Experiencing poor Sucrose elution

[C.Bourque]

Hi all

This is my first time using this forum, so please bear with me if my topic has already been covered. I searched for information regarding my issue, but couldn't find anything in previous posts. Hope someone has some insight!

So, the issue: (sorry for such along post...just wanted to be as detailed as possible) I am currently trying to separate and quantify various sugars in aqueous solution using a Benson Polymeric 806 BP-100 Ag+ Carbohydrate Column with 806G BP-100 Ag+ Carbohydrate Guard Column, operating at 90 degrees Celsius as recommended, and filtered, degassed water as mobile phase, running at 0.3 mL/min. We are using a Waters 2414 RI for detection.

So far, I have had fairly good success in resolving well defined peaks using standard solutions of Maltose, Maltotriose, Glucose, and Fructose. I am also trying to separate Sucrose in order to obtain a standard curve for that sugar, however the peaks are not well defined. Sucrose consistently shows a sharp increase followed by a long, sloppy decline (see attached images). All the solutions I am running are roughly 0.001 g/mL concentration for comparative purposes.

Please find attached two chromatograms of Sucrose alone illustrating what is happening with the peaks. The first illustrates the elution of Sucrose before I washed the column with a 0.01 M EDTA disodium dihydrate solution (under the recommendations of a BP technician), while the second illustrates how Sucrose elutes following the EDTA washing. It is puzzling why Sucrose does not resolve well, yet Maltose (a very similar disaccharide) resolves quite well.

I have tried a number of possible solutions, and nothing seems to resolve the peak better. I have tried ordering high purity sucrose (>=99.9%) and running that through, but the problem persists (the chromatograms attached for Sucrose are the >=99.9% sample). I have tried increasing and lowering concentration of my standard solutions, changing the flow rate of the system, we have even bought a brand new Waters differential refractometer (as our old one did not have the best sensitivity at even the highest concentrations) and the problem still persists. Despite the issues of sensitivity with our older RI detector, this phenomenon was still displayed, which makes me think it is not a setting on the detector which needs to be adjusted, especially considering all my other sugars separate fine. For these reasons I believe this to be a column problem.

Does anyone have any suggestions on what might be happening with the column, or why this separation of Sucrose looks so sloppy? We have been through all the troubleshooting guides we can find and still cannot find any solution.

Please advise, and thanks so much for taking the time to read this!

-Chris

p.s. I apologize for the size of the images... I tried saving them on my computer as about 400 x 400 pixels, however they still seem to be showing up quite large in the preview. I'd be very appreciative if anyone has any suggestions on how to prevent that as well!

[HPLCCONSULT]

Lots of comments:
Did you consult with the column manufacture about what sucrose looks like under these conditions (do they have any application notes with Sucrose) ? Did something happen to the column ? Is it loaded up with detergent (EDTA)?

Could you tell us which sugars you would like to separate ? Looks like you are using the 30 cm x 7.8mm long column with the 50mm x 4.6mm guard column ? *It would help to know why you selected this column as there are so many other good carbohydrate HPLC methods and columns on the market.

I have never used the specific resin column you reference in your post (and am puzzled why you selected it as there are so many other choices). The chromatogram supplied using a pure water mobile phase at 90C with just pure sucrose looks like that of a long chain sugar, not a simple sugar (or of a high Mw component plus Sucrose). I do know that if you heat sucrose enough (~ 190C) it will break down into glucose and fructose. That decomposition can result in caramelization (high Mw products), but you are no where near that temp.

What does glucose and fructose look like using your method ? Where do they elute ? The more I look at your chromatograms, the more I think you have loaded it up with a high Mw material. If we could see what the other sugars look like (and if you still see the saw problem), I think we can figure this out quickly.

Is the water clean ? I would think that running pure water would create a breeding ground for bacteria in both the column and mobile phase. Filtering the mobile phase is not enough, the column and system should be "decontaminated". I would consult with the manufacturer on this.

I also noticed that you are heating your column to 90C, but have your detector set at 25C. RI is super sensitive to temp changes. You may want to run at the same temp and insulate the line.

If you only want to resolve some basic sugars apart you can just use an amino column and a simple mobile phase of ACN/Water [(This example uses an ELSD, but RI is similar) http://www.hplctools.com/Sugars_in_Oran ... C_ELSD.pdf]

[C.Bourque]

Thanks for the reply!

I have been in contact with the manufacturer about this issue, and their only suggestion was to wash the column with 0.01 M EDTA disodium dihydrate to see if that can "mop up" any possible contaminating metals. Unfortunately, after two washes the sucrose elution seems worse than before. I have just received word back today from BP (after emailing these findings) and unfortunately their only recommendation is to choose another column, as they have little literature pertaining to sucrose separation with these Ag columns.

You are correct about the dimensions of the column.

