Polyamide/cellulose low pressure chromatography

[carlo.annaratone]

Hi all,

I am attempting to purify the following compounds starting from belgian endive material:

lactucin
lactucin oxalate
deoxylactucin
deoxylactucin oxalate
lactucopicrin
lactucopicrin oxalate

I already have an analytical protocol running in reverse phase, however I do not own the equipment for running prep hplc. One protocol found in literature uses Sephadex LH-20 eluting isocratically with H2O : EtOH : CH3OOH (49.99 : 50 : 0.01). However I would prefer to use two steps in order to achieve higher final purity and yield.

Bare silica in normal phase nicely separates the free sequiterpenes, but there is untolerable tailing and low loading for the oxalates (already verified experimentally). My idea was to use a low pressure column running with cellulose or polyamide 6.

Any comment on the feasibility of this? Any suggestion for a good starting point for a protocol?

[tom jupille]

If you're starting from scratch, get (or make) TLC plates with the same material to do your initial mobile phase screening.

Re the tailing on silica, water content of the mobile phase can make a *huge* differnce in tailing & loadability. If the mobile phase was organic, make sure it was fully water-saturated.

[carlo.annaratone]

Hi Thomas,

regarding the bare silica, I did not check the water content of the mobile phase, but I am quite convinced that it is a hopeless separation. The peak of lactucin oxalate (of which I obtained a small standard by repeating analytical C18 runs) is barely moving with EtOAc:IPA:formic acid 8:1:1

Regarding the TLC, this is a sensible suggestion, however I was a bit put off by the price of the TLC plates. I am the only one in my department doing liquid chromatography, therefore i don't like paying $250+ for 50 polyamide plates that will sit unused forever if they don't do the job for me. For the same price I can get polyamide (or cellulose) bulk material and run directly a couple of test columns.

Another alternative would be self packing an MPLC column from Pharmacia that I got from ebay and using it with my Agilent 1100 as a makeshift flash system. In this case I could use any medium I like, and use the UV detector for monitoring fractions. The flow-rate would be lower than ideal, but I think it could work for my needs (the system goes up to 5 ml/min and the diameter of my column is 16 mm)

[tom jupille]

Packing your own column sounds good to me. I wouldn't get commercial TLC plates in any case, but rather make my own (microscope slide size) with the same stationary phase I would propose for the column. I like TLC for this type of screening because it's quick and you can see *everything*, including what stays at the origin.

[carlo.annaratone]

Hi Tom,

any hint on how to make TLCs out of glass slides?

[Hollow]

Hi Tom,
any hint on how to make TLCs out of glass slides?

yes please, I'm interessted too

[Andy Alpert]

Back in 1968 we used to dip the slides into a slurry of the stationary phase material in methylene chloride and slowly pull it up. This left a fairly uniform layer of the material on the slide which was good enough for qualitative analysis. Don't expect a high degree of reproducibility from slide to slide, though.

[tom jupille]

Back in 1968 we used to dip the slides into a slurry of the stationary phase material in methylene chloride and slowly pull it up.

We used diethyl ether, but same idea. We ran the chromatography in baby food jars. As Andy said, not great reproducibility, but adequate for quick-and-dirty screening.

[carlo.annaratone]

Hi all,

here is a quick update. I managed to pack by myself the MPLC column with a makeshift tool of my invention (after messing up the whole lab with splats of cellulose powder). It is connected now to the Agilent 1100 system acting as pump, injector and detector - it runs pretty smoothly at 5 ml/min.

I started with cellulose and a solvent composed of 75% ACN / 25% water. I see only one peak, nice and symmetrical. Any idea on how to change the solvent system? Which solvents are goo starting points? I never ran on cellulose before. My guess is that it behaves like a normal phase system\

[Andy Alpert]

When you operate a stationary phase as polar as cellulose in 75% ACN, then you're in the HILIC mode. Any gradient would involve a decreasing % ACN. By the time you get to 40% ACN, you'll probably have eluted everything.

[JMB]

I have used microscope slides (silica gel) to monitor reaction mixtures of polyols, partially to fully derivatized.
Usual mobile phase was EtOAc/EtOH/H2O in 10:3:2 or 45:5:3 v/v/v mixtures.

I would typically develop the plate, dry under warm air gun, develop again, re-dry and develop again; then spray to detect spots. This multiple development (3-5 runs) was intended to approximate the expected behavior of the mixture on a dry-packed silica gel column.

The lab. had a commercial do-it-yourself kit, purchased I believe from Whatman.

If you can get hold of a copy of Thin Layer Chromatography by Egon Stahl (especially an older edition ca.1970) it may show the apparatus.

[carlo.annaratone]

So far the cellulose separation gave no usable results, i only had one single peak eluting close to void volume. The following step as been trying polyamide. A water/MeOH gradient will only elute the free sesquiterpenes but the esterified forms are retained on the column. How can I elute them? Literature suggests to increase solvent strength with high % of acetic acid. Any suggestions?