fixing a reso problem with Refractive Index Detection method

[Meerkat]

Trying to run some sucrose laurate using an ostensibly non-complicated method;
just involves dissolving 100 mg of sucr laur in THF, then using THF as the mobile phase run the elution for 10 minutes @ Column temp = 40 C; RID temp = 40C; flow rate = 0.4 ml/min.
Column used = "TSOH_TSK Gel Superh2500 15cm x 6mm x 3u.

Wish I could post a chrom image (admins help me out here) but it shows 2 peaks at 6.1 min (small) and 6.8 mins (large)--the 2 peaks are not well resolved per baseline.
Any ideas on how to fix this? Anyone with RID experience? Working with carbohydrates?

[tom jupille]

That's a GPC column, so you don't have much leeway in terms of volume, so the first place to look is for extra-column volume (tubing too big and/or too long, or injection volume too high). If all that checks out, the next possibility is a bad column. You should be able to confirm/reject that possibility by re-running Tosoh's test procedure.

The instructions for linking to chromatograms are here:
viewtopic.php?f=1&t=2617

[Gerhard Kratz]

In Addition to Tom's comment, may I ask you what RI detector flow cell volume has your detector. Inlet and outlet capillaries should have a small ID and should be as short as possible. Do you use a guard column, and if yes, do you use what Fittings?

[Meerkat]

detector = 2414 waters, all tubing seems to be normal type of clear plastic typically used for LC work.
will post a chrom pic today.

[tom jupille]

normal type of clear plastic typically used for LC work

What it's made of is of secondary importance here (so long as it holds up to your mobile phase, of course). What you need to know is the internal diameter and the length.

[Meerkat]

That's a GPC column, so you don't have much leeway in terms of volume, so the first place to look is for extra-column volume (tubing too big and/or too long, or injection volume too high). If all that checks out, the next possibility is a bad column. You should be able to confirm/reject that possibility by re-running Tosoh's test procedure.

*Column is brand new, so not a factor.

I may try flow rate, polarity adjustments to the m.p.

In Addition to Tom's comment, may I ask you what RI detector flow cell volume has your detector. Inlet and outlet capillaries should have a small ID and should be as short as possible. Do you use a guard column, and if yes, do you use what Fittings?

*The rid is of the standard type, I purge the flow cell for 5 mins @ 5 ml min, let sys equil for 30 mins on the operational flow, autozero it, then commence the injs.

Will look into measuring the tubing dimensions though, not sure why i'm doing this.

Will try some injs using modifications to flow, m.p. composition towards less polar.

[tom jupille]

Wy is the tubing size important? Because peak width depends not only on what happens in the column, but also in the tubing, fitings, injector, and detector. All of that other stuff is collectively refered to as "extra-column" band broadening. GPC columns generate narrow (concentrated) peaks which can broaden (dilute) significantly if you have a lot of tubing volume. For the size column you are using, you should have no more than about 30cm of tubing with a maximum id of 0.010".

[Turret_Error]

0.4ml/min and you're only equilibrating for 30 minutes?!

maybe it's overkill but we equilibrate our carbohydrate columns for ~4 hrs.

we run mainly on agilent instruments and our methods have pretty good resolution. we got a couple dionex machines that have bigger tubing and a lot of our methods aren't suitable on them because we see a lot of coelution, so diameter is pretty important here

[Meerkat]

I measured the steel lines betw inj and head of column to be 42 cm, the lines are covered with red urethane covering which has diameter of 2 mm with a central pore of 1 mm.

Regarding equil times, 3 hrs is the norm for such gel/ sec columns.

New result: down-adjusting / setting the flow to .1 did improve the reso from 1 to 1.4.
Will try in-tandem cols next? But we only can afford one such gel column as cost was >$500 USD