[coquim]

i think is not possible but...it never knows...if somebody got some minimal information about it, i´ll appreciate so much...
 
Thanks!!!!!!

[Arkcon]

The real question is, what is your detector?  A short RP column may work, for a suitably clean matrix.

[coquim]

You´re right, i´ve forgotten.....i got an UV detector.....what do you think? i´ve found nothing on the net....

[Arkcon]

You´re right, i´ve forgotten.....i got an UV detector.....what do you think? i´ve found nothing on the net....

You will not find an efficient wavelength for a carbohydrate.  That is such a given, most people will facepalm at the mention of it.  A refractive index detector will work, but the process is hardly smooth, given the various limitations of that instrument.  There is also pre- and post- column derivitization, but likewise, that's a technique for experts.

If you really want to learn these sort of techniques, a good place to start is the US (B, JP, EU -- whatever flavor you like) Pharmacopoeia.  There are simple, old-fashioned, HPLC techniques written there.  You can then look to individual vendors for their refinements.

As usual, you have to walk before you can run, and your particular analysis involves the metaphorical walking and running on metaphorical quicksand with weights on your legs.

[JGK]

Sugar analyses  generally require IR or ELSD detection. 

However, if you do not have access to these detectors, there is a technique (indirect Chromatography) where you add a molecule with a chromophore (at a fixed concentration) to your mobile phase and measure the negative peak produced when the analyte (which does not have a chromophore) passes through the UV cell.

http://books.google.com/books?id=6agCAq0yHJYC&pg=PA490&lpg=PA490&dq=indirect+chromatography&source=web&ots=1SVb9GV6hc&sig=sgNAtCHuUDEtx0aLsPy8-vtnO4U#PPA490,M1

[Arkcon]

Like JGK says, you can add a chromophore to the mobile phase, and get a negative peak.  Most modern software allows you to flip the signal, for a more typical chromatogram, and they can integrate negative peaks, just fine anyway.  You do surrender sensitivity when you do this however.  It was a common, old-fashioned way to detect ions, when you didn't have an IC.

Check out this PowerPoint I found while Googling, it explains several of the concepts I've been talking about.

{clicky}

*Note* This is an Google HTML version of a PowerPoint document.  I have the Powerpoint saved, but I can't attach it.  It is on the Grace server, a company that makes a number of column types.  Notice that they seem to favor bonded normal phase columns for carbohydrates.

[coquim]

Hey guys!!!!!!  you are the best!....thanks for all the information you´ve given me, it seems you have a lot experience or you both are a sort of genius...
Now i must read the info and try to find out what kind of chromophore i could use....
thousend of thanks again!!!!

[coquim]

Hi!!
I´ve read the article but i couldn´t find a chromophore solution in order to use so, i´m here again....i´ve thinking if i could use potassium permanganate....i really have no idea....so.....i´m all ears....i mean...eyes....
thanks!!!

[ARGOS++]

Dear Coquim;

“Permanganate” is something of the worst you can do!

“Permanganate” is a strong Oxidiser, but you need something inert as possible!

I agree anyway much more with JGK, because “negative”/inverted Signals are mostly weaker by several factors and influenced by a lot of noise.


Good Luck!
                    ARGOS⁺⁺

[Arkcon]

Uhhh.. when in HPLC we say "chromaphore" we don't necessarily mean in the visual range.  Permanganate is a good way to color water in a flask purple, assuming you like solutions of oxidizer that look like weak grape Kool-Aide, but, for RP or normal phase HPLC, permanganate would be a bad choice, it's not really soluble in solvents.

What you're looking for is something with a UV-absorbing group, like a benzoate salt for RP, or a quinoline for normal phase, that will raise your HPLC detector's baseline up high.  As the fructose comes off, it will dilute your highly absorbing mobile phase, and cause a negative peak.

*[EDIT]*

Like ARGOS++ reminds you, this is the bass-ackwards way of doing detection, and is more susceptible to detector noise.  It'll work in a pinch, but you'd like to get you hands on an RI detector, if you can.

The article I linked will probably not mention this chromaphore spiking method.  I browsed the HTML version, it seems to talk about a good variety of HPLC topics.  I don't have powerpoint, so I can't see it in all of it's slide show glory.

[coquim]

sniff sniff....ok...hit me hit me!!...i feel no pain....what a shame!.
Thanks...i´m gonna dream with chromophore solution tonight...