Chromatography Forum

Hi, I'm running simple sugars like sucrose on a HPLC with a RID. My mobile phase is water. My problem is I get a mysterious peak (of varying sizes) at a retention time that coelutes with my fructose. The peak always elutes even with just a plain water blank injection. The peak even elutes with just an air injection even though the peak is negative. Since their is a peak even with just air can i assume the problem is related to my injection?

Column fouling.has occurred (normal). You will need to follow the column manufacturer's suggested washing instructions. As you did not say what column you are using (Note: When asking advice, please include all of the method details needed. Ask someone at your school for help), we can not make a specific column wash suggestion. Many "sugar" columns (e.g. Amino, "sugar" columns, etc) should be washed down with IPA to prevent surface fouling over time. Maybe every 5 injections to start with, then re-equilibrate? Check with the supplier before using any specific wash solution. In any case, your chromatograms show clear fouling so clean the column regularly to restore resolution. *NOTE: Also check your injector for seal damage as "carryover" may also be one of your issues (easy to solve with proper servicing of the injector valve. "Air" injections are irrational as you are using a RID ! RID shows a difference for everything injected. Carryover would cause problems at the retention time(s) you describe, not a Tzero. Injector testing should always be done using pure mobile phase, never "air: in HPLC).

BTW: In general, "sugar" analysis in pure water by RID is not formally used or may be considered an invalid method of analysis for simple sugars. The detector (RID) is non-specific and using pure water only allows for poor selectivity, poor resolution (and frequent fouling) with co-elution and poor reproducibility. High temp analysis with aqueous/ACN mixtures on "sugar" columns can yield better results with RID. Many poor quality HPLC application notes or "article's" found online as a resource show good and bad examples. You can see these types of poor quality "methods" in many of those new open-access and questionable online "journals".. There are hundreds of bad methods for "sugars" and alcohols. We recently reviewed a few for a customer that were provided by a beverage "Tax Authority" and another one from a "Customs" dept overseas. All showed misinformation and invalid methods.

Thank you. Sorry for the lack of specifics. The column is a Shodex SP0810 packed column. I'm using the Shodex SP-G guard column with it also.

Hi Mcantu,

Could we see a picture of the chromatogram? Can you describe the retention time, preferably the retention factor, of your fructose peak? To me this sounds like fructose eluting at the dead-time and coeluting with your tzero peak. If this so called "peak" is, as I am understanding it, still there but negative upon a air injection, then that may be the blip from the injection itself.

Like multi said, providing us more information will help us help you. Personally, I'm not familiar with this separation, however I do find some of the descriptions a little surprising. As Multi mentioned, running this method in exclusively water will come with a lot of drawbacks (I wont rehash).

Please let us know more about your analysis!
TS

Update: Sorry, I just looked up the application note for this column and if your chromatogram looks like the application note you listed (SP0810) then the problem is certainly not a injection issue that manifests at tzero. Fructose comes out in the middle of the chromatogram (if based on the application note) and this would not manifest from an injection issue.

Thank you sir. Yes, you found the correct application. I'm working on uploading some chromatograms. Keep you posted.

"Fructose comes out in the middle of the chromatogram (if based on the application note) and this would not manifest from an injection issuee."

Incorrect, a worn rotary valve seal in the injection valve results in sample peaks observed during the run, not at Tzero. So one can certainly have an injector "issue". Understanding the HPLC system flow path and operation are required to troubleshoot correctly.

Absolutely, I mischaracterized that. I should have referred to the "injection issue" as a sample-prep/diluent issue.