Shark-Fin peak from biomass samples

[Noser222]

Why sodium azide? Your column is in the lead form, so you should only be using a mobile phase of water or a lead salt.

[Bart]

Sodium azide is not compatible with this column as Noser222 noted. The sodium will compete with the lead counter-ion on the sulfonated polymer and eventually displace some of the lead, changing the retention characteristics of the column (or worse). I did not see any reference to sodium azide in the LAPs and see no need for it?

I understand why you would not want to inject a pH = 1 sample. Is it possible to dilute with water up to the same level as the neutralized standard and inject, or is the pH still too low?

[jase1337]

Hey Bruce -

Given that you have no buffering ability in the mobile phase, and the peaks moves around, I suspect that your samples may be varying in pH, or carrying enough ions to cause a similar effect.

The literature for this column says to use only water, with up to up to a 20% max of a polar organic solvent. We have tried going up to this limit, but it has not helped. We were thinking of running 50mM Na-Cit, pH5.5 as the mobile phase, (same buffer used for a lot of the samples we shoot), but shodex tech support said it would irreparablydamage the column.

My question about spiking a sample remains. what happens when add some standard to a sample, hydrolyse both, compare against results for hydrolysis of sample and standard alone?.

I will try this today. Should have results by Monday.

Noser222 -

Why sodium azide? Your column is in the lead form, so you should only be using a mobile phase of water or a lead salt.

To prevent any microbial growth in the system; it's also in most of my samples to prevent anything feasting on the free sugars.

I just wanted to add a Thank You, to everyone for your comments, questions, and suggestions.

[jase1337]

Hi Bart,
I haven't seen any column degradation with using azide, but I will try to switch the mobile pahse to pure water, and just keep a more careful eye on HPLC cleanliness. It is not possible to neutralize with just water. It would be too high of a dilution.

[Bart]

You don't need to neutralize, just reduce the acidity. The guard should protect the column. It would be no worse than sending a constant stream of sodium into the column! Although the polymer has a stronger affinity for the Pb ion, under pressure and heat, the sodium will start to strip the lead from the polymer, although slowly under these concentrations. You will notice a drop in retention times as this happens. Considering your samples, it would be a good idea to have a Pb form guard column in front of the column. Water is the recommended storage solvent for these types of columns, although it is a good idea to take them out and flush them every once in awhile if not in constant use. I just have a feeling the neutralization step with the calcium carbonate may be the culprit. If not, I would continue with Bruce's suggestions.

[Bart]

You don't need to neutralize, just reduce the acidity. The guard should protect the column. It would be no worse than sending a constant stream of sodium into the column! Although the polymer has a stronger affinity for the Pb ion, under pressure and heat, the sodium will start to strip the lead from the polymer, although slowly under these concentrations. You will notice a drop in retention times as this happens. Considering your samples, it would be a good idea to have a Pb form guard column in front of the column. Water is the recommended storage solvent for these types of columns, although it is a good idea to take them out and flush them every once in awhile if not in constant use. I just have a feeling the neutralization step with the calcium carbonate may be the culprit. If not, I would continue with Bruce's suggestions.

[jase1337]

ok, I've switched the mobile phase to water only a few hours ago, and will try some samples shortly.

I'm also pretty sure that the CaCO3 neutralization is the cause of my troubles, but I'm just confused that in our discussions with NREL, they never mentioned that they had any HPLC issues due to this step.

I'm also scrounging around the lab for more resins for sample clean-up.

[jase1337]

So after flushing the system of the previous mobile phase, (water with 1mM NaN3) with pure water only, I ran a few plates of samples, and it appears that the "shark-fin" artifact peak is gone.

However, these samples were shot with new guards in place. Even so, I usually only get 20-40 samples through before I see the interfering peak. This time I was able to load almost 70 samples, and the chromatograms all looked pretty good.

I will keep posting on the situation, as I have a backlog of samples that need to be run, (and re-run) to get repeatable data.

Thanks to everyone for their assistance, (esp. Bart, Bruce, & Noser222 - in no particular order)!