overlapping peak problem, help!

[katydinh]

My problem is my HPLC results has 2-3 peaks are overlapping.
Can anyone tell me how to separate these peaks?
Thank you so much!

[Don_Hilton]

For someone to help you out, a few more details would be helpful -like instrument, instrument conditions, and, what it is you are trying to separate.

[katydinh]

I analyze glutamate and gaba by OPA derivatization
Condition as follow:

Column: Optimapak C18 150x4.6mm
Flow rate: 0.8ml/min
Column temp: 50 C

Eluent:
A buffer (2 liter) :
1. Na2HPO4. 12H20: 33.5g
2. Na-acetate.3H2O: 13.4 g
3. Acetic acid : 2ml
dissolved 1, 2, 3 by water then make them fit 2L . Take out 130ml then replace by 130ml THF
filter through 0.45 um A.F filter

B buffer
MeOH: CH3CN: DDW = 450: 100: 450
filter through 0.5 um F.A filter

Mobile phase: A: B = 1:1
sonication 15 min before use

Reagent :
1. Potassium borate solution (2L)
a) Boric acid : 61.8g
b) Potassium hydroxide : 52.5 g
a and b mix together and dissolve by water, adjust pH= 10.4 and final fit 2L
2. OPA
0.25 g OPA + 3ml MeOH then mix
Then add 0.4ml 2 mercaptoethanol
Then add Potassium borate solution to fit 50ml

Injection method:
5 ul borate buffer + 1ul sample + 1ul OPA
let them reaction for 2min before injection to the column

In this condition gaba peak is good and is not overlap by any other peak but
my problem is that I can't distinguish which peak is from glutamate because at retention time from 2 to 3 min there are about 3 peak overlap. (it is said from the protocol that glutamate retention time is about 2 min)

Therefore I really want to separate these peak in order to get a glutamate peak which is not overlap.

Please help me and thank you so much!

[tom jupille]

Resolution is a function of differences in retention (selectivity) and peak width (efficiency).

Selectivity can (sometimes) be tweaked by changing the gradient steepness, the buffer pH, and/or the temperature. You can try making small changes and see what happens.

Efficiency is generally a function of particle size and column length (flow rate has a small effect), but can be diminished if the column is old or damaged.

What you need to do is look at a "reference" chromatogram to see what the separation *should* look like; that will let you identify whether you have a selectivity problem or an efficiency problem.

[Perreman]

Depending on detector and software, you may change some parameters to gain resolution. For example, with a PDA you may change the slit width to a lower value which would generally increase the resolution slightly(this might also be possible with a UV-detector, but im not sure). However, this depends on what detector you are using.

[HW Mueller]

Perreman, I am having trouble seeing how slit width adjustment will improve chromatographic resolution.

[Perreman]

Unfortunately, I dont have an explanation for it. It is stated in our software manual and it seems to be correct given the results I have received using different valus of slit width. However as I said, I cannot verify this by theory.

[Perreman]

Okay, I have read a manual about the topic I refered to in the software.
They state: "A wide slit width will increase sensitivity but decrease the resolution". My own thoughts about this is if you increase the width of the slit, more light will be able to pass through the cell and then hit the detector, hence the increased sensitivity. If slit width is reduced, you would then receive lower peak areas (and thereby narrower peaks) which would increase the resolution between peaks.

Or is my mind all "cuckoo pants" about this?

Sorry for any inconvenience if im way off with this.

[HW Mueller]

When the manual talks about "decrease the resolution" they are talking about UV spectra, not a chromatogram.
You can not influence the chromatography by light intensity of the detector.

[tom jupille]

To expand a bit on HW's post:

The resolution in question is *spectral* resolution, not *chromatographic* resolution. Spectral resolution is the measure of how well the detector optics can discriminate between different wavelengths of light.

This is an unfortunate case of the same word being used for two different things.

[Perreman]

Aha, I see! Thank you for clearing that up for me.

[John Lim]

I agree with Tom in comparing your results to a reference chromatogram as a first start. Is it possible to inject a monostandard of glutamate and then compare by overlaying the results to your sample acquisition data? This way you might be able to discriminate among the three overlapping peaks; you will, of course, still need to resolve the peaks.

Are the other two peaks that are interfering with your glutamate are also *normally* present in your analysis where they were separated previously (and composition known), or are they new peaks? If new peaks then might be contaminants (then you should troubleshoot the source).

For some general troubleshooting recommendations: if these overlapping were previously present (and separated) then consider the following recommendations in order from easiest to more complex:

1. Check your method program; is it the correct one?

2. Recheck your eluent composition; make some new eluent, flush out the lines, equilibrate your system and re-run the analysis.

3. Issue with your column? Without knowing more about your analysis, is it possible the overlapping peaks are the result of column capacity loss? Perform a column cleaning operation per the column product manual. Better yet, replace with new column set, condition + equilibrate and re-run your analysis.

4. Possible hardware issue? Run an instrument PQ to rule out/isolate hardware problems. This should include a gradient performance check if your method uses a gradient (I could not tell from your earlier information). This might be best performed by your instrument service engineer.


If this is a new analysis and you have never separated these peaks previously, then follow Tom's recommendations for adjustment of your method and gradient conditions to separate them as part of your method development effort for resolution, selectivity and efficiency parameters.

Some chromatograms and method conditions might be helpful if you can present them to us; perhaps some of our other Chromforum colleagues might have some additional recommendations with more information.

Good luck!