Better peak resolution HPLC

Hey all…I need some advice on how to get better resolution of these peaks. I run an isocratic method with 29% water,71 % ACN 0.1% FA , 10mM Amm Formate at 228 nm.

Thanks in advance!

We need about 1000x the information you’ve provided to even attempt to formulate a response to this. Analytical separation can be achieved through multiple different options but without knowing even basic information, it would be a waste of time to make suggestions.

Can you provide the following information:
All relevant HPLC parameters (column temp, sample temp, injection volume, flow rate, column type and length, stationary phase, what the peaks are or, at the very least, general information about the sample type we are working with)

Also what have you done so far to get where you are at would be a big help. Maybe you tried different pH values, or different mobile phase ratio’s that didn’t work very well…so we don’t repeat things you’ve already tried.

I would be happy to help - I have done analytical method development and validation for 10 years, but a picture of a chromatogram, mobile phase and wavelength is not sufficient to be able to give you real advice or suggestions.

Use a gradient elution if you can. Can start at 70% ACN:30% and increase from there.

Zoraku…first of all thanks for replying!
The column temp is 40C
The sample temp is 20C
The injection volume is 10ul
the flow rate is 1 ml/min
My column is a brownlee SPP 2.7um C18 3.0x150mm
The peaks you were looking at are 16 cannabinoids at 25ppm in ACN.
Im running a Perkin Elmer Flexar
The only thing ive tried to date was suggested by the service engineers which was to run several different methods and vary the water/ACN ratio. I started with a 27/73 through 33/67. I thought that 29%water/71% ACN looked to be about the best.
Im a 25 year analytical chemist but my background is in elemental analysis(ICP-OES,ICP-MS). This is my first go around with this type of instrument and this application.

1 Like

Just not a great method. Try shimadzu’s high throughput method which is listed online for free. And if you want an even better one i can give you mine for a consult fee.

1 Like

I could tell it was a flexar once I saw you were using totalchrom. I hate totalchrom and PerkinElmer…at least their Flexar instrument. Anyway that’s neither here nor there.

So, first the thing I would ask is how important is run time? If you are not concerned about run time, the first thing I would suggest would to take the same exact column and try a 250 or 300mm column. This will increase your solvent use, increase your run time, but you will have more contact time on the column and thus increasing resolution between all peaks. This might help for some of the peaks that don’t need too much more resolving.

Second, if you want to keep the same column length then I would suggest decreasing flow rate although this may or may not have negative impacts on peak shape.

You can always look at trying a different stationary phase but if you already have a pretty decent chromatogram you’re better off playing around with the mobile phase. Another option is to set up a gradient program, linear or stepwise. This would be the most time intensive option at it’s a lot if trial and error on your end to see what works.

Or, like Magister said, I would suggest looking up a published method and then use that as a baseline. Most of your parameters are pretty common for a lot of methods, so there’s nothing that stands out there. I’d also be happy to help with the development of you feel it’s something you want to develop the ability to do yourself opposed to just paying for a method.

Perkin Elmer gave me a method and helped me set it up but I feel like I might need more than 2 days of training and numerous emails which is why I tried my luck here.
Its unlikely that Ill be given any more funding for improvement so Ill need to make due with what I have in house.
I too feel like Im very close and it has to do more with chemistry than instrumentation.
A little more background…this instrument is less than 6 months old and has only analyzed cannabinoids for in house QC purposes in its short lifetime.
I am trying to bring an EPA level/style of legitimacy to our processes including analysis protocols.
With all of that in mind,one thought I had was…Do I need to be so concerned with resolution if the instrument is just measuring peak height?

1 Like

The resolution, truly speaking, isn’t abysmal. Only the 3rd and 4th peaks really have big problems. What cannabinoids are those?

In elution order:
Thats what makes me think I should cool my jets about maybe going down this rabbit hole. I just thought a brand new instrument should be able to do this no problem and I dont think my technique is THAT bad.

The instrument itself really doesn’t impact the resolution, you can use an old instrument the same as a new and get the same results you have. What is the goal? Quantification? I would suggest peak area not height.

Again, I would highly suggest you resolve all peaks of interest if you really do want to try to get more stringent and try to get to EPA or any other regulatory agency standard for chromatography.

1 Like

The red line in your chromatogram is the software constructed baseline ( bottom of the peaks ).
After the negative deflection in the first minute you need to re-attach it to the data line to get accurate quantification of the peak areas. As shown in your chromatogram the peaks areas are going to be much bigger than they really are, if you don’t correct the baseline.
Also, you say it s the peak height that’s important. I don’t think that is true. Its the area under the curve that determines the amount not the height.


The good news/bad news about this industry so far IMHO is that its the wild,wild west which is why Ive been taking what I can from the EPA style analyses Im used to and apply it here. Method blanks,dups,spikes,LCS,etc.
Thanks srihugh1…Ill look into how to edit that in the software. Im new to HPLC.

I’ve had decent resolution using 95% MeOH 4.99% HPLC grade H2O, and 0.01% TFA on a reverse phase column

It’s really not the wild west. Chromatography has been pretty standard in many industries. Cannabinoids are just another set of small molecules using the same technology that’s been around. The goal is to resolve and identify or quantify peaks of interest, that can easily be achieved with the set-up you have if you start playing around with the mobile phase composition.

I would suggest looking for chromatography forums. There are a lot of great information sites out there that you can self teach a lot of the basics if you have a general understanding of the chemistry going on. Feel free to DM me if you want some assistance I would be happy to help. I have over 50 GLP studies submitted to the EPA in support of pesticide registrations, many of them are analytical method validations.

It’s not technique, or instrument. It is all an issue of column and solvent method. It may be your column is just inadequate for isocratic method. Try a different column or try a gradient method.

Hi Pauly. What’s your pressure at? Honestly your chromatography isn’t that bad. I would start with trying to change your mobile phase. Try 0.1% Formic Acid, and ACN with 0.085% Formic Acid. I wouldn’t use Ammonium Formate either.

I run isocratic to get good CBDA/CBGA separation then ramp the gradient to get out everything else. The more polar cannabinoids are eluting pretty fast, try either lowering the temperature or going to 40:60 to start.

Hope that helps and good luck.

Level the signal floor- there’s a utility in there somewhere that does that, that might not get better separation, but the peaks will be the correct amplitude. It does a signal floor calibration

Didn’t see your comment before I commented but yeah there’s a noise floor calibration that happens and then you can edit the baseline in that utility

The answer is to turn off integration from the start, and turn it on just before your first peak elutes, at around 1 minute or so. Right now, your software is integrating as if the baseline starts from a point at your inverted peak (probably from your eluent system changeover), which will screw up all of your integrations.