Sucrose analysis

[tom jupille]

The "split peaks" problem shouldn't be an issue for you. That problem most commonly occurs in the analysis of glucose on the calcium form cation exchange resins (e.g., the Aminex HPX87C). That column is capable of separating the alpha- and beta- anomers of glucose, but the equilibration ("mutarotation") between the two forms at room temperature is fairly slow, so that you get significant interconversion during the chromatography; the result is a broad, or flat-top, or split peak (depending on the exact temperature and on the effiiency of the column) raising the temperature speeds up the equilibration so that you end up with one reasonably sharp peak representing the "average". You're not analyzing glucose, and the HILIC columns don't separate the anomers anyway.

For best results, the detector and oven temperatures should match. The "operating temperature" in the specs is for the electronics.

[miro2009]

The Tosoh amide is my favorite column for this app. You might also try the new Waters
UHPLC amide phase if you want high throughput. I have heard that amino phases tend
to form a schiff base with the sugar and performance degrades pretty rapidly.

Not to worry about temperature control of the cell. I think. Check with your sales rep
but your RI must be able to both heat and cool to obtain the desired cell temperature.
The RI will control the temperature that you need in the cell. The column can be 80C
but the RI cell will be some other controlled temp. i.e. no need for both column and RI
to be set at the same temp.

Thanks Mardexis for your input, today I got I reply from the support team at Tosoh where I was asking about their amide column and also about operating temperatures, here's my question to them followed by their reply that really shocks me:
Q: As we are still gaining experience with this new detection (RI) system, can it be operated a temperature different than that of the column oven?
A: "Auto sampler, column oven and detector cell should have the same temperature."

Now what?? Obviously I won't set my autosampler at 80oC!

[mardexis]

As Tom suggested in the last post... it is best to have everything set to the same temperature. As a practical matter though, we had ten hplc running 24 hours a day and all had the biorad 87H running at 80C and the RI detectors were typically
set at 50C. You should be able to monitor the actual temperature of the cell. If you are using an Agilent column heater you can set a second control temperature to match your RI cell and run the column effluent through the second column heater prior to going into your RI cell.

[carls]

Amino columns (e.g. Luna Amino) with 80/20 ACN/H2O isocratic generally work well at 30C. Anomer peaks are not observed presumably due to slightly alkaline envioronment inside the pores of the sorbent due to the amine functionalities on the silica surface. I have found the best results when using continuously mixed, premixed mobile phase. This helps reduce mobile phase artifact peaks and/or drift with RI detection. This is easily accomplished by placing your premixed mobile phase on a stir plate.

[Gerhard Kratz]

I like these problems with sugar analysis!
Premixed mobile phase is much better
column and detector temperature MUST be the same
I got excellent results even with an old Knauer RI detector
Isolate the tubings from column to detector very well
Flush the column 2 x than recommended
and always purge the reference cell in your RI detector before you start
And place your RI not too close to the AC
miro2009: obviously the most experienced tech support guy at Tosoh had a day off when you called them
Standard Amino columns at 30°C I would not use if I want to get a robust and reproducible method.
Good luck

[miro2009]

Standard Amino columns at 30°C I would not use if I want to get a robust and reproducible method.
Good luck

Dear Gerhard, thanks for the input; I will take in account all these considerations, although the choice of where to start for a newbie like me is getting overwhelming The more I screen literature and finding a lot of column choices of different types: amino/amide/polyamine, so confusing, but I need to start somewhere, so I narrowed down the choices to the following, and hope you tell me your opinion which to purchase as a start point:
1- YMC pack amino column (http://www.ymc.de/ymceurope/products/an ... Amino.html)
2- YMC pack polyamineII (presumably more stable)
(http://www.ymc-europe.com/ymceurope/pro ... minII.html)
This link also has a part of their catalog where they describe a method for disaccharides separation with operating temp. of only 26C?? no split peaks? http://www.ymc-europe.com/ymceurope/fil ... alytes.pdf
3- Tosoh TSKgel NH2-100 http://www.separations.us.tosohbioscien ... H2-100.htm
4- Tosoh TSKgel amide 80 http://www.separations.us.tosohbioscien ... ide-80.htm

