column selection

[duantech]

I have to separate a series of compounds with polarity ranging from a sugar compound to a moderate non-polar compound. The detection will be CAD. Mobile phase just water/ACN. I have tried phenomenex synergi fusion-rp, and Hrdrobond AQ. Neither could retent the sugar well. Any suggestion or lead on what column I shall try next?

thanks

[YMC-Kune]

Hi duantech,

For suggars in particular we would recoment an Amino or Polyamin II column. For highly polar compounds we would recommend a YMC-Hydrosphere column. It can be run at 100% water with good retention for polar compounds.

O.K. I don't want this to become a YMC advertisement, so if you need more information, please do not hesitate to contact me.

But for my information, what are your experiences with detecting sugars with the charged aerosol detector? I used the CAD for pharmaceuticals APIs with varying success.

Regards,
Daniel.

[Uwe Neue]

You can retain sugars on a suitable (water wettable) RP column using a high salt concentration (salting out), but even under these circumstances the retention is weak. Anyway, this is not an option due to CAD. I recommend HILIC columns for something as polar as sugars. If your sugars can form anomers, you need a suitable amino column to get single peaks, or a weakly basic mobile phase. Otherwise, silica columns are more stable for HILIC. Both packings (amino or silica) provide ion-exchange properties that may allow to retain your other analytes, depending on their nature, but you need mobile phase additives beyond acetonitrile / water to accomplish this.

[Koen Hollebekkers]

by my experience the Bio-rad Aminex and the Shodex.... (I don't remeber the type) are the standard for sugar analysis

[Bryan Evans]

Can you be more specific about the sugar / drug compound?
We can post data on this forum if you can give us more information.

[duantech]

thanks all for your info and suggestion.

We decide to go for Shodex Asahipak NH2P (amino) column to retain the sugar, hoping it can also retain its parent compound and others. More info here. The sugar (5 carbon Monosaccharide) is one of the products from acid hydrolysis of a compound with three sugar units to lose. The parent compound and other hydrolysis products (parent compound losing 1, 2, or 3 sugar units) can be retained and seprated on a C18.

From the literature, we know that it is quite likely that the column can retain the sugar. The question is, will it retain the parent compound and other compounds? Which factor will be in play for rentention, polarity (high organic Mp for better retention of all compounds with sugar unit) or non-polarity (low organic MP for better retention of a compound without any sugar unit)?

UWE, what type of additive shall I start with to achive retention of all compounds?

thanks

duantech

[Bryan Evans]

We recommend high organic (ACN)/water mixture.
If your drug (or sugar) compounds are ionized, than you'll
need buffer in your mobile phase: 10-100 mM NH4AcOH or
NH4HCOOH. You may also get better peak shape if you increase
column temperature to 50-60C.

Below are LC-ELSD of mono, di, and trisaccharides on Unison UK-Amino (3uM silica):

http://www.silvertonesciences.com/files/TI325E.pdf
http://www.silvertonesciences.com/files/TI337E.pdf

[Uwe Neue]

For the sugar, the best approach is to use about 70% acetonitrile and 30% water. This will give you some reasonable retention. More acetonitrile increases retention, more water decreases retention.

I do not have sufficient information to make any statements about the rest of your analytes. If they are acids, they will be strongly retained via ion-exchange with the amino group. If they are bases, or neutral compounds, you should get less retention on this column, or they even may elute unretained. As above for the sugar, you can increase retention by using a higher concentration of acetonitrile.

Considering that the other analytes contain multiple sugar residues, chances are good that you will get some retention.

If they are acidic compounds, you have several options, which we will need to discuss in more detail, since this can affect the sugar as well. First question: what is your sugar? (Underlying this is the question if your sugar can form anomers.)