- smaller particle size
- longer column
- less tailing

For better selectivity:
- different gradient steepness
- different column
- different additive concentration
- different solvent
- different temperature

Out of these possibilities, the longer column and/or smaller particle size would be the easiest to implement in the sense of having th least chance of changing other parts of the chromatogram. They would, of course, necessitate and increase in pressure and/or run time.

Given that you have already explored gradient steepness and additive concentration effects, if this were my problem, I'd try the following:
- increase the temperature
- get a better column (newer "type B", "third generation", "high purity", whatever, silica) to reduce the tailing

[Mark Tracy]

What is the sample dissolved in and how much do you inject? If the sample solvent is too strong and/or too large an injection, it will cause poor peak shapes in the beginning of the run. When you begin with 99.95% aqueous, this is something to be careful of.

[rajeev]

The sample is dissolved in solvent A (100% water +0.05% HFBA). Injection volume is 150ul (injection loop size is 200ul)

rajeev