Chromatography Forum

Hi all,

The last few weeks I have been working on the analysis of monosacharides and disacharides on a Dionex ICS-3000 system. Initially the system worked fine, but the performance of the system seems to get worse over time.

The system configuration is as follows:
- Dionex ICS-3000
- Chromeleon 6.8
- Column temperature: 20°C
- Carbopac PA1 4x250 column
- Mobile phase A: 18mM NaOH
- Mobile phase B: 150mM NaOH
- Samples contain proteins that are denatured wich acetonitrile. After dilution, samples contain 0.2% acetonitrile.

Gradient:
min----%A-----%B
0-------100-------0
20-----100--------0
39--------0-----100
55--------0-----100
55.1---100--------0
65-----100--------0

Detector program:
0.050 v 100 ms integration off
0.050 v 100 ms integration on
0.750 v 200 ms integration off
-0.150 v 400 ms integration off

Basically I have two problems:
- bumpy baseline, even when I inject MQ water
- Peak area decreasing over time. Disacharide peak areas seem to be decreasing more than the monosacharide peak areas.

I guessed that either the column or the detector might be slowly getting polluted so I rinsed the whole system with MQ water, but that didn't solve the problem. Is there another way to regenerate the system? I am fairly new to both ion chromatography and Chromeleon software, so any help would be welcome.

Hi Tjalling,

Thank you for your posting...please contact me directly so that we may assist you in resolving your carbohydrate background/sensitivity issues.

A few quick questions, observations and comments based on your report:

1. Please let us know what Dionex Application Note or documented source your method is based upon? Your conditions (Temp, gradient and waveform) may be customized somewhat from the general conditions for carbohydrates/mono-, disaccharides.

2. The primary symptom you described for peak area decrease as a function of time is frequently attributable to system contamination. The most common cause for high background in ECD (electrochemical detectors) are contaminants used in the eluent preparation. This will include both the DI water and the hydroxide. Please ensure that the degassed, filtered DI water is ASTM Type I (18.0 MOhm-cm or better) and that the hydroxide solution is prepared from this DI water and from certified 50% w/w NaOH (e.g., Fisher)...absolutely never prepare from hydroxide pellets!

3. The recommended waveform (WF) is the 'Quadruple Potential' WF for the ICS-3000 ECD...your WF looks different and this could definitely have an impact on the sensitivity; an incorrectly programmed WF may apply too much or too little current and thereby reduce the efficiency of your working electrode. In your case it appears to be a modification of the 'Triple Potential' WF which is not recommended for the ICS-3000 (this is an older WF version that has been improved with the Quadruple Potential WF)

4. Other factors that may affect your background and sensitivity, but are not limited to the following:
* pH reference electrode condition
* working electrode condition (are you using the conventional (CWE) or disposable (DWE) working electrode?...the CWE may need to be polished or replaced and if using DWE, may need to be replaced)
* electrode gasket condition (a worn gasket may result in microleaks / channeling) and in my observation often yields an high background
* column condition (capacity): may need to clean/condition or replace your column set

5. A system cleanup using 2M NaOH (prepared from 50% w/w NaOH) bypassing the column and detector followed by copious flushing with DI water, then reconnecting the column(s) and detector and equilibrating to initial conditions (with the detector cell powered on) will clean most contaminants and microbial agents if present

6. In some cases (heavy) metal contamination may interfere with your analysis and will require system cleaning with a chelating agent

7. For monosaccharide analysis, an AminoTrap column may benefit in matrices containing amino acids; however, you should investigate items #1-6 above first as I believe your problems are more fundamental than this

8. Also for monosaccharide analysis a BorateTrap might be useful if peak symmetry (esp. for mannose) is an issue (borate is one of the first breakthrough ions in a DI water system); again, please investigate #1-6 above first

9. Dionex also offers a Test Mixture (P/N 043162, a neutral mixture of 6 monosaccharide standards) that can be used for troubleshooting both separation and peak sensitivity in conjunction with a CarboPac PA20 (P/N 060144 guard + 060142 separator) column...this will narrow down chemistry vs. hardware/consumables issues

10. In some cases, a pump degas conversion kit may be installed (if using an EG module with RFIC)...looks like in your case you are only using manually prepared eluents so this may not be applicable


Again, please contact me directly and we can assist you and if necessary can help you with a dispatch by a Dionex service engineer or specialist for an on-site visit. Representative Chromeleon Backup files (CMB) will also be helpful to review your PGM and other acquisition conditions.

If you are in North America and in certain global regions, Dionex offers Applications, Hardware and Software training directly to operators through their Centers of Excellence Training facilities.