Analysis of sugar(Galactose) by HPLC-RID

[mt_fong_01_13]

Dear All,
I have conduct experiment by analysis of 6 sugars which are fructose, glucose, sucrose, maltose, lactose and galactose in general foodstuff.

Can anyone suggest a single column to seperate above six sugar by single HPLC column(if any)?

I currently use the amine column (4.6x150mm, 4um) for anlaysis the five sugar.
For the last sugar(galactose), I use a polymeric sulphonate resin column (8mmx300mm, 7um, Pb Ligand; SP0810)
for analysis.
Actually, the SP0810 column are seldom use.
And, I left the column with mobile phase of water for storage for a year.

After a year, I use the column for analysis on the same standard solution.
Experimental condition:
Injection volumn: 10uL
Flow rate: 500uL/min
Column: Shodex SP-0810
Column temp:80'C
RID detector temp:55'C

refer to the above chromatogram.
I have found that the column seems deteriorated. The peak becomes board peak and tailed with compared with the old chromatogram. The column has not injected any thing for a year...Am I use a wrong mobile phase for storage and therefore deteriorate the column.

I have tried to regenerated the column as the column care manual instructed.
3-4 times 50uL 0.2M lead(II) acetate solution is injected to the column.
However, I cannot restore the column at all.

Can anyone give me advice(s) for this case.

If regeneration is not practical, I shall buy a new column.
Then, how can I keep the column in goods performance apart from using guard column.

By the ways, are there any alternative columns can be used instead when purchase new one.

[M Farooq]

First of all never store any silica based column in water. All forms of silica dissolve in water to some extent. Amine columns are even more unstable than the rest. Anyway, buy a new column. All major manufacturers provide a long term storage conditions for columns, which should be strictly followed.

There is one and only one column material which is one of the ultra-stable chromatographic materials I have ever seen. Even boiling acids, bases and solvents do not affect it. This is porous graphitic carbon (PGC) material. One company sells it under the name of Hypercarb. This column works great for the separation of sugars in general.

[tom jupille]

All forms of silica dissolve in water to some extent.

This is true but not directly relevant to the problem, since the column in question is a PS-DVB based cation exchange resin in the Pb form.

From the look of the chromatogram, my guess is that the column dried out over the year that it was in storage and the resin bed shrank a bit as a consequence with a resulting headspace. The next thing to try would be reverse-flushing the column at a low flow rate (about 1/5 of normal).

The polystyrene resins are quite robust chemically (the conversion to a cation exchanger is done by cooking the resin with fuming sulfuric acid, for example) but they *do* shrink and swell in response to changes in their chemical environment.

Different ionic forms provide different selectivity. Commonly used are H, Ca, Pb, and Ag. You might want to check the BioRad catalog to see which is the best fit for you.

[Gerhard Kratz]

When the column was not in use for 1 year and is dried out also the Hypercarb column is killed. Dried out means that air, oxygen is in contact with the packing material. That will kill the packing material.
Before you spend now too much time, solvents my advice and recommendation is to purchase a new column. Amino columns are very cheap, ok the Aminex column costs more, but still compared to the labor costs of 1 day it is cheap. Good luck.

[M Farooq]

When the column was not in use for 1 year and is dried out also the Hypercarb column is killed. Dried out means that air, oxygen is in contact with the packing material. That will kill the packing material

Drying out may disturb the bed by shrinking (if it is a polymer) or a functional group is oxidizable somehow. I am afraid that the assumption about air damage to Hypercarb is not correct. Just to give an example about its inertness: If Hypercarb material is heated in chlorosulfonic acid, it still remains pure carbon without any particle morphology damage.

Ron Majors wrote a very nice article about top 10 column myths and one them is about drying

"Myth 10: Columns Always Should Be Capped Tightly to Prevent Packing Damage from Contact with the Atmosphere

False. In general, the tiny hole in the endfitting, which is probably not bigger than 0.02 in. in diameter, has such a small cross-sectional area that damage to the column resulting from air coming in to contact with the packing and the solvent inside the column evaporating is minimal. Even if a small amount of air entered the column, it would probably have a hard time diffusing through the microparticulate packed bed and coming in to contact with enough packing material to cause detrimental effects. If there was a small amount of air in the column, as soon as it was pressurized when it was next installed into the HPLC instrument and mobile phase pumped through it, the small amount of air would probably dissolve at high pressure or be flushed out in the initial fluid in a short time and not cause any problems with later use. However, if you would feel more secure by capping the endfittings by all means do so. Most columns come equipped with male compression fitting caps that can be tightened to prevent any possibility of storage solvent evaporation or air entering the packed bed."

[Gerhard Kratz]

I agree with the publication of Ron. But on the other side the Shandon packing procedure states to avoid Oxygen gets in contact with the packing material.

[Perreman]

I'm not sure how well lactose and sucrose will separate from other peaks, however a Phenomenex Rezex RPM (Pb2+) was used in one of their applications. https://phenomenex.blob.core.windows.ne ... RL=/Search.

I've had good experience using their Rezex RCM (Ca2+) when analyzing sugars, so it could be worth a try.

[mt_fong_01_13]

Thnaks for all of your reply.
I have purchased a new column. It is same for previous one.
Shall I ask some of the information about the sample prepration procedure when I use the Sodex SP0810 column, a Pb2+ ligand excahnge column.
I extract sugars from sample just using water only.
For example, I place 10g or 10mL sample in beaker with a stirrer bar, add 50mL water, then add 5mL of each Carrez Solution 1 and 2, and finally add water to final solution weight to 100g. Allow the mixture to stir 30minutes on a manestic stirrer plate. Allow it settle for 30 minutes. Filter clear supernatant to nylon membrane filter prior to LC anlaysis.
The extract is analysis by both amine column and the above polymeric column for 6 sugars of fructose, glucose, sucrose, maltose, lactose and lactose by HPLC-RID.
My questions is ...
Is the SP0810 column can tolerate my final filter extract?
I have use the Carrez solution for sample preparation. Do it demage the SP 0810 column?

Should I need to further clean-up my final extract with the sodium type cationic exchagne SPE prior to HPLC analysis wheing using the ligand excange column?