Chromatography Forum

I am Chetan Gaonkar, I am using a Agilent 1200 series LC system. I used an Zorbax C18 column for separation of free sugars from water samples using Acetonitrile:Water (85:15) and RI detector. I had created a method for separation of free sugars and analysed few of my samples using the following method.

After my analysis i purged my column using same ACN:Water. After a few month I had to analyse few other samples so I started with the sample analysis, but I was not able to detect any peaks with standard nor samples.

To solve the problem I

1) Used fresh Standards
2) Prepared fresh Mobile Phase
3) Filtered my samples and increased the concentrations
4) Re-write the completed method
5) I also tried changing the column to assess if my column had saturated (But it didnt work)

But Nothing helped


While using the instrument, it showed an errorr (Unbalanced diodes).

I dont know whats wrong, Please do the needfull to solve my problem

Thanking you in advance

Hi, no panic.
Clear system by hot water, sugars solved in the it.
From column- what is column ( tipe of phase?)

Sugars are typically analyzed using an amino column with these mobile phase conditions, not a C18. Are you sure you used the same/correct column for the second analysis?

If this were my problem, the first thing I would do is to get the service manual from Agilent and re-run the performance qualification test on the detector (the fact that you are getting an error message points toward a detector failure, as does the fact that you saw *no* peaks when you re-ran standards). If the detector fails, proceed accordingly to fix the problem (as DSP suggests, it may simply be junk from evaporated solvent in the flow cell).

As carls suggests, your conditions are somewhat unusual. Sugars are commonly run using ACN/water on amino bonded phase columns. I would expect them to be unretained on a reversed-phase method. If that were the major problem, however, I would expect to see a large peak at the dead time.

Finally, check the rest of the system. Although unlikely, something like an autosampler failure *is* a possibility.

The error "Unbalanced diodes" probably means that sample and reference cells do not contain the same liquid. Purged both cells with your solvent before optical balance is performed.

You seem to have had plenty of good advice for this post. But if you still need some advice please let me know. We are Chromatography specialists, willing to give technical support if required. We supply chromatography consumables, including Columns, 30 years experience of chromatography may just give you some assistance. Have a look at our website http://www.greyhoundchrom.com
Good luck with your research


sue@greyhoundchrom.com
http://www.greyhoundchrom.com

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DR

I tried to clean the column with 100% ACN.
But still the error comes Unbalanced Diodes.

And i have used the same column for carbohydrate analysis, and i had got results for my previous analysis. so i dont know where i m gone wrong.

Hi
Have you purged the reference cell as FOR said? After this is done zero / balance the detector. Now does the unbalanced diodes error remain or does it dissappear only to reappear at a later date?
If it remains then you may have an breakdown. If it returns later this tends to suggest the system has not settled down yet.

Ya i purged the reference cell too to remove any contaminants. the Error comes only when it detects the mobile phase. later there is no detectable peak

What problem am i really facing

Hi

Are you pumping mobile phase when you purge the detector?
Have you consulted the manual for steps on purging correctly?

Ya i did, after purging for 2hr i am not getting the error. But still i am not able to get the peaks.
I asked my service engineer to provide me a manual now.