The aim of our method is quantification of only sucrose present in a 0.05M sodium phosphate matrix, there are no other sugars in our sample, so no complex separation is needed. But we would prefer a robust reproducible stable column (I assume there is no expected formation of schiff's base as sucrose is non reducing??), and would high operating temperatures still be needed in our case (probably not if I choose option 2??)

So what would you all think? which option of the above is best to start with (or if you other column suggestions), based on our above info and preferances??

Awaiting your replies, and thanks in advance!

[tom jupille]

I think you may be over-agonizing about the problem. This is *not* a difficult analysis.

Any of those columns should work -- just make sure they give you an application note showing conditions for sucrose.

Peak splitting should not be a problem with sucrose in these HILIC-type separations, so pick a convenient temperature (unless your column oven has a chiller, you should probably be about 10 degrees above ambient to maintain good temperature control).

Your sugar level is high enough that detector sensitivity should *not* be an issue, so don't worry too much about temperature and flow stability.

Go for it!

[carls]

Here's an application note showing the analysis of simple sugars using Luna 5um Amino. A simple mobile phase (80/20 ACN/H2O) and RI Detection are used. Temperature of 40 C was used to ensure stable baseline, RI cell was also 40C, not to suppress anomer separation.

Hope this helps

http://www.phenomenex.com/cms400min/lit ... sugars.pdf

[alfordclinton]

Sucrose analysis includes the following things. They are as follows:-
1. Determination of protein: a modification of the Lowry method that gives a linear photometric response.
2. Determination of end point of primary drying in freeze-drying process control.
3. Determination of acrolein and related compounds with m-aminophenol.

[JamieAlison]

The Tosoh amide is my favorite column for this app. You might also try the new Waters
UHPLC amide phase if you want high throughput. I have heard that amino phases tend
to form a schiff base with the sugar and performance degrades pretty rapidly.

Mardexis - I am curious how fast a schiff base forms in amide columns. I am having a very short column life, while trying to analyze sugar standards on the BEH amide 2.1 x 100 mm column. ~100 samples of sugar monomer standards and the column irreversibly over pressurizes. Do you know of any good literature about schiff base formation? I was wondering if there was a chemical reaction between the column and the sugar. Do you have personal experience running sugar analysis on the BEH column or know of anyone who does? Is there a way to slow down the schiff base formation? Conversely, are there conditions that make the schiff base worse? Temp, media components et cetera?

Thanks so much!!

[Karen01]

The Tosoh amide is my favorite column for this app. You might also try the new Waters
UHPLC amide phase if you want high throughput. I have heard that amino phases tend
to form a schiff base with the sugar and performance degrades pretty rapidly.

Mardexis - I am curious how fast a schiff base forms in amide columns.

I don't think they do.

I am having a very short column life, while trying to analyze sugar standards on the BEH amide 2.1 x 100 mm column. ~100 samples of sugar monomer standards and the column irreversibly over pressurizes.

We have been doing sugar analysis with UPLC BEH amide columns fro 2 years now with very different experience. Coudl you have particulates in your standards? Do you filter your solutions? If so how?

What is your mobile phase and column temperature (We run at 35C)? What is your LC system?

[HPLCaddict]

The Tosoh amide is my favorite column for this app. You might also try the new Waters
UHPLC amide phase if you want high throughput. I have heard that amino phases tend
to form a schiff base with the sugar and performance degrades pretty rapidly.

