Dionex Carbohydrate Troubleshooting

[korkster]

Hello Everyone,

I'm an annoyed novice when it comes to Dionex. Unfortunately, I have to use the equipment to analyze some carbohydrates. So here's my current problem:

The chromatography for my sugar standards are bad. You can't see the peaks, the ones you do see aren't repeatable, and the baseline is really high.

I ran this analysis back in December and everything worked out with the standards. Before I shut down the system (until now), I purged the column with 150mM NaOH and then capped it. I then flushed 18M-Ohm water through the pump and the electrodes. I then stored the reference electrode in its KI solution.

Before I ran the standards yesterday, here's what I did:
1) Collected fresh 18M-Ohm water. Purged with He for 30 min. (Eluent A)
2) Made fresh 300mM NaOH. Filtered with a 0.2um nylon and purged with He for 30 min. (Eluent C)
3) Both Eluents are stored under blanketed Helium.
4) Flushed the system with water (no column in) for 30 min.
5) Calibrated my reference electrode- went well.
6) Polished my working electrode (Gold, non-disposable).
7) Uncapped column.
Changed column filters in guard (both) and analytical (front). The guard was a bit orange brown in the beginning, but the end was the nice cream color. The analytical was nicely cream.
9) Connected freshly-trimmed black tubing from guard to analytical.
10) Hooked the columns to the system (not the cell).
11) Flushed with Eluent A (water) to remove any junk (30 min).
12) Attached columns to cell.
13) Using Manual Acquisition to collect the baseline, ran the system at starting conditions (100% Eluent A- water) for about an hour.

***This is where the problems started. It looks like for about 20 minutes I had a nice baseline, but from 20 to 60 minutes, the baseline just kept creeping up.***

Concerned with this, thinking that I futzed with the column by using water, I ran the standards overnight hoping to see an improvement over the course of the run.

Unfortunately, there was no such luck. The chromatograms look just as bad as they did in the beginning. I'm trying to figure out what I did wrong, or if it's the column. If it is the column, do I need to regenerate or purchase a new one? The data is due on Monday and my time is running out.

Any help you guys could give would be great. From my previous calls with Dionex, it's hard to find someone who can actually help you. Thanks again!

[John Guajardo]

Hi korkster,

You mentioned the baseline is really high. What are the actual background and pH readings? Have you verified that your settings are correct for the "mode" (IntAmp vs. DCAmp), "reference electrode" (pH vs. Ag) and also the "waveform"? What happens to your background if you remove the column set and install a piece of yellow backpressure tubing instead? Finally, if the system sat idle for 5-6 months, I would recommend flushing it with 2M NaOH. This includes flushing all eluent lines thoroughly (prime each line with at least 40mL of eluent).

Hope this helps.

[lmh]

a friend of mine had a similar problem a couple of years ago. I have vague memories that it was a problem with the water. Despite having a very high resistance, it contained something that gradually messed up the detector electrodes (it was "ordinary" lab RO 18MegaOhm). But I might be completely wrong on this.

[korkster]

Hi John Guajardo,

You mentioned the baseline is really high. What are the actual background and pH readings? Have you verified that your settings are correct for the "mode" (IntAmp vs. DCAmp), "reference electrode" (pH vs. Ag) and also the "waveform"? What happens to your background if you remove the column set and install a piece of yellow backpressure tubing instead? Finally, if the system sat idle for 5-6 months, I would recommend flushing it with 2M NaOH. This includes flushing all eluent lines thoroughly (prime each line with at least 40mL of eluent).

I'm going to check the items you suggested, but I had a question first about the 2M NaOH flush- should that be done with the column in or out? And I'm the guessing the cell is disconnected at this point?

[John Guajardo]

I'm going to check the items you suggested, but I had a question first about the 2M NaOH flush- should that be done with the column in or out? And I'm the guessing the cell is disconnected at this point?

Start with no column installed and see if that does the trick. You may need to install backpressure tubing to stay above the low pressure limit setting. After flushing with 2M NaOH, you'll want to follow it up with water, then your usual eluent. If installing the column at this point causes the background to shoot up again, flush the column with 2M NaOH for ~1 hr.

Leaving your cell inline should be ok since you're not using disposable electrodes.

Also, make sure you are using good, fresh 50% NaOH as your source. If you see white floaties in the bottle, it's time to open a new one.