Chromatography Forum

Hello,

Yesterday I was given some sausages and was told that I have to give the % of polyols in them with HPLC. As we have a method for separation of sugars which is also used for glycerol determination it seemed suitable. The compunds whcich I had to sum up were: glycerol, L-arabitol, xylitol, pentaerythritol, manitol, sorbitol, dulcitol and müo-inositol.


As can be seen from structures it is probably impossible to separate them using the equipment I have but I was told that as we only need sum of the compounds it doesn´t matter if they are separated or not. The column I had was zorbax carbohydrate (4.6mm, 250mm, 5um), temperature 30C, eluent was ACN:water (70:30) and flow rate 1ml/min.
If I understand correctly the column contains aminopropyl groups. What I´m not sure about is what the retention mechanism is. Does the compound interact only with van der waals forces or is there something else?
Firstly I injected all compounds(2g/l) at once and got following chromatogram


Then I injected them on by one and found that the peaks consist of following compounds:


This also raises the problem that some compounds don´t have same sensitivity in RID and therefore can´t be used to quantify as one peak if you are not sure that there can be only one compound.
As I had been analyzing sugars before it seemed that the retention times could overlap with sugars and injected 2g/l fructose, glycose, galactose, saccharose and maltose


It can be seen that peak 3 can´t be used (xylitole,L-arabitole overlap with glycose and galactose) in my opinion and peaks 2 and 4 are very questionable, as this is ment for food analysis and samples can contain sugars.
So I took my sample, did the sample preparation(same as with sugars) and got the following picture


As can be seen from here the sausage contained peak 1 (glycerole, pentaerythrite(probably can´t be that) and myo-inositole. I am fairly OK with analysing these 2 compounds with the method but not with other peaks.
So my questions are:
1. Is there any way to separate using these compounds with HPLC
2. What is the retention mechanism between stationary phase and analytes(why is this column better for carbohydrates than C18)
3. Can I quantitate one peak as sum of compounds, even if the sensitivities seem to be different
I would be very grateful for help.

1. Is there any way to separate using these compounds with HPLC

Between the sugar alcohols and the sugars, you're talking about something over a dozen compounds. You *are* seeing selectivity differences, so even a more efficient column (smaller particle size) would help. You can also exploit temperature and column chemistry differences to change the selectivity,

2. What is the retention mechanism between stationary phase and analytes(why is this column better for carbohydrates than C18)

It's called HILIC (hydrophilic interaction chromatography). To grossly oversimplify: water preferentially sticks to that polar packing and analytes partition between the water-rich surface layer and the (relatively) water-poor mobile phase.

3. Can I quantitate one peak as sum of compounds, even if the sensitivities seem to be different.

No.

Me - I would've tried GC after trimethylsilyl derivatization first.

However, what about doing this assay gravimetrically by passing through a mixed bed ion chromatography column (like a large buret), then blowing off the solvent and weighing. We do this for nonionic content of consumer products.

Thank you for your answers.

Me - I would've tried GC after trimethylsilyl derivatization first.

However, what about doing this assay gravimetrically by passing through a mixed bed ion chromatography column (like a large buret), then blowing off the solvent and weighing. We do this for nonionic content of consumer products.

I´m not familiar with this method and don´t understand how do you guarantee selectivity from sugars for example?