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44.4.13 AOAC Official Method 977.

20 Separation of Sugars in Honey

Liquid Chromatographic Method First Action 1977 Final Action A. Apparatus

1.0 mL/min (3.45 Mpa; ca 500 psi); temperature, ambient (ca 23C); detector set so that 380 mg fructose gives full-scale peak. Mono-, di-, and trisaccharides are eluted from column in order of MW.
D. Preparation of Test Solution

(a) Liquid chromatograph.Equipped with isocratic solvent delivery system, manual injector, refractive index detector, and recording/computing integrator. (b) Column.300 4 (id) mm m-Bondapak/Carbohy drate (Waters Associates, No. 84038). (c) Test solution clarification kit.Available in kit form from chromatography suppliers; 0.45 mm filters stable in organic solvents are suitable. (d) Syringes.10 mL (No. 701-N) point style No. 1, 2 0.020 in. od, 25 gauge needle (Hamilton Co.).
B. Reagents

Weigh 5.000 g test portion in small beaker and transfer to 50 mL volumetric flask with 25 mL H2O. Immediately dilute to volume with CH3CN and filter through 0.45 mm filter, using clarification kit.
E. Chromatography

Inject 10 mL standard solution into chromatograph. Establish retention times, measure peak heights, and check reproducibility. Repeat for test solution. Calculate glucose, fructose, and sucrose from integrator values or from peak heights as follows:

Weight percent sugar = 100 (PH/PH) (V/V) (W/W) where PH and PH = peak heights (or integrator values) of test solution and standard, respectively; V and V = mL test and standard (50 and 100) solutions, respectively; and W and W = g test portion (5.000) and standard, respectively. References: JAOAC 60, 838(1977); 62, 515(1979). CAS-57-48-7 (fructose) CAS-50-99-7 (glucose) CAS-57-50-1 (sucrose)

(a) Mobile phase.LC grade acetonitrile diluted with H2O (83 + 17). Degas mobile phase daily by magnetic stirring 15 min under vacuum. (b) Sugar standard solution.Place 3.804 g fructose, 3.010 g glucose, and 0.602 g sucrose into 100 mL volumetric flask, dissolve in 50 mL H2O, and add CH3CN to volume. Composition of standard approximates 5 g honey dissolved in 50 mL aqueous CH3CN (1 + 1).
C. Operating Conditions

Fructose, glucose, and sucrose are baseline separated and quantitated in 20 min under following conditions: flow rate,