HPLC Method tranfer

[kiriakos]

Hello to everyone. I'm new in HPLC method transfer and I'll need some help.
I'm trying to determine prothioconazole in commercial pesticide formulations based on CIPAC method.
Below are the gradient conditions for a Zorbax Extend C18 column
(50*4.6 (i.d)mm*3.5μm).
Time 10mM Phosphoric Acid AcN/THF/MeOH (50/25/25) Flow (ml/min)
0.0 50 50 2
2.7 50 50 2
2.71 05 95 3
3.6 05 95 3
3.61 50 50 3
4.0 50 50 2
4.4 50 50 2

I have the same column , with the same dimensions but with smaller particle size, 1.8μm.
I think that with the same gradient i will have high pressure in the system so i tried the same gradient but i double the time in each step and i reduced the flow by 1. (i.e 2ml/min to 1ml/min and 3ml/min to 2 ml/min). The peak i get was splitting.
So itried to reduce the flow more by 0.5ml/min. The peak i get was wider.
I Don't know if i'm dealing with it right.
If anyone can give me some advice.
Thanks in advance.

[kiriakos]

Gradient condition
https://ibb.co/RNm7WZX

[TylerSmith123]

Hello Kiriakos,

First I'll answer your latter question with a simple explanation curtesy of Tom: "Assuming a concentration-sensitive detector (such as UV), the peak area is obtained by reading the signal (absorbance) at regular time intervals, and adding up all the readings from the beginning to the end of the peak.

To a first approximation, if you cut the flow rate in half, the sample spends twice as much time in the flow cell, you get twice as many readings, and the area becomes twice as large. What you are seeing is perfectly normal."

In all honesty, I would recommend you look up a method transfer tool. Retention is not in a direct relationship with the flow-rate as you've probably seen by now. Albeit, I am not sure what exactly your goals are for this assay. Are you trying to quantify the amount or just confirm it's presence in the samples? If your analyte is acidic, I would recommend upping the acid content of your mobile phase-- maybe to something like 0.15-0.2% (v/v)?

But try a gradient method transfer tool with the CIPAC method onto your column and it should start you off at a pretty good place. It will allow you to manually change the flow rate as well if you have issues with your pressures.

Try a few of those suggestions and let me know if your peak shape, or method, improves to what you'd like

[Consumer Products Guy]

First thing I'd ever try whenever I had peak shape issues: injecting a SMALLER VOLUME to see if shape got better/worse/same.

[vmu]

The method is essentially isocratic, but with a column wash step at an increased flow rate and a re-equilibration step after the elution of the compound of interest (approx. tR = 2.0 min, VR = 4.0 ml). The volume of the mobile phase pumped through the column is what actually governs the retention. To keep the eluent volume unchanged at each stage you should increase the stage duration by the same multiplier as that by which you decrease the flow rate. Do you obtain the peak at approx. 4.0 min (VR is still 4.0 ml) when the flow rate in the first (isocratic) stage is 1.0 ml/min? Is tR 8.0 min (VR is still 4.0 ml) when the flow rate in the first stage is 0.5 ml/min? What is the magnitude of the back pressure?

When switching to a smaller particle column, several issues are to be taken into account. Smaller particles lead to higher plate numbers (but require higher flow rates for higher efficiency) and narrower (and higher) peaks. This leads to more stringent requirements to the extra-column band broadening (lower injection volume, lower capillary lengths and diameters, lower detector cell volume). The data collection rate may need to be increased as well. The response linearity may need to be checked.

F = 0.5-1.0 ml/min is on the left (B-term-dominated) branch of the van Deemter curve for the 1.8-μm column; therefore, the decrease in flow rate results in the decrease in efficiency and the increase in the peak width (additional to the obvious peak broadening (in time units) strictly reciprocal to the flow rate).

[Hollow]

vmu summarized everything quite well.
The separation is under isocratic condition, so that should not be too hard to transfer.
With mobile and stationary phase staying the same, the retention factor of the substance should stay the same too.
If you're working with the pure susbtance for the transfer only, you may even ommit the column flush and take care of that transfer later.
The main "rule" is to keep the ratio of the (pumped volume) / (column volume) the same for each gradient segment [(tsegment * F) / Vcolumn)]

But peak spliiting shouldn't occur by the transfer of the isocratic separation, so there are other issues here.

One thing could be the injection solvent, its elution strength or its pH, to be exact.
What is you're compound dissolved in? If it's more than ACN 50% or the pH is not acidic, then weird peak shapes can occur. Therefore, like CPGuy suggested, try to:
- dissolve it in initial mobile phase
- inject less of you sample
- if possible dilute it with water (to get below the elution strength of the initial mobile phase)
- adjust the pH to at least below 2 unit of the pKa value of your compound

If that's not going to improve the peak shape, then maybe also think of having a bad connection (dead volume) somewhere in the sample path.
Maybe even caused by the higher pressure, even if there is no leak (slipping back ot the tubing inside the fitting).
So maybe reconnect every fitting (start with the easiest ones at the column).

A third option may be, that there is a second substance, that you separate with the higher plate counts, that before just coelutes with less resolution power (not very likely)