Mardexis - I am curious how fast a schiff base forms in amide columns. I am having a very short column life, while trying to analyze sugar standards on the BEH amide 2.1 x 100 mm column. ~100 samples of sugar monomer standards and the column irreversibly over pressurizes. Do you know of any good literature about schiff base formation? I was wondering if there was a chemical reaction between the column and the sugar. Do you have personal experience running sugar analysis on the BEH column or know of anyone who does? Is there a way to slow down the schiff base formation? Conversely, are there conditions that make the schiff base worse? Temp, media components et cetera?

Thanks so much!!

I'm afraid Schiff base formation is not the source of your problem. This is an issue that's specific to AMINO columns. You are using an AMIDE column, which is something completely different. The amide group is practically absolutely inert concerning chemical modifications.

[Andy Alpert]

Schiff base formation with amino columns:
Between 1975-1989, people used amino columns to separate sugars in the presence of a lot of organic solvent in the mobile phase. Few people gave much thought to the mechanism of retention. In several papers there was speculation (or uncritical pronouncement) that it involved transient formation of a Schiff base between the aldehyde group of the sugar and the amine groups in the stationary phase. This line of speculation lived on despite the fact that the chromatography worked just as well with sugars that lacked reducing ends (and which couldn't form a Schiff base). In 1990 I published a paper (J. Chromatogr. 499 (1990) 177) that introduced hydrophilic interaction chromatography as a general-purpose mode. Any polar stationary phase works for HILIC if you use enough organic solvent in the mobile phase. The paper also demonstrated that basic compounds are the most hydrophilic ones. That accounts for the good performance of the amino columns: the amine groups just happen to be quite polar. They have the additional benefit of not separating anomers of reducing sugars, as has been commented on in numerous threads in this forum. You can get the same benefit by adding 0.1% triethylamine to the mobile phase when using a neutral material for HILIC; it accelerates the rate of mutarotation. That's why people use it with the Waters BEH Amide material (and every other neutral stationary phase).
I did in fact read one paper that did have pretty good evidence for formation of a Schiff base between reducing sugars and amino columns. It involved low concentrations of the sugar and prolonged incubation, and was clearly irrelevant to the real conditions of the chromatography. Schiff bases can't form with amide groups. This reaction does not account for your problems with the lifetime of the Waters BEH amide columns.

[JamieAlison]

Thanks, all.
I am new to this type of chromatography, so your explanation that Schiff bases cannot form in these columns is very helpful.
We have changed from 0.2% TEA to 0.2% NH4OH to see if the TEA was causing a problem. As I indicated, we haven't even injected real world samples yet. So far, we have had 270 injections of the same standards without an overpressurization event. My fingers are crossed.

This forum has been incredibly helpful!! I wish I had discovered it sooner.

Thanks again.

Jamie

[JamieAlison]

We have been doing sugar analysis with UPLC BEH amide columns fro 2 years now with very different experience. Coudl you have particulates in your standards? Do you filter your solutions? If so how?

What is your mobile phase and column temperature (We run at 35C)? What is your LC system?[/quote]

Karen - so glad to hear that you have been able to use the BEH amide for sugar analysis for 2 years. Our standards are filtered, and we haven't even run real-world samples yet. Our mobile phase is A:80/20 ACN/H2O + 0.2% TEA and B:70/30 ACN/H2O + 0.2% TEA run at 85C. This is based upon a Waters application note for the analysis of beer fermentation broth. Our application is similar, and the method affords us the necessary resolution between monomers and oligomers. Our system is the Acquity Hclass. We tried to switch the 0.2% TEA with 0.2% NH4OH, as many of the papers using the method for oligosaccharide analysis seem to utilize the NH4OH instead. So far, the system has run all weekend without fail. 270+ injections. Our goal right now is to see if for some reason the TEA was part of the problem, although I have a hard time understanding why that would be. There are also many successful application of the BEH amide column analyzing monomeric sugars with TEA as a mobile phase component in the literature as well. What do you think about the temperature? 35C doesn't quite give us the resolution we need. Do you have any experience running at higher temps?