Also, you could be right, something could have happened to the column before I started using it, as it was left over from a previous PhD student in our lab, but to my knowledge nothing is wrong with it. In fact, I don't think it was even used much previously because, from speaking with this previous student, they were not able to analyze sugars in a timely fashion. That being said, however, is why I am using this particular column... not so much by choice as by availability (it was already in the lab). Also, I believe one of our collaborating labs was using the same column and so we purchased it under their recommendations.

There should not be any EDTA left in the column, it was washed for an hour and then allowed to rinse with water until baseline was steadily maintained. Sucrose elution was poor prior to washing with EDTA anyway.

I have attached a chromatogram of the other four sugars I am interested in monitoring (Maltotriose, Maltose, Glucose, and Fructose). As you can see, they all elute very well. I know I've heard some bad things about this particular column, however aside from sucrose, the other sugars do separate nicely! This makes the situation quite puzzling indeed, as you would expect bacterial or other forms of contamination to effect all the sugars, not just one. Perhaps I am just not familiar enough with HPLC though.

As for the water being clean, unfortunately that is something we do not have a great deal of control over, aside from filtration in the lab, and filtered once again through a 0.45 um filter when degassing. Perhaps I will explore this option further after consulting with BP. I wouldn't think that the combination of constant 0.3 mL/min flow coupled with 90oC operating temperatures would allow for much bacterial growth, but it is a possibility.

As for the sucrose eluting with a high MW material, this seems unlikely. As stated, I have purchased high purity sucrose in order to create these standards. Given that a blank shows no visible peaks, the only material coming through should be sucrose.

You are correct about RI being very sensitive to temp. changes, as I have seen first hand. Unfortunately with the Waters 2414 RI you can only set the detector to max of 50 or 55oC, which is still a far cry from the operating temp. of 90oC at the column. So I decided to leave it at the default 25oC. I would think that the 90/25oC temp. change would simply take care of itself, assuming that all my samples continue to experience the same temperatures. Indeed this seems true, as the other sugars are very consistent, run after run. We do insulate the line as well.

[HPLCCONSULT]

Thank you, you answered many of my questions (such as what a blank looked like and where the other sugars eluted). Many of my comments did not deal directly with the problem you were describing, but instead were aimed at general HPLC recommendations.

Now, back to your observations/comments. Based on what you have stated: (1) A blank run shows a flat baseline with no issues. *No problems with the HPLC system. (2) The other four sugars all elute as one would expect. **Again, no issues with the HPLC system and also this implies the column is just fine. (3) Sucrose is the only sample which does not elute as you would hope. *** Sounds like sucrose is interacting with the column in an unexpected way.

It appears that this column chemistry is not appropriate for resolving sucrose. Something about the silver chemistry used is interacting with the sucrose (or your sample of sucrose) causing the profile of a large Mw material to appear. I took a quick look at the column company's web site (which has very little info) and saw only one example of that column being used. That example did not show sucrose, but it did show other sugars like the ones you have used. I also searched the web for any examples of the column being used to resolve sucrose (a rather common sugar), but could find none. Since the column works fine with all of your other samples and appears to have never worked to separate sucrose you have two options. (1) Try a NEW column OR (2) change the method to a mainstream method using an amino column and simple mobile phase.

I do not like things that do not make sense, but sometimes you solve problems by utilizing a different solution to the problem. The column does not appear to work so I would develop a method using an inexpensive amino column.

[C.Bourque]

Thanks very much for all your help and suggestions. I think buying a new column will be the only option at this point, because as you said, there seems to be no other problem with any other aspect of the HPLC system. Indeed you have come to the same conclusion as the technician I have been emailing at Benson Polymeric, that this Ag column must be reacting with the sucrose in an unforeseen way (unforeseen even to them!), and that use of another type of column is probably warranted.

After double checking the "instructions" that came with the column I noticed the example chromatogram on the back. It must be the same one you saw online, as Sucrose is not shown. Perhaps they have not tested this column for such separation.

It is a shame that I'll have to re-calibrate, however there is really no other option.

That being said, would you recommend any particular column or method which utilizes RI? We don't have an ELSD or CAD in my lab, and it would be a shame to not use our shiny new RI detector.

[HPLCCONSULT]

From what I saw on their web site and searching the literature, I would use a different column and method.

The method example I provided can be used with an RI detector. It was developed with an ELSD, but would work fine. The best column and method for your samples should be selected by searching the literature for a method which can separate all of the compounds you are interested in on one column. You should be able to find hundreds of application notes on simple sugars. *I usually look for a method which uses a simple mobile phase, lower temp (not 90C) and column that will not break the bank in cost. For carbohydrate methods, their are many that would work well. As I said before, I would just start with a quality amino column and run a mixture of YOUR sugar standards through it at different mobile phase mixtures to see which one worked best. It should not take you long to find a good method. Good luck.

