Baseline Problems in NREL Sugar Analysis

[Obidan]

Dear colleagues,

I am new in this forum and I hope that I can count on your assistance with the following problem.

In my lab we are trying to perform a sugar analysis according to NRELs guideline for "Determination of Carbohydrates in Biomass by HPLC";. I did everything according to their lap-02 protocol. The analytical setup we are using is as follows:

column: Phenomenex RPM-Monosaccharide (allegedly an analogue to Bio-Rads HPX-87P – a lead column)
guard: Rezex Carbo-Pb 8
sample vol:10 myl
eluant: HPLC-grade Water
Flow rate: 0.5 ml/min
Column Temp: 70 degrees C
Detector: RI

As you can see in the chromatogram we have two problems:
1st - There is a baseline ramp from 8~16 min or so interfering with the sugar peaks.

2nd - Mannose and Arabinose are not separated

Concerning the first problem we figured out by doing a blank injection of neutralized H2SO4 (with CaCO3) thatit is due to the CaCO3. Now I read that you could avoid that phenomenon using a Bio-Rad de-ashing guard column. After I called the Bio-Rad customer support and I was told the price of that (cartridge holder+c 1 deashing and 1 Pb cartridge for 1500 Eur!) I decided not to try unless it would be absolutely necessary (For my Phenomenex Pb column alone I paid 900 Eur including a set of matching guard columns and holder.). We also tried SPE with Waters Sep-Pak C18 without any success. Concerning the 2nd Problem I do not really have a clue how to proceed. I read that this is a common problem. But still, is there any chance for me to achieve a separation of these two sugars? Although problem number one is the bigger one =)

Has anyone in this forum ever had a similar experience with the sugar analysis and could help me out? I would appreciate any kind of advice. Thx

[Noser222]

I have a little bit of experience using this column, so I will help as I can. First, Arabinose and Mannose are not separated by this column. If you look in their catalog they list most sugars of interest with retention times on each Rezex column. The simple H+ form column does separate those two.
As for sample prep, C18 won't help you for deashing sugars, but you could get cation and anion phases for a similar price. I would try that before I spent that much on a deashing cartridges.
Does the hump get worse over time, or is the same with each run? You may want to regenerate your column with Lead Nitrate, and the instructionons that came with the column explain how.

[Obidan]

The column is brand new – maybe 50 injections were performed on it. But yeah, I thought also of regenerating and already ordered lead nitrate. I can not definitively say if the hump is getting worse over time – but I have the feeling that it is of a constant magnitude. If I inject pure sugar samples (without the H2SO4 hydrolysis and CaCO3 neutralization) I don’t have any problems at all. Today I tried to do an SPE with on an amino cartridge (equilibrating with H2O,MeOH and washing with a MeOH/H2O mix, eluting with water, this time someone told me to dilute my sample 1:1 with MeOH and set it to a slight basic pH with CaCO3) But again this extraction was useless, there were no sugars at all in the water fraction and the only peak I could make out on the chromatogram was that of MeOH.

@noser222: You say I could try cation and anion phases for the deashing effect. How would you approach that? Should I perform an IE chromatography in series? First cationic and then anionic? Which strength and material would you recommend? Thanks for your help!

[Noser222]

Those sugars won't bind to an amino phase unless you have a mix of at least 95% acetonitrile/5% water, maybe even 100% acetonitrile. Methanol is too strong.

I would just find a strong anion-exchange phase and a strong cation-exchange phase and you can run your neutralized sample through directly...the sugars will not bind. Any supplier of SPE phases should have a guide that will help choose the right one.

The cation-exchange phase by itself may do the job if it is the calcium that is giving you trouble. To avoid any leftover carbonate, maybe calcium hydroxide would be better since it only generates water.

[sball]

Obidan,

I am not sure if this will help you or not but I have run a similar separation on a Hi-Plex Pb column (manufactured by Polymer Laboratories), under slightly different conditions to those you described, and achieved a partial separation of Arabinose and Mannose. If you don't need complete separation of these two sugars, then perhaps you could try the conditions below, and see whether it works any better for you:

Mobile Phase: Water
Temp: 80°C (or 85°C)
Flow: 0.4ml

Best Regards,

Steve.

[Bart]

Hello Obidan,

Your analysis was discussed in a previous thread. Perhaps you'll find the information useful.

http://www.sepsci.com/chromforum/viewto ... highlight=

Topic: Shark-fin peak from biomass samples

It is my experience that the typical ligand exchange Pb based columns on the market do not separate mannose and arabinose. We (Transgenomic) do offer some Pb based columns that do separate mannose and arabinose. Please feel free to contact me if you are interested.

[Obidan]

Thanks for your help so far. Yesterday we ordered that Bio-Rad de-ashing guard column and hope for an improvement with that upgrade.