Repromer Ag, HPLC column, 9 μm, 300 x 8 mm

Chromservis launched new line of HPLC columns intended to separate sugars, sugar alcohols and organic acids. There are many columns in the market for the analysis of saccharides with various selectivity and separation principle. There are four main methods for the separation of sugars, sugar alcohols and organic acids.

Important information

The differences under these 4 column types are mainly in elution order of individual peaks and in separation modes and elements used in the separation. Two useful rules exist:

• Reprosil Carbohydrate, Repromer RCX and Reprosil-Pur C18-AQ columns – the first eluting compounds are small sugar molecules, and then higher polysaccharides
• Reprogel a Repromer (H, Ca, K, Na, Pb) polymeric columns – the first eluting compounds are in opposite, polysaccharides.

Separation of monosaccharides improves on Repromer columns from Na to Ca and to Pb form. For oligosaccharides, there is just opposite rule – the separation improves on columns in following order: Pb, Ca and Na.

Separation modes:

1. Separation on silica gel-based phases with amino group (USP L8) works on Reprosil Carbohydrate column at temperatures between 20 and 40 °C. The typical mobile phase is acetonitrile/water (approx. 70/30). The column stability is in the range of pH 3 and 7.
2. Separation on polymeric phases with ion-exchange sorbent works on Repromer columns (formerly ReproGel), which use a sorbent fully sulfonated styrene-divinyl benzene copolymer crosslinked with 8% strong cation-exchanger. Here are few ionic forms suitable for separation of sugars, sugar alcohols and organic acids:

• Repromer H (USP L17) sugars, sugar alcohols, organic acids;
• Repromer Ca (USP L19) disaccharides, monosaccharides and sugar alcohols;
• Repromer Pb (USP L34) monosaccharides, milk samples with lactose a lactulose;
• Repromer Na (USP L58) mono and oligosaccharides;
• Repromer K.

Typical mobile phase is water at higher temperatures (60 to 90 °C) for all phases or 5mM sulphuric acid (H2SO4) for Repromer H column at lower temperatures. The detection is RI or UV (192–200 nm), the pressure below 100 bars.

3. Separation on polymeric anion-exchange styrene-divinyl benzene phases at higher pH pH (USP L47) in isocratic or gradient mode with detection PAD (pulsed amperometric detector), CDD (conductivity detector) or RI.

• Repromer 10-RCX for lower sugars and sugar alcohols to DP8;
• Repromer 30 RCX for higher sugars to DP15.

Typical mobile phase is 10–100mM sodium hydroxide (NaOH) or sodium acetate at gradient separation, the pressure below 100 bar.

4. Separation on silica gel-based phases with C18 group (USP L1) is on Reprosil-Pur C18-AQ column at temperatures between 20 and 40 °C. A typical mobile phase is water for sugars and 0,2M trihydrogenphos­phoric acid (H3PO4) for organic acids, the pressure up to 400 bars.