Simultaneous Determination of Inorganic Anions, Organic Acids, Amino Acids and Carbohydrates by Capillary Electrophoresis

Transcription

1 Simultaneous Deteration of Inorganic Anions, Organic Acids, Ao Acids and Carbohydrates by Capillary Electrophoresis Tomoyoshi Soga Yokogawa Analytical Systems Inc., Tokyo, Japan Gordon A. Ross Hewlett-Packard GmbH, Waldbronn, Germany International Symposium on Analysis of Carbohydrates Sep Stockholm, Sweden

2 Summary A capillary electrophoretic method for the simultaneous deteration of inorganic anions, organic acids, ao acids and carbohydrates was developed. A highly alkaline ph condition was used to confer a negative charge on every compound, and to promote their migration towards the anode. Outstanding separations were obtained and 43 species were well separated in a single run. This method could be readily applied to the simultaneous analysis of these anionic compounds in soy sauce, sake, nutritious tonic and pineapple samples.

3 Experimental Instrument HP 3D CE System (Hewlett-Packard) HP Basic Anion, ph 12.1 (PN ) Capillary fused-silica l: effective length 14 cm L: total length cm i.d.: 5 µm Temp. C Injection 5 mbar, 6 sec Voltage - 3 kv

4 Compounds typically analyzed... ph > 12 All compounds were negatively charged Compound pka NO Lactic acid 3.86 Acetic acid 4.76 Asp 2.77 Gly 5.97 Arg Galactose 12.35

5 Effects of ph mobility (X 1 cm V s ) ph Asp Glu CysCys Tyr Gly Ala Ser Pro Met Leu Ile His Trp NGNA NANA Ribose Xylose Fructose Mannose Glucosae Galactosae Galactose Fucose Sucrose

6 Mobilities of Anions at ph 12.1 (2 C) Compound Mobility Compound Mobility Compound Mobility Compound Mobility ( X1-4 cm2/vs) ( X1-4 cm2/vs) ( X1-4 cm2/vs) ( X1-4 cm2/vs) Bromide Chloride Nitrite Nitrate Sulfate Oxalate Ascorbate Malonate Fluoride Formate Citrate Pyrophosphate Phosphate Tartarate Succinate Malate a-ketoglutarate Asp Glutarate Glu Adipate Acetate Pyruvate CysCys Glycorate Tyr Gly n-propionate Lactate Borate Ala Ser n-butyrate Levulinate Mannuronic Pyroglutamate n-pentanoate Glucuronic Pro Met n-hexanoate Galacturonic His Leu Ile n-heptanoate Gluconate Trp NGNA n-octanoate NANA ManNAc Ribose Fructose GlcNAc Mannose Xylose GalNAc Rhamnose Glucosae Mannosae Lactose Arabinose Maltose Galactosae Lactulose Galactose Fucose Sucrose Raffinose Mannitol Trehalose Sorbitol Galactitol Xylitol Erythritol Inositol

7 mau Effect of Temperature on Separation Br NO2 Cl NO3 SO4 Oxalate Tyr Gly LactateAla Ser Trp F Citrate PO4 Malate Asp Glu Acetate His NGNA NANA Pro Met Leu+Ile Ribose Mannose Xylose Glucosae Galactosae Galactose Fucose Sucrose Br Cl NO2 NO3SO4 Oxalate Citrate Malate Asp Glu Acetate Tyr Gly Lactate Ala Ser F PO4 Pro+ Met His+Leu+Ile Trp NGNA NANA Ribose Mannose Xylose Glucosae Galactosae Galactose Fucose Sucrose Mannitol Sorbitol Xylitol Inositol Mannitol Sorbitol Xylitol Inositol mau C C

8 Analysis of Inorganic Anions,Organic Acids, Ao Acids and Carbohydrates mau Br Cl NO3 SO4 Oxalate NO2 F Citrate PO4 Malate Asp Glu Acetate Pro Met Tyr Gly Lactate Ala Ser Leu+Ile His Trp NGNA NANA Ribose Mannose Xylose Glucosae Galactosae Galactose Fucose Sucrose Mannitol Sorbitol Xylitol Inositol HP HP Basic Basic Anion Anion (PN ) (PN ) Sample Sample Cl Cl mg/l, mg/l, carbohydrates carbohydrates 2 2 mg/l mg/l each, each, others others 5mg/l 5mg/l each each Capillary Capillary fused fused silica, silica, l l 14cm, 14cm, L L cm, cm, id id 5 5 µm µm Injection Injection 3 3 mbar*s mbar*s Temperature Temperature C C Voltage Voltage -3-3 kv kv,, (reversed (reversed polarity) polarity) Detection Detection Sig. Sig. 35/2nm, 35/2nm, Ref. Ref. 23/1nm 23/1nm

9 Reproducibility of Anion Analysis Time %RSD.1-.5 % (n5) Area %RSD.8-4.9% except NO2, NO3, Trp and Ribose mau

10 Reproducibility of Migration Time Compound Bromide Chloride Nitrite Nitrate Sulfate Oxalate Fluoride Citrate Phosphate Malate Asp Glu Acetate Tyr Gly Lactate Ala Ser R.S.D. (n5)(%) R.S.D. (n5)(%) Compound Pro Met His Leu+Ile Trp NGNA NANA Ribose Mannose Xylose Glucosae Galactosae Galactose Fucose Sucrose

11 Reproducibility of Peak Area Compound Bromide Chloride Nitrite Nitrate Sulfate Oxalate Fluoride Citrate Phosphate Malate Asp Glu Acetate Tyr Gly Lactate Ala Ser R.S.D. (n5)(%) R.S.D. (n5)(%) Compound Pro Met His Leu+Ile Trp NGNA NANA Ribose Mannose Xylose Glucosae Galactosae Galactose Fucose Sucrose

