Samples with high sucrose concentrations

[Louise_R]

We have a student who is analyzing small organic molecules from enzymatic reactions by reverse phase HPLC (DAD detector), using a C18 column with water and acetonitrile (75:25). They would like to study the effect of different concentrations of sucrose in the samples (anywhere between 10-50%); is there any concern with analyzing such samples? I assume that the sucrose would not be retained by the C18 phase and as long as the concentration of acetonitrile was kept reasonably low, the sucrose should remain soluble and just pass through the HPLC? The overall viscosity of the samples should increase with sucrose concentration but given the low injection volumes of 5 to 10 uL, I wouldn't think that would cause any issues with the injection system, reproducibility, or separation? My background is strictly organic, so I have never worked with sugars, enzymes, etc. Are we missing something?

[uzman]

For sugar analysis , RID detector is suitable .
C-18 column also not a suitable choice , there are carbohydrate columns for such analysis ; either in Pb++ or H+ form.

[itspip]

Start with this paper. These researchers used a CAD, but an RID is also good.

https://www.mdpi.com/1420-3049/24/23/4333

[Louise_R]

I'm sorry I should have clarified a bit more I guess - we are NOT trying to analyze the sucrose itself, we are analyzing small organic molecules which is why we are using C18 and a DAD detector (we don't want to see the sucrose). The question was more about if there were any issues instrument/column wise to be aware off when injecting samples that contain significant amount of sucrose as part of the matrix...

[Hollow]

If not I am wrong, the two contributors before seem to have missed the point of your question.

You don't want to analyze the sugar; it's just in the matrix, right?
But in kinda high concentrations.

I wouldn't be too concerned at 10-15%, which is somehow like in normal beverages.
But I don't know about 50%: guess the viscosity aspect may be to be considered, as it may affect the injection precision. Maybe you need to adjust the drawing/injecting speed parameters.

If the concentrations of the analytes are not very low, I would try with just diluting with water or even with something like acetonitrile, which may precipitate the sugars and enzymes, which then can be removed by centrifugation or filtration.

Depending on the ionizability of the analytes, you may want to use some formic acid (or alkali) in the diluent. Be sure to use enough to change the pH, as the matrix may be buffered for the enzymatic reaction.

If dilution is not feasible due to low concentration, but the analytes are well retained on C18 phase, then I would tend to go with a solid phase extraction procedure:

load,
wash with plain aqueous solvent,
elute; with the possible advantage of a preconcentration


edit: was already typing when Louise_R replied

[lmh]

shouldn't be a problem if it's not too viscous. If you dilute with water to reduce viscosity, then of course you're diluting with a low-strength solvent, so you can also inject more volume to retain sensitivity, without losing your peak-shape.

Incidentally, if your student goes on to use LC-MS, you can benefit from the analytical column to keep your mass spec clean! Just divert the first minute of your run to waste, and then the sugars won't end up being converted to toffee in the spray chamber (which smells nice, but isn't great for the long-term health of the instrument).

[itspip]

I'm sorry I should have clarified a bit more I guess - we are NOT trying to analyze the sucrose itself, we are analyzing small organic molecules ...

Thanks for the clarification. You wont see the sucrose with DAD and it should have little to no retention. I don't think that the amount in a soft drink will change the viscosity so much that you would really need to mess around with the injection procedure. I would make a note, as suggested, that if you do move to MS to reject the void volume to keep it easier to clean the MS.