How to separate similar retention times? Trial and error

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Hi everybody,

I'll like some help trying to separate these two unknown compounds from ammonium fiber expansion pretreatment (AFEX) of pulp mill sludge. I ran the slurry through a couple of sizing columns and collected 10 mL fractions. A couple of the fractions are very clean (image below), but the detected unknown peaks are on top of each other. Here's the problem below (I figured out how to properly print now for better overlays comparisons):

Image

Details:
Isocratic
Flow Rate: 0.65 mL/min
Oven Temp: 45 C
UV Cell Temp: 45 C

Column: Rezex ROA-Organic Acid 8% Cross-Linked Hydrogen
Mobile Phase: 1 mM H2SO4

The peaks don't match any of the standards in the run (about 225, sugars, organic acids, sugar alcohols, alcohols, many others). I would like to collect the fractions for LCMS but I need to separate them out first.

I tried:

- Increasing the temp from 45 to 75 in 10-degrees steps. Little change.
- Flow rate of 0.4 to 0.8 in 0.1 steps. Little change.

My next thoughts were to start playing with the mobile phase concentration, but I thought I'd ask the forum first see what anybody thought.

Thanks, y'all!!
I would try the following,

1) lower the flow rate

2) lower the acid concentration

3) change the acid

4) lower the column temperature

Make only ONE change per experiment.

5) Report back on Tuesday!

Good luck,

Regards
JMB
JMB wrote:
I would try the following,
1) lower the flow rate
2) lower the acid concentration
3) change the acid
4) lower the column temperature
Make only ONE change per experiment.

5) Report back on Tuesday!


Sounds like the "Design of Experiments" class that all household and laundry had to attend a couple of decades ago. A good way to learn what the largest effects are due to.
Exactly!!

Regards,
JMB
Hi JMB,

Since these are truly unknowns and you would like to identify them, I would actually recommend starting with a different column. Like you said, these compounds could be anything in that mill-sludge (within reason), so to start my method development, I'd first investigate the separation with a C18 column. I'd also keep the acid out of my first injection's mobile phase just to asses if these compounds are actually acidic (although this can be done easily without the HPLC too).

But for the largest changes in selectivity, I'd typically start with the column, ie stationary phase (if I have others on and hand the method changing won't forgo some policy).

When you were changing temperature, however, do you imply that the temperature was changing over the course of the run, or does your statement mean that you equillibrated the column at the incremental temperatures, and then injected the sample? This may help with those marginal changes that you're seeing.
I'm getting closer!!!!

So much trial and error. All the DoEs... You guys... I think I need to write a paper about this. I've done 39+ different mobile phases... That just wouldn't separate, until now!!!!! I want to freaking dance, only I don't dance so there's that...

Image

Seriously, maybe this is manuscript worthy...
TylerSmith123 wrote:
Hi JMB,

Since these are truly unknowns and you would like to identify them, I would actually recommend starting with a different column. Like you said, these compounds could be anything in that mill-sludge (within reason), so to start my method development, I'd first investigate the separation with a C18 column. I'd also keep the acid out of my first injection's mobile phase just to asses if these compounds are actually acidic (although this can be done easily without the HPLC too).

But for the largest changes in selectivity, I'd typically start with the column, ie stationary phase (if I have others on and hand the method changing won't forgo some policy).

When you were changing temperature, however, do you imply that the temperature was changing over the course of the run, or does your statement mean that you equillibrated the column at the incremental temperatures, and then injected the sample? This may help with those marginal changes that you're seeing.


Hey Tyler - Thanks for all your replies.

I tried in a few different C18 columns with various mobile phases and I simply couldn't separate them. But after a bit of "DoE" and just following the data, I got them a bit more separated. I will continue to tweak the protocol and see if I can get them further apart. But until then, I wanted to report back to the forum that I am making headway.

Cheers!!!!

P.S. No. I don't know what they are yet
I find changing the column temperature can be a highly effective and oddly not well known parameter to resolve coelutions on the HPLC.
MSCHemist wrote:
I find changing the column temperature can be a highly effective and oddly not well known parameter to resolve coelutions on the HPLC.


In my own experience, we typically tried different temperatures first, especially once column compartments with cooling capacity became available.

Of course, we looked for better/worse/same before going to other parameters.

In the old days I had a pointy-haired boss who believed that speeding up the chart paper speed helped resolution !!!! I can't make this stuff up !
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