Chromatography Forum

Hi

Im fairly inexperienced at HPLC but im trying to develop a method for testing for sucralose, I have an RID detector but im having problems with the peak. i have been running sucralose standards with different levels in them but getting the same peak area , i have it in negative polarity and i am using a C18 biphenyl column and i've tried mobile phases with both methanol and acetonitrile but i am getting no further just wondering if anyone could help me, thanks.

Does the retention time of your peak change when you change the aqueous/organic ratio in your mobile phase (solvent). If the retention time stays constant, then you are probably not seeing your peak at all; what you are seeing is "t0 noise" (sometimes also called the "solvent front").

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Im getting different retension times but struggling with peak area differences. I have no problem with sucrose as a negative peak just struggle with sucralose with different concentrations.

Dissolve sucralose in the mobile phase and inject without column. peak are without column should match you peak with the column. It is a little bit trickier with RI detector. O would not expect too much retention of sucralose on RP column

propably the substance have no retention at all on the C18 column, so the peak comes in the dead volume. The difference in area respect to concentration is not that big, so there no way to see the discrimination between different standards when the peak is in dead volume.

I would go with C18 column that could withstand 100% water, and using 100% water hoping that the sucralose have some retention. Or maybe changing to different selectivity... HILIC maybe.

Im getting different retension times but struggling with peak area differences.

I thought the problem was that you were getting the *same* peak area?

And how different were the retention times? In general, for reversed-phase, a 10% difference in the aqueous/organic ratio should change retention by a factor of about 3.

propably the substance have no retention at all on the C18 column, so the peak comes in the dead volume. The difference in area respect to concentration is not that big, so there no way to see the discrimination between different standards when the peak is in dead volume.

I would go with C18 column that could withstand 100% water, and using 100% water hoping that the sucralose have some retention. Or maybe changing to different selectivity... HILIC maybe.

Ive just had a look our biphenyl can use 100% aqueous phase so i will try this. Thank you

Im getting different retension times but struggling with peak area differences.

I thought the problem was that you were getting the *same* peak area?

And how different were the retention times? In general, for reversed-phase, a 10% difference in the aqueous/organic ratio should change retention by a factor of about 3.

Sorry yes everytime i run sucralose whatever the concentration i get exactly the same peak area.
As for retention times, i have only changed between 15% methanol or acetonitrile for now acetonitrile had better separation between sucrose and sucralose but i got 1/6th difference retention time which is around 30 seconds

It sounds like you might be looking at a "system peak" generated by your injection, not actually sucralose at all.what happens when you inject a diluent blank?

I think i may need to look at a different column possibly

I think i may need to look at a different column possibly

You may, but that's like saying you may need to look at a new car because your current one is making a funny noise. It's a lot easier (and cheaper) to change something like the aqueous/organic ratio than it is to change the column.

So, first thing: are you getting any retention at all with your current mobile phases? You will need a k' value greater than 2 in order to quantitate effectively (i.e. your retention time should be more than 3x the dead time). You can estimate the dead time (t0) from the column dimensions and the flow rate t0 ≈ (0.5 * L * dc^2) / F where:

• - L is column length in cm
  - dc is column inner diameter in cm
  - F is flow in mL/min.

If the peak you are looking at is near that estimated t0, then either you have no retention or your sucralose peak is totally retained. [By the way, sucralose should give a positive peak with RI (see this application note for an example: http://www2.shimadzu.com/applications/lc/l297.pdf )]. If it is unretained, you could try cutting your organic solvent to 5% (or even zero, as has been suggested). If it's totally retained, you could try increasing the organic solvent concentration (10% increments usually work fairly well - try 25%, 35%, 45% and 55% and see what happens). If you *still* don't see a peak for sucralose, *then* it's time for a different column.