[C.Bourque]

Ok, sounds good! Thanks again

[lmh]

do you have access to a UV spectrophotometer able to measure continuously at 340nm? If time and money for development are short, and you really need sucrose, you may do better to look at completely different analytical techniques. There are a number of very simple, good methods for sucrose. My favourite is an enzyme-coupled assay measuring sucrose, glucose and fructose in a single run, requiring a handful of enzymes and substrates all readily available from Sigma...
(waits for cries of "shame, shame" for mentioning a non-chromatographic method)

[C.Bourque]

We do have a UV spectrophotometer, however I don't think it is equipped to measure continuously. We have been using it to measure turbidity of our samples, each time we have to fill up a little 3.5 mL cuvette with sample, load it into the cuvette chamber of the spec. and press a button each time we want to measure.

Would this method work for Maltose and Maltotriose as well do you suppose? As those are sugars I need to measure also.

It should be noted as well that I will be running quite a few samples... somewhere in the order of 150, which is why HPLC is so appealing, giving me the ability to load up the system with a bunch of samples to run automatically overnight. Would this enzyme assay be more labor intensive?

[lmh]

to be honest, it probably would be more work.

The principle is that you put all the chemicals needed for conversion of (assorted sugars) -> glucose -> G6P -> 6Pgluconate in the cuvette (i.e. buffer, ATP, NAD(P)). You then add the coupling enzymes in reverse order, in tiny volumes that don't affect the overall absorbance (which you can check by doing a blank). On adding G6PdeHase, only G6P can react (and is measured by conversion of NAD(P) -> NAD(P)H at 340nm). On adding hexokinase, glucose can react (fructose can too, but can't get further than F6P). The absorbance change tells you how much glucose was present. Phosphoglucoisomerase now links F6P to G6P, so the further absorbance change on adding PGI tells you fructose. Then adding invertase links sucrose into the system, and tells you sucrose.

For maltose and maltotriose, I don't have a recipe, so you'd be in the realms of method development. I have a feeling that alpha-glucosidase and amylase would be helpful enzymes. I can't remember off the top of my head, but have a feeling that alpha amylase won't cleave G2, but glucosidase will. Measuring G3 in the presence of G4 and upwards would probably be impossible enzymatically. Checking this is all working is the major thing that would put me off trying to do your samples spectrophotometrically. Originally my thought was that for limited numbers of samples, if you've got the right spec, you could do sucrose by spec to supplement the good results you're getting for the others by chromatography, assuming you don't have the money to buy columns guaranteed to do the entire job, and are stuck with what a student has left behind..

For the number of samples you're talking about, it helps either to have a spec with a sample holder that can cycle between multiple samples. Years ago we used to do 100 measurements routinely, using two Kontron specs with 10 positions each. Even allowing 20min per assay, 20 samples in 20min isn't bad going (quicker than UPLC or CE!). Alternatively nowadays if you have a microtitre plate reader, you can do phenomenal numbers at once.

In theory if you can't measure continually, you can leave a cuvette until the reaction is finished, and make a single measurement. The problems are two-fold: (1) how do you know the reaction is finished? (2) it's hard to handle the situation where, for other reasons, there is a gradual steadily climbing base-line. If you have continuous measurements you can find the steady climb and allow for it (at a simple level by printing the resulting curve, drawing through the straight bits with a ruler, hoping for two parallel lines, and measuring the distance between them).

[C.Bourque]

Thanks for the idea! It does sound like an interesting way to measure sugars. But like you say, I would have to get into a new method development for maltose and maltotriose, coupled with a fairly rudimentary spec and having to order in all the necessary enzymes, makes this a less attractive alternative to HPLC. As well I need to measure ethanol concentration, so that would add another method to the mix!

It may be that I simply work with what I have (perhaps I'll exclude Sucrose from my analysis) and only measure the other sugars in my sample. Sucrose is actually in quite small concentration anyway, so perhaps I can simply work around it. Or perhaps I'll just order a new type of column.

Again, thank you to you, and to everyone who have helped me out, it's much appreciated!

[HPLCCONSULT]

Just stick with using HPLC and your RI detector. Sugars (esp sucrose) are rather simple to resolve and there are tons of examples in the literature to help you get started (just do a search). No need to invent new techniques for something when many of the existing techniques work fine. Keep it simple and use the equipment you have to solve the problem.

[tom jupille]

The cation exchange based carbohydrate analysis columns come in different ionic forms tailored for different analyses. These were originally commercialized by Larry Cummings at BioRad when I was there in the late 70's. The calcium or lead form column would be a better choice for sucrose rather than the silver form column. There are some sucrose examples in BioRad's bulletin:
http://www.bio-rad.com/webroot/web/pdf/ ... n_1928.pdf

[Ting Wu]

It is better to use lead ionic form for Sucrose separation. I have a good application data here on Sucrose and Maltose Analysis, using Sepax Carbomix Pb column and HP-NH2 column. Please contact me twu@sepax-tech.com and I can send it over to you for your review.

[unmgvar]

you could also use a column called
Sugar D from Nacalai
http://www.nacalai.co.jp/cosmosil/data/Web/AP-0335.htm
http://www.nacalai.co.jp/cosmosil/data/Web/AP-0331.htm
it works like in HILIC mode and you can also play around with the solvents ration and temp if you need it for a better separation.
I prefer it to using those carbo columns. they either do it or not.