Chromatography Forum

I have developed a short assay method and found the peak was really broad. See other conditions in the chromatogram. What is your suggestion to make this peak sharp?

I would adjust the ammonium acetate buffer to pH=4.8 (pKa of acetic acid, you are just out side of the useful buffer zone for acetate) and try a higher separation temperature (if possible). Maybe 40°C.

My first question would be - have you used this short/narrow of a column before on this system with better results, or have you only used larger columns like 150 x 4.6 mm?

The Alliance systems, as shipped, have a lot of extra column volume. Doesn't matter much on a 150x 4.6, but you have 1/25 of the column volume with a 30 x 2 mm. You may already know this, but I will review anyway.

1) If it hasn't been done already, replumb with as short of tubing as possible using 0.005" (red PEEK) tubing.

2) Make sure all fitting are at the proper depth. The stainless nuts on the tubing when shipped are swaged to the port depth of a Waters column, which is different than all other manufacturers. I would recommend PEEK nuts so that this can easily be changed when switching columns. All connections should be zero dead volume.

3) Check the detector cell volume. It should be <10 uL for adequate performance. You will get better performance with these small columns with a cell volume of 2 uL or less.


Chromatographically (in order of what I would do):

1) increase temperature, I would even go up to 60 C.

2) Acid or basic analyte? You would ideally like your mobile phase pH to be >1 pH unit from the pKa of the analyte and < 1 pH unit of the pKa of your buffer. Your pH is probably good, but you may want to go a little lower, say to 5, for better buffering.

Even better, if both pKa's are for basic groups, increase the pH above the pKa. Use an ammonium bicarbonate buffer at a pH of 10. For adequate column lifetime, you will need to use a high pH column, such as a Phenomenex Gemini-NX or Waters XBridge. Will probably need a higher % organic to elute.

3) Very analyte dependent, but I prefer acetonitrile over methanol - subjectively, I believe it tends to give better efficiency.

4) Go to a smaller particle size. I believe the Luna your using is available in a 2.5 um particle.

1. extra column effects as already mentioned: tubing and flow cell need attention.

2. flow rate is not optimal for a 2mm dia column optimal flow rate is about 0.2ml a min.

A 10 x 0.46cm column would almost certainly give much better results

if you want to use a smaller dia column 3mm is better with a flow rate of 0.5ml min

As already said - look at extra column effects

Just keep in mind the big picture here.
If this method is just used for internal R&D applicaions - optimizing the
instrument for Fast LC is great.

If this method is to be validated and transferred to
labs around the world - it might be easier to leave things as is
and stay with standard (100-250mm) x 4.6mm columns.

I will agree with all previous suggestions and add a single one to the “collectionâ€

Thanks to everyone!

It seems the conclusion is kind boring: the broad peak may be due to the nature of the molecule, the system and a bit overloading. maybe.

I did try 40mM phophate buffer pH2.5 and 40 mM carbonate buffer pH 9.5. Neither one did provide significant sharper peak.

I also tried a new brand new luna with the same demension and a new Sonoma with the same demension. Neither did much better too.

The retention time has a great effect to the peak width. such as from 0.8 to 2 min, the width was almost doubled with the same buffer and organic(different ratio).

The same LC and column gave a peak, in another project, less than half of the width I got now in.

When dilute the solution with a factor of 10, the peak width decreased to 2/3.

I did not try all suggestions. Please let me know if you believe you have the key for me.

Thanks again,

Any noticable difference if you increase the temp - maybe 45C?

Hi ym3142,

Here are some suggestions that might improve the situation:

1. Keep the 40 mM buffer concentration (pH 2.5)
2. Set the flow rate to 0.3 mL/min
3. Increase the ACN share of the mobile phase so that the retention time remains as it is currently.

If it is not enough to fulfill your requirements, try increasing the temperature as Bryan suggests by a couple of degrees at the time. If the temperature increase results in significant improvement, maybe you won’t need to increase the ACN percentage as the higher temperature will most probably shorten the retention times for your peaks (apart from the narrowing the peaks of course).

Best Regards

What about using a gradient from about 40 to about 60 in lets say 3 minutes, flowrate 1.5 ml/minute?

Let me clarify some:

Most of the results were obtained at 0.4mL/min, which was not significant better than 0.7mL/min.

The result for the other project with sharper peak was obtained at 1mL/min (ACN though)

ACN gives much worse separation than MEOH in my case. (pH 5.7 is better than lower pH for separation too)

when my flow at 0.7ml/min the pressure is ~4000psi so I do like to go up further. additionally, I need a 2-3 min method and can't gradient.

I may try Temp when got time. thanks

I'm serious here: I had a boss once that felt we should sharpen up peaks by decreasing the chart speed !!! And increase resolution by slowing down the chart.

I'm serious here: I had a boss once that felt we should sharpen up peaks by decreasing the chart speed !!! And increase resolution by slowing down the chart.

Bingo!-Problem solved.

</irony>

I'm serious here: I had a boss once that felt we should sharpen up peaks by decreasing the chart speed !!! And increase resolution by slowing down the chart.

A former boss would have expected me to increase resolution by speeding up the chart, sharpen peaks by increasing the overall run time, and keep the absorbance range from -200 to 1200 mAU just to make all chromatogram major peaks look pure with flat baselines and easy to compare.

I assume that knowing those things is a critical aspect to becoming a good boss

Please keep having fun,

Bruce Hamilton

Hi ym3142,

ACN gives much worse separation than MEOH in my case.

Sorry, I meant to write organic solvent, which in your case is MeOH.

The rationale I had in mind was, to move to more comfortable region of the van Deemter curve for the utilized particle size (taking into account the column diameter) by reducing the flow rate, which should improve the column efficiency. The increase of the organic should adjust the retention to the “original oneâ€