12 Linearity and Sensitivity Compound Linearity 2-1 (mg/l) Correlation Detection limit (mg/l) S/N3 Sulfate Citrate Glu Lactate Fructose Galactose Sucrose Sorbitol

13 Analysis of Soy Sauce mau 35 Cl Succinate Glu 1 5 Citrate Malate Asp Acetate Gly Lactate Ala Ser Pro Leu+Ile Met Galactose HP HP Basic Basic Anion Anion (PN ) (PN ) Sample Sample Soy Soy sauce, sauce, 1:5 1:5 diluted diluted with with water, water, ultrafilteration ultrafilteration with with 3kDa 3kDa cutoff cutoff filter filter Capillary Capillary fused fused silica, silica, l l 14cm, 14cm, L L cm, cm, id id 5 5 µm µm Injection Injection 3 3 mbar*s mbar*s Temperature Temperature C C Voltage Voltage -3-3 kv kv,, (reversed (reversed polarity) polarity) Detection Detection Sig. Sig. 35/2nm, 35/2nm, Ref. Ref. 23/1nm 23/1nm

14 Reproducibility of Soy Sauce mau 35 Time %RSD.1-.3 % (n5) Area %RSD.6-5.4%

15 Reproducibility of Soy Sauce Chloride Citrate Succinate Malate Asp Glu Acetate Tyr Gly Lactate Ala Ser Pyroglutamate Pro Met Leu+Ile Galactose Compound R.S.D. (n5)(%) Migration time Peak area

16 Analysis of Sake mau 25 2 Malate 1 5 Cl Citrate PO4 Asp Glu Acetate Tyr Gly Lactate Ala Ser Pro Leu + Ile His Fructose Isomaltose Arg HP HP Basic Basic Anion Anion (PN ) (PN ) Sample Sample Sake, Sake, 1:2 1:2 diluted diluted with with water, water, Capillary Capillary fused fused silica, silica, l l 14cm, 14cm, L L cm, cm, id id 5 5 µm µm Injection Injection 3 3 mbar*s mbar*s Temperature Temperature C C Voltage Voltage -3-3 kv kv,, (reversed (reversed polarity) polarity) Detection Detection Sig. Sig. 35/2nm, 35/2nm, Ref. Ref. 23/1nm 23/1nm

17 F Analysis of Nutritious Tonic mau 25 2 Malate Sucrose 1 Leu+Ile Fructose 5 Cl Citrate Met Trp HP HP Basic Basic Anion Anion (PN ) (PN ) Sample Sample Nutritious Nutritious tonic, tonic, 1:1 1:1 diluted diluted with with water water Cl, Cl,.3;.3; Citrate, Citrate,.2;.2; Malate, Malate, 3.1; 3.1;,,.5;.5;,,.9;.9; Met, Met, 1.1; 1.1; Leu+Ile, Leu+Ile, 3.; 3.;,, 1.; 1.;,,.7;.7; Tyr, Tyr,.2;.2; Fructose, Fructose, 4.3; 4.3;,, 4.4; 4.4; Sucrose, Sucrose, g/l g/l Capillary Capillary fused fused silica, silica, l l 14cm, 14cm, L L cm, cm, id id 5 5 µm µm Injection Injection 3 3 mbar*s mbar*s Temperature Temperature C C Voltage Voltage -3-3 kv kv,, (reversed (reversed polarity) polarity) Detection Detection Sig. Sig. 35/2nm, 35/2nm, Ref. Ref. 23/1nm 23/1nm

18 Reproducibility of Nutritious Tonic mau 2 Time %RSD % (n5) Area %RSD %

19 Reproducibility of Nutritious Tonic Compound R.S.D. (n5)(%) Migration time Peak area Chloride Citrate Malate Met Leu+Ile Try Fructose Sucrose

20 Analysis of Pineapple mau Ctrate.51 % Malate.1 % Fructose 1.3 % 1.3 % Sucrose 4.2 % HP HP Basic Basic Anion Anion Sample Sample Pineapple Pineapple 1:5 1:5 diluted diluted with with water water Preconditioning Preconditioning 4 4 at at run run buffer buffer Capillary Capillary fused fused silica silica l l 72cm 72cm L L cm cm id id 5 5 µm µm Injection Injection 3 3 mbar*s mbar*s Temperature Temperature 2 2 C C Voltage Voltage kv kv,, (reversed (reversed polarity) polarity) Detection Detection Sig. Sig. 35/2nm 35/2nm Ref. Ref. 275/1nm 275/1nm

21 Reproducibility of Pineapple mau 3 25 Time %RSD.1-.2% (n5) Area %RSD %

22 Conclusion 1 Method for for the simultaneous analysis of of inorganic anions, organic acids, ao acids and carbohydrates. 43 compounds can be analyzed in in 38. Detection limit 5-5 mg/l (S/N3) Wide linearity range 2-1 mg/l Good reproducibility

23 References [1] T. Tsuda J. High Resolut. Chromatogr. Chromatogr. Commun. 1 (1987) 622. [2] T. Soga et al. J. Chromatogr. A 767 (1997) [3] T. Soga et al. Anal. Biochem. 261 (1998) [4] S.H. Pine et al. Organic Chemistry 4th ed. McGraw-Hill, New York, 1981, p.785. [5] T. Soga et al. J. Chromatogr. A 834 (1999) [6] T. Soga et al. J. Chromatogr. A 837 (1999)