Sucrose Shoulder/Trailing Peak with HPLC-RID

[kjordan]

We have a new Shimadzu HPLC and have been developing our methods. We are analyzing samples for sugars and using water as our mobile phase. We started with a 0.4 mL/min flow rate based on a protocol we are following. In between our sucrose and fructose peaks, we are seeing the baseline remain elevated with a little hump sometimes in between. When we run a sucrose standard (rather than the mixed sugars one), we still see a low shoulder peak/trailing peak on the right side of the sucrose peak. We were instructed to increase our flow rate to 0.8 mL/min and see what it looked like then. It looks better, but there is still something there. We are unsure if we need to increase our flow rate for the sucrose peak to absorb this or if something else is happening here, and we should not include this in our area calculated. We were integrating the mixed sugars standards by omitting it at first.

[Multidimensional]

"We were integrating the mixed sugars standards by omitting it at first." -
Please do not do that as it will invalidate your method and data (it is unscientific). The solution to the problem will not be found by changing the flow rate either. Use the correct LINEAR flow rate for the method (developed based on the column type, dimensions and particle size, which you did not share with us, but are VERY important to know). Speeding up the flow often just 'hides' problems (selecting the correct flow rate and conditions are things that an experienced local chromatographer could help you with).

Using a mobile phase that is based on pure water has MANY basic drawbacks. Much of the material in your sample will slowly absorb onto the support over time, and not get flushed out or washed off (leads to column fouling and invalid results). All columns must be periodically washed with some type of solution that is STRONGER than the mobile phase to elute off retained material that accumulates over time. Depending on the purity of your samples, sometime a column can be fouled from just ONE injection. Refer to the formal literature supplied with the column regarding which chemicals are safe to use (and at what pressure limits) and develop a 'wash' method that will be used after each analysis (then wash, then re-equilibrate the column before the next injection. Note: RID take a VERY long time to equilibrate so be patient).
Most importantly of all, please contact an experienced chromatographer for help running these samples. It may seem simple, but it is not. An experienced chromatographer can help you develop proper wash and analysis methods appropriate for your samples.

[Multidimensional]

"HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)"; https://hplctips.blogspot.com/2018/02/h ... s-for.html

[Hollow]

you don't provide much details about your method, so it's hard to make educated suggestions.

But maybe have a look/search on separation of "anomers" in combination with "sugars" and "HPLC".
Maybe you need to play with the column temperature...?

[LuccasLN]

Hi kjordan,

By the information you have given i will understand you are working with LEX. Just as Hollow has said, column oven temperature might give us a clue on what is happening. As an addition, the type of column oven should give a possible clue on what is happening (ex. heating block or heated air).

additionally, since we are talking about LEX, you should have a clear cleaning protocol for cleaning the column in the manual. it should be either use a mobile phase with a certain salt or make a few injections with a solution containing a specific salt. You do not mention how many injection you have made, but if it's been a few and then this is happening, this should be something interesting to look into. you might need to "replenish" those counter ions in the stationary phase!