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Shiseido's Carbohydrate Analysis
Analysis of carbohydrate phosphate is shown in Fig. 4 as an example of gradient analysis that cannot be conducted with an RI detector. Since SUCREBEAD I is a strong anion-exchanging column, the order of elution is roughly determined by the net charge of phosphate group and dissociated hydroxyl groups. As shown in (5) and (6) of Fig. 4, even with the same number of hydroxyl groups, difference in phosphorylation position caused a sensitivity difference. A molecular structure seems to influence the contact between hydroxyl groups and the gold electrode of PAD. Roughly speaking, when neutral glucose are monophosphorylated, the detection sensitivity is lowered by 1/10, and when biphosphorylated, the detection sensitivity is further lowered by 1/10. These values may depend on the type of carbohydrates.
 (a)
 (a)
【Fig. 4】  Analysis of carbohydrate phosphates
【Fig. 4】 Analysis of carbohydrate phosphates
 (b)
 (b)
Acidic carbohydrates, such as carbohydrate phosphates, N-acetylneuraminic acid, and galacturonic acid, can be concentrated on the top of SUCREBEAD under a neutral mobile phase, and thereafter analyzed by switching it to a basic one. Selectivity can be obtained from the column as well as the PAD. Various ways on carbohydrate analysis can be possible by the use of PAD and SCREGEAD.
As another interesting attempt, Prof. Sawada and coworkers (Hirosaki University) reported a system for analyzing phosphorylated carbohydrates and nucleotides related to photosynthesis by the combination of various detectors including PAD with post-column reaction, based on separation by anion exchange chromatography. They succeeded in analyzing eight types of phosphorylated carbohydrates and 11 nucleotides.
In this attempt, the electrochemical activity of hydroxyl groups in a basic condition played an important part5. Fig. 5 shows the constructed system and its results (reproduced from quoted reference 5).
Provided by Prof. Shinichi Sawada, Bionomics, Agronomic Life Science Dept., Hirosaki University
【Fig. 5】 Analysis of carbohydrate phosphates and nucleotides related to photosynthesis
【Fig. 5】 Analysis of
     carbohydrate phosphates
    and nucleotides related to
    photosynthesis
Chromatograms of mixtures of authentic suger phosphates and nucleotides and the program of gradient elution. The mixtures of suger phosphates and nucleotides contained 3.6-5.1 nmol(DHAP, 100 nmol; RuBP, 75.4 nmol) and 1.9-2.5 nmol of each component, respectively. Glucose was 6.5 nmol. Other details are given in the text.
 (f)
NANOSPACE PAD is expected to have various applications. One possible application6 is the detection of amino acids together, using the function for freely setting waveforms at a linearly varying potential.
Analysis of carbohydrates in food not requiring particularly high sensitivity can be carried out by combining separation in a normal phase system by CAPCELL PAK NH2 with an RI detector (Fig. 6). CAPCELL PAK NH2 is synthesized by applying a polymer coating to silica through the unique process, and has been proven superior to conventional amino-phases7. Detection by PAD is possible by post-column adding a base solution to the mobile phase. This method can provide increased sensitivity of dozens of times, compared to RI detectors.
 (g)
 (h)
   【Fig. 6】 Analysis of oligosaccharide gradient
【References】
[1] O.Shirota, Trends Glycosci. Glycotechnol., 5 (1993) 41
[2] William R, LaCourse and Dennis C. Johnson, Anal. Chem., 65 (1993) 50
[3] A.Ohkubo et al., J.Microcolumn Separations, 13(1) 8-12 (2001)
[4] T.Kimura, O.Shirota, and Y.Ohtsu, J. Pharm. Biomed. Anal., 15 (1997) 1521
[5] S. Sawada, et al., Anal. Biochem., (2002) Mar 1;314(1) : 63-9
[6] Petr Jandik et al., J. Chromatogra. B, 732 (1999) 193-201
Alan P. Clarke et al., Anal. Chem. 71 (1999) 2774-2781
[7] H. Kutsuna, Y. Ohtsu and M.Yamaguchi, J. Chromatogr., 635 (1993) 187-193
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