peak separation issues

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Hey all,

I've banging my head against the wall with this separation for a while. I've been bouncing between columns, eluents and gradients and ALWAYS get the same results--4 closley eluting peaks with no separation. The most I've managed to do is swap the positions of two of the peaks. Running on an agilent 1100.
Imagehttps://imgur.com/a/sqjuk

My analyte of interest is at 2.195.

Flowrate: 1.2 ml/min
Column: YMC-Pack ODS-AQ; 250x4.6 S-5um 12nm
Temp: 40 C (Yes, I've tried at lower temps)
Mobile phase: A 50mM Phosphate buffer @ 2.5 ph; B ACN
Gradient of 82:18 to 70:30 over 5 min (0-5min, 6min hold).
UV/VIS @ 218 nm.

The sample is cornflour extracted with 50% 10mM TCEP in water 50% EtOH. I'm trying to pull out cysteine. If I use 100% EtOH I have a nice clean cysteine peak, but the introduction of water gives me the bonus coeluting peaks (which is expected). The problem is that I have a 30% loss of cysteins in 100% EtOH. Cleanup with SPE has been largely unsuccessful.

I'm sure I'm missing something obvious, though I'm starting to think a new column may be needed. From digging around the literature it looks as though the ODS-AQ column is pretty similar to whats being used (eg primsep 100), so I'm hesitant to dump $1k on something that likely won't even work. Any input would be great. Thanks!

PS Precolumn derivitization doesn't work for some reason and postcolumn is not an option.
We are missing some basic info here. What is your FLOW RATE? Also, what is the gradient program because you state it is 5 minutes in length, but we do not know when it starts. Please provide this essential info.

You do not appear to have a peak separation problem. You appear to have a retention issue. Retention is needed before you work on separation. Am I correct that your use of a 4.6 x 250 mm column with the current method results in all of your peaks eluting out between 2.0 and 2.5 minutes! If so, your gradient has not started yet. Best guess is that you have no method yet, just samples eluting around the column's void volume (knowing the flow rate would confirm this). Normally, we start with more aqueous phase (for RP methods like you have). Perhaps derivatization, ion-pairing or HILIC. Have you looked through the literature for examples? I saw many methods with similar techniques.
Add in increments ~5% Methanol to Mobile A and slow down your gradient ramp to <1% per minute. This will tease apart your peaks because it is very rare to have molecules with a similar solubility in 3 solvents.
Updated to add flowrate of 1.2 ml/min. The retention is just as bad at lower flow rates. The gradient begins right at time 0. Then from 5-11 it holds.

Derivitization does not work this with this sample matrix for some reason (controls come out, samples do not). HILIC was off by another 30% and the peaks were fronting really bad (looked smeared). Ion pair was my next venture.

I've been over and over the literature; the same conditions just don't seem to work with this for some reason. The best I've had is 5%ACN isocratic, but after a couple of runs the column seems to crash and I wind up with the same as you see above.
HPLC chemist wrote:
Add in increments ~5% Methanol to Mobile A and slow down your gradient ramp to <1% per minute. This will tease apart your peaks because it is very rare to have molecules with a similar solubility in 3 solvents.


This idea interests me because I thought the biggest issue was going to be the differences between the mobile phase and the injection solvent. I'll give it a whorl and see if it pans out. Thanks.
Drop your flowrate to 1.0 mL/minute too. Use every trick you have previously used (different column...) and measure the difference in retention time between these peaks. Good luck!
As I recall (its been years since I developed an HPLC method), baseline separation between peaks is a resolution of 1.5 The 1100 can calculate it for you, if you use the 'comprehensive report' style.
Try a shorter column too since 250 mm (25 cm) columns are too sluggish to changes in the mobile phase composition. Try a 5 cm, 10 cm, or 15 cm column.
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