Sugar Separation Problems

[josebenjamin]

Friends,

I have the following problems using an Aminex HP87C column. The standard solution of sugars that comes with each column looks like this;

http://tinypic.com/view/?pic=es6ovc

Several sugars (first two) look as if they are being separated in two forms, and the efficiency is lower than expected. The third signal is glucose, an extran chromatogram with glucose alone showed the same problem.

I hope someone can help explaining the results and finding a solution.

The conditions were H2O at 0.6 ml/min and 80C. These are very close to those recommended except that the temperature is 5C lower.

Thanks,

josebenjamin

[Rob Burgess]

What is the history of the column? is it brand new? It looks like your column may be degarded possibly.

Are you sure that your column is operating at the stated temerature. You need this high temperature to ensure a fast equilibration of anomeric forms for your sugar molecules, otherwise you may be seeing a separation of the anomeric forms.

[josebenjamin]

Robb,

The column is brand new. It has been stored in refrigeration for some time before the first use. I know we are working at 80C instead of 85C, but it is hard to believe that 5C can make such difference. Our instrument can only reach 80C.

If you think of something else, please let me know,

josebenjamin

[tom jupille]

The HPX-87C column is a calcium-form cation exchange resin. As Rob indicated, it can, indeed, separate the α- and β- anomers of glucose. The catch is, of course, that the two forms equilibrate slowly in aqueous solution. The peak shape improves at higher temperature because of faster interconversion (effectively, you see the "average" of the two anomers). If your instrument can run sub-ambient, take the temperature down to 4°C and you will split the glucose into two nice peaks.

That said, your chromatogram looks like what I'd expect to see at a much lower temperature, and it also looks like you are having problems with some of the other peaks. Check to make sure that you have good preheating of your mobile phase before it enters the column. As I recall, that's a big column (300 x 6.8 mm ?), and you could well have a significant radial temperature gradient as heat flows in from the outside of thc column.

[Bart]

I'm responsible for the product support for our line of columns, and we have essentially the same type of column (Coregel 87C). You are right, 5C lower will not make any real difference in the separation. If you have a similar type of column, I'd plug that in to check for any system related causes first. Also, dilute your sample to see if that improves things. The column is also very sensitive to water quality, so check your source water, and you may need to do the wash procedure recommended by the manufacturer. But if I had to make a guess by the look of the chromatogram, I'm afraid you either have a void volume, or cracked (channeling) bed. Gel type columns are extremely pressure sensitive, and are easily compressed if the maximum recommended pressure is exceeded. You should contact Bio-Rad immediately for some support.

[Fabiano]

I always wanted to know why we need 85 ºC for this columns. I imagined that was for accelerate the difusion into the pores of packing.
Someone have some reference (review paper) that discuss the details of this mechanism of separation (ligand exchange for sugars)?

Thanks

[tom jupille]

The technique has been kicking around for over 30 years. If memory serves, it was documented in papers by Harold Walton in the late 60's.

[HW Mueller]

You are remembering ~ correctly, Tom, just checked Walton´s Book (Principles and Methods of Chemical Analysis, prentice Hall, Englewood Cliffs, 1964, p. 159, used in his course 516 which I took ~1969). Some refs are given there: Example: Helfferich, Nature, 189, 1001 (1961); Walton, Latterel, Analytical Chemistry 1962, Symposium Volume, 356, (NY: Elsevier 1963).

[josebenjamin]

Thans to you all for your comments,


I doubdt very much tha channeling would be the problem, that usually gives a distorted or split signal for all the peaks not just a selected few. This column is brand new and has not suffered high pressures. Also, diluted samples give the same proble.

We have done some work and it seems like the problem is temperature related. Even though 5oC may not make such a difference, larger gradients will.

Our oven is set at the maximum of 80C, but testing the oven with a thermocouple we have detected severe gradients. The column area seems to be operating at no more than 60C. I have found this problem in several instruments. LC manufacturers are not yet very good at controlling and designing LC column ovens.

Thanks again to you all.

josebenjamin

[Rob Burgess]

If you are still having a problem with not getting to the required temperature I would suggest trying either of the the following:

Using a water / oil bath for your column
A direct contact block heater
A strong forced air oven (as you would find in a GC oven)

[Rob Burgess]

The Shodex website offers a good explanation on how a Ligand Exchnage mechnaism operates:

http://www.shodex.com/english/dc030104.html

[josebenjamin]

Rob,

Thanks for your suggestions. None of them is really practical to us. We have none of the equipment required and the space and connections are very restricting.

What we are trying to find is a heating tape. This hopefully will make it possible to reach 85C.

thanks,

josebenjamin

[Bart]

Dear Jose,

Thanks for the information on the oven problem. That could explain why we occasionally get back a "defective" column, only to find it is in perfect working order. Keep me posted on your results.

For crude applications, we have used aluminum wool or foil to improve heat transfer. But it sounds as if you want to not just improve it but actually increase the temperature.

[Uwe Neue]

I am not an expert on this type of separation, but when I hear that there are double peaks on sugars, it triggers in my mind the thought of a separation of anomers. If that is the case, a proper temperature will speed up this equilibrium. However, you may not get to the correct temperature, unless you preheat the solvent before it hits the column.

So my question is, if you are using some length of tubing to preheat the mobile phase before it hits the column.

The experts may correct me, if this is a silly idea.

[josebenjamin]

Uwe;

In this instrument the autosampler and column oven are a single module. The injection valve is very close to the heated zones and as a matter of fact that has been a point of concern for this unit. Therefore all the olines coming in and out of the valve are at a somewhat higher than ambien temperature when the oven is operated.

I do not believe mobile phase preheating is a big concern in this case. The flowarte is low, 0.4-0.6 ml/min and the oven is set at a maximum point. Unfortunately as I mentioned before the oven settings are incorrect and the temperature is not uniform. In other words, the oven design is not really good.

josebenjamin