31.5.
04 PC 353 stirrer (replaced by PC 510); and filtration apparatus for
AOAC Official Method 980.13
Fructose, Glucose, Lactose,
Maltose, and Sucrose in Milk Chocolate
Liquid Chromatographic Method
First Action 1980
Final Action
A. Apparatus
(a) Liquid chromatograph.—With Waters Associates, Inc.
M6000A pump, R401 refractive index detector, or equivalent, and
10 mV recorder.
(b) Column packing.—µ-Bondapak carbohydrate column, 300 ×
4 (id) mm (Waters Associates, Inc.).
Column must meet following criteria:
Capacity factor for fructose = K′ = (tR − t0)/t0 ≥ 5
where tR = retention time for fructose = time from injection to
maximum peak height of fructose; t0 = retention time for solvent =
time from injection to maximum peak height of first baseline
distortion or solvent peak.
Resolution factor (distance between 2 band centers divided by
average band width) =
Rs = (t2 − t1)/0.5(tw1 + tw2)
where t2 and t1 = times from injection to maximum peak heights of
second peak (glucose) and first peak (fructose), respectively; and tw1
and tw2 = baseline widths (in time units) of first and second peaks,
respectively. For fructose:glucose ratios of 2.0–0.5, Rs ≥ 1.0; for
ratios ≥2, Rs ≥ 1.25.
Replace column when either or both criteria are not met.
(c) Injection valve.—7120 LC injector with 50 µL loop (Waters
Associates, Inc.), or equivalent.
(d) An cil lary equip ment.—Bransonic 12 ul tra sonic bath
(Branson Ultrasonics Corp., Eagle Rd, Danbury CT 06810-1961,
USA; No. B1210 MT), or equivalent, to degas solvents; Corning
solvent purification.
B. Reagents
(a) Sugar standard solution.—10 µg/mL. Dry individual sugar
stan dards (fruc tose, glu cose, su crose, lac tose, and malt ose;
available from Sigma Chemical Co.) 12 h at 60°C under vacuum.
Dissolve in H2O and serially dilute to concentration of 10 µg/mL.
Prepare daily.
(b) Mo bile phase.—CH 3 CN (No. 2442; Mallinckrodt
Nanograde, or equivalent) + H2O (charcoal filtered) (80 + 20). Filter
through Whatman GF/F 0.7 µm glass fiber filter, and degas in
ultrasonic bath before use.
C. Preparation of Test Solution
Weigh 10.0 g finely divided milk chocolate into ≥100 mL
centrifuge bottle and add 50 mL petroleum ether. Centrifuge ca
15 min at ca 1800 rpm. Decant and discard supernate. Repeat
extraction.
Pulverize residue with glass rod, add 100 g H2O, and weigh.
Place in 85°–90°C water bath 25 min. Cool to room temperature
and add H2O to original weight. Centrifuge 10 min at 2000 rpm,
withdraw portion of clear supernate, and filter through 0.45 µm
Swinney syringe filter.
D. Determination
Fill 50 µL injection loop with test solution and inject into column
with mo bile sol vent flow ing at 1.5–2.0 mL/min. Cal cu late
concentrations of each sugar by comparing peak heights or areas of
each sugar peak from test solution with corresponding height or area of
standard. Use same method of measurement (area or height)
throughout.
Reference: JAOAC 63, 595(1980).
CAS-57-48-7 (fructose)
CAS-50-99-7 (glucose)
CAS-63-42-3 (lactose)
CAS-69-79-4 (maltose)
CAS-57-50-1 (sucrose)
 2005 AOAC INTERNATIONAL