sugar separation RID detector propylamino column

[lisanaftali]

hi, my name is Lisa naftali, for my final assignment i'm trying to determine inulin in syrup. I'm looking for a method for separation inulin using propylamino column. what is the best column between Waters carbohydrate column 10 um or lichrosorb NH2 merck 5 um?
i'm trying to separate inulin from other sugars (glucose and sucrose) with acetonitril and water as mobile phase, what is the best composition for inulin?

thank you

[tom jupille]

There probably is no "best" column. I would suggest doing a Google search on "inulin" + "hplc". That will turn up lots of possibilities.

[lisanaftali]

thank you for your reply. yes, there are many better possibilities, but the only available column in my laboratory is propilamino column.
i had already tried using acetonitrile-water in various composition but the result is not satisfied. please advice. thank you.

[tom jupille]

the result is not satisfied

Can you be more specific?
No retention? if so, what ACN/water ratios did you try?
Retention, but poor efficiency (wide peaks)?

Another question:
- are you trying to measure the total amount of inulin, or are you trying to establish the distribution of oligosaccharides?
If you're looking for the former, you should be aware that most HPLC columns will at least partially separate the various dp's of inulin, so that you are not likely to see a single peak, but rather a family of peaks characterizing the distribution of oligomers. Determination of "total" inulin typically requires hydrolysis and measurement of the resulting fructose.

[lisanaftali]

i had tried ACN-water (20:80), ACN-water (40:60), ACN-water(80:20), and water (100).
inulin peak only appear in water(100) but glucose is appear at the same retention time as inulin,
and no inulin dan glucose retention in ACN-water (20:80), ACN-water (40:60).
only glucose appear in ACN-water(80:20).

yes, i'm trying to measure total amount of inulin. based on Inulin Determination for Food Labeling journal by Angela Zuleta, and Marı´a E. Sambucetti, using the anion column they can measure a total amount of inulin in a single peak. can i do that with propylamino column?

I have read a journal about determination total inulin using propylamino column with hydrolysis of inulin, but i really don't understand how to count % inulin from fructose and i have no inulase enzyme for hydrolysis inulin.
but maybe it's the only way to determine inulin using propylamino column,isn't it?what can i do?

I really appreciate your reply. thank you

[tom jupille]

using the anion column they can measure a total amount of inulin in a single peak. can i do that with propylamino column?

I'm sorry to say that the short answer is "no". Those are two quite different separation mechanisms.

[lisanaftali]

deeply appreciate your immediate reply

I'm sorry to say that the short answer is "no". Those are two quite different separation mechanisms.

no possibilities at all?
how about increasing the column temperature?

i heard that using TFA i can hydrolysis inulin. is that right?
can you give me a hand with some literature that can help me understand how to count inulin from total fructose?

because of limited subscribtion, it's rather difficult for me to get some good journal.thank you for your support.

[tom jupille]

no possibilities at all?
how about increasing the column temperature?

What part of "no" is unclear?

Inulin is essentially an oligosaccharide of fructose (with one glucose at the end). The amino bonded phase column will separate the oligos by chain length. No matter what you do, you will not get a single peak for "inulin" on that column. You can find more background material here:
http://www.wiley-vch.de/books/biopoly/p ... 39_448.pdf

Inulin can be hydrolyzed to yield fructose under acidic conditions, but it typically requires strong acid and high temperature (for example: http://www.ejpau.media.pl/volume9/issue4/art-38.html ). I don't know whether TFA will work, but you can always try.

By the way, I have never worked with inulin. Both of those links came from a Google search. There is a *lot* of information out there.

[lisanaftali]

What part of "no" is unclear?

i mean inulin determination with propylamino column without hydrolysis is impossible ?

The amino bonded phase column will separate the oligos by chain length.

what kind of chemistry interaction between that separation?

thank you so much for your help, i'll try to search more information.^^

[tom jupille]

i mean inulin determination with propylamino column without hydrolysis is impossible ?

Very few things are impossible. This one is very, very, very improbable.

what kind of chemistry interaction between that separation?

Carbohydrate separations on amino bonded-phase columns are based on what would be called "HILIC" (hydrophilic interaction chromatography); when they were first done 30+ years ago, it was simply called "normal phase". In essence the analyte molecules partition between a water-rich layer on the surface of the stationary phase (hydrated amino groups) and a relatively water-poor mobile phase (typically 25-35% water). The separation mechanism is based on differential solubility. Since each oligosaccharide has a slightly different solubility, they separate from one another, so you do not get a single peak for "inulin". If the column is efficient enough to completely resolve the oligos, you get a "picket fence" type pattern; it it is not very efficient, you get a "blob" that represents the envelope of the various unresolved oligos.

What I found searching the web was that separations of inulin as a single peak are done by anion exchange at high pH. Since only one end of the molecule is ionized, all of the oligos have the same charge, and they all elute as a single peak.

[lisanaftali]

thank you for your explanation.
but, this part is quite difficult for me to understand. what is "blob" means?thank you.

If the column is efficient enough to completely resolve the oligos, you get a "picket fence" type pattern; it it is not very efficient, you get a "blob" that represents the envelope of the various unresolved oligos.