44.4.13
45 Mpa; ca 500 psi); temperature, ambient (ca 23°C);
AOAC Official Method 977.20
Separation of Sugars in Honey
Liquid Chromatographic Method
First Action 1977
Final Action
A. Apparatus
(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
(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,
1.0 mL/min (3.
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
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)
2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL