What solution do you use to inject your sample in? Do you take the liquid from your mobile phase bottle or from another source?

• Is your sample retained on the column? Is this an SEC application or RP ? What is the column name and type? What are the column dimensions (exactly) and the flow rate used? Your initial description sounds like you may be looking at the tzero peak and have no retention.

Using pure water for analysis is never a good idea in liquid chromatography. Contamination and fouling of the column and entire flow path is also the result. I have analyzed many water soluble polyols and polymers over the years and we never used pure water as a mobile phase with any column.

Can you speak with your teacher or the person who has HPLC experience at your school to help you setup the method? Basic training problems of this type are much easier to solve in-person, not on the web. This is a very hands-on technique where you need to 'see' what is going on and how things are set up. You will learn more and be able solve the problem much faster with direct help and support.

@william Letter, My sample (blank) came from the same source as my mobile phase. I am using USP monograph, Mannitol, related substance.The column is a carbosep coregel (L 30cm, id 7.8, 9µm); flow rate 1.5 mL/min. The interferent peak is at 21 min. we've tried a lot of troubleshooting and we're running out of ideas.

OK, that info is helpful, but please understand that these types of problems are easy to resolve on-site, not via the web.

Two things need to be checked: (1) A fouled or contaminated column would show similar problems as you describe. Replace the column with a NEW one or fully clean and wash the column you have (Using the column manufacturer's procedure as these columns are very delicate!), then wash down the flow path, flush the RI cell and injector, then test the column with a blank and standard (not your sample) to verify the column is acceptable. (2) Which Coregel column are you using (many types are available)? Most are ion-exchange columns, very delicate, easily overloaded and damaged. The use of the pure DI water is a red flag and bound to be a problem when using it. Maybe you should consider a different column for your application. The column is one of the least expensive items used in chromatography (a consumable), but is also the most important part too. Selecting the correct column first is the most important step in method development. BTW: That is not a column I would suggest for most sugars or small polyols. Too many problems.

If you could provide DETAILED information about your actual application, then we may be able to provide more help, but w/o more info, it sounds like you may be overloading the column and have fouled it (?).

William Letter thanks for your help, we were using carbosep coregel 87c column ( Ca++ Form) .we have washed the flow path, injector and RI cell with (MeOh, ACN, Water, Isopro) mix and tried a new equivalent colmn (Aminex HPX-87C) after but nothing changed. Maybe the mix is not strong enough to wash the HPLC sys ! i'm thinking of using other solvents (like Hexane and dichloromethane)

Could you explain to me why the use of of the pure DI water is a red flag ? what type of colmn do you suggest for sugars or small polyols ?

• **If you could provide DETAILED information about your actual application, then we may be able to provide more help, but w/o more info, it sounds like you may be overloading the column and/or have fouled it (?).

What have you done to the column? Are you operating at 85C, as recommended by the manufacturer? Have you washed it per their instructions (never more than 20% ACN. Never use Hexane or other organics).

• "Column Regeneration. Prepare an aqueous 1% solution of calcium nitrate containing 0 .25% EDTA. Filter this solution through a 0.45 micron membrane and degas. With the column in the inverted direction, pump at 0.1 ml/min at 90° C overnight. Retest the column under normal operating conditions. Note that it may take some time for the baseline to stabilize."

William Letter

The method is used to analyze traces of Isomalt, Maltitol, Mannitol and Sorbitol. The column (carbosep coregel 87c) is dedicated to this application. Yes, we are operating at 85 °C; the column is stored with pure DI water in a refrigerator, washed with pure DI water and NEVER been used with organic solvents (following the manufacturer recommendation). Does storing the column in pure DI water could be the problem? Is it possible for an algae growth to occur in the column?

We tried a new equivalent column (Aminex HPX-87C) but the peak is remaining (could it be because we used pure DI water?)

As you recommended we are trying using drinking water as mobile phase and as a blank.

for information: The HPLC instrument have been serviced and qualified.

Any HPLC column stored in only water will grow bacteria, algae and perhaps even mold. This is one of many reasons why you should follow the manufacturer's suggested wash and flush methods ( 1% solution of calcium nitrate containing 0 .25% EDTA) plus have a qualified in-house column validation method for periodic testing of the column for fitness. As noted by the manufacturer, some organic solvent may be beneficial in washing too.

Nassamer wrote: "Does storing the column in pure DI water could be the problem? Is it possible for an algae growth to occur in the column? "

• Have you ever seen what can and does grow in a refrigerator, in water? Many types of undesirable 'critters' thrive in such an environment.