Chromatography Forum

Hello
I'm working on diol column in order to get separation of polar compounds - proanthocyanidins. My problem is that so far everything what I injected was eluted in one peak at the very begining - 3.7 min. It seems that nothing is retained on the column.

Do you have any suggestions?
Do my column need to be regenearted?

diol analytical column (250 x 4.6 mm, 5u particle size)
Flow rate: 1 ml/min
Binary mobile phase:
A: acetonitrile: acetic acid (98:2)
B: MeOH:H2O:acetic acid(95:3:2).
Mobile phase A (100 ~ 60%): Mobile phase B (0 ~ 40%)

Elution at 3.7 minutes indicates very slight retention.

If you're trying to get a previously documented method up and running, then the most likely explanation is that your column is dead or that you are not using the exact column packing that was cited in the method.

If you are starting de novo, 2% acetic acid is a fairly high concentration, and acetonitrile is fairly strong as a normal phase solvent. I'd get rid of the acetic acid and consider a weaker (less polar) solvent.

HPLC-MS Analysis of Proanthocyanidin Oligomers and Other
Phenolics in 15 Strawberry Cultivars
Journal of Agricultural and Food Chemistry
Publication Date (Web): December 28, 2009
DOI:10.1021/jf9030597

"Normal Phase HPLC-MS of Proanthocyanidin Extracts. To
separate proanthocyanidins, normal phase analysis was carried out as
reported previously (19). The same HPLC-MS equipment described above
was used. The column used was a Develosil diol column (250 x 4.6 mm
i.d., 5 μm) (Phenomenex, Torrance CA). The solvents used as mobile
phase were CH3CN/HOAc (98:2, v/v) (A) and CH3OH/H2O:HOAc
(95:3:2, v/v/v) (B). A linear gradient elution with a flow rate of 1 mL/
min and an injection volume of 20 μL was used. The gradient started with
0% B to reach 40%B after 35 min. The elution became isocratic between
35 and 45min.UVdetection was set at 280 nm, and fluorescence detection
used excitation at 276 nmand emission at 316 nm(19,20). The system was
also coupled to the MS detector under the conditions described above for
the HPLC analyses."


Dead column (or some lack of conditioning of the column?) seems likely.

has anything to do with your sample solvent?

Hello
I'm working on diol column in order to get separation of polar compounds - proanthocyanidins. My problem is that so far everything what I injected was eluted in one peak at the very begining - 3.7 min. It seems that nothing is retained on the column.

Do you have any suggestions?
Do my column need to be regenearted?

diol analytical column (250 x 4.6 mm, 5u particle size)
Flow rate: 1 ml/min
Binary mobile phase:
A: acetonitrile: acetic acid (98:2)
B: MeOH:H2O:acetic acid(95:3:2).
Mobile phase A (100 ~ 60%): Mobile phase B (0 ~ 40%)

I don't think so, I dissolved samples in mobile phase A, acetonitrile, so it should not affect seperation.

has anything to do with your sample solvent?

Hello
I'm working on diol column in order to get separation of polar compounds - proanthocyanidins. My problem is that so far everything what I injected was eluted in one peak at the very begining - 3.7 min. It seems that nothing is retained on the column.

Do you have any suggestions?
Do my column need to be regenearted?

diol analytical column (250 x 4.6 mm, 5u particle size)
Flow rate: 1 ml/min
Binary mobile phase:
A: acetonitrile: acetic acid (98:2)
B: MeOH:H2O:acetic acid(95:3:2).
Mobile phase A (100 ~ 60%): Mobile phase B (0 ~ 40%)

Some columns exhibit so inconsistent performance that you can achieve retention, or separation, or whatever you expect from it, on one column and achieve nothing on the next you buy – even though the same brand and specifications.
If I were you I’d purchase (potentially borrow) another (new) column and try the analysis with otherwise the current parameters. It’s the easiest way to eliminate the column as the troublemaker and you’ll probably need another column sooner or later – it is, after all, a consumable and does not live forever. Although some products are more stable and perform more consistently than others.

Best Regards