44.7.13 AOAC Of fi cial Method 979.23 - AOAC International

44.7.13
AOAC Of fi cial Method 979.23
Sac cha rides (Ma jor) in Corn Syrup
Liq uid Chromatograhic Method
First Ac tion 1979
Corn In dus tries Re search Foundation–AOAC Method
A. Prin ci ple
Corn syrup so lu tion is passed through cat ion ex change col umn.
Sugars are sep a rated by mo lec u lar ex clu sion and se lec tive
ad sorp tion, and measured with dif fer en tial refractometer. Peaks are
quantitated against ap pro pri ate stan dard with dig i tal com put ing
in te gra tor. Psicose, fruc tose, dex trose, maltulose, other
disaccharides (DP 2), DP 3, DP 4, etc. are de ter mined in corn syrup
with 50W-X4 resin. Al ter na tive Q15-S col umn is suit able for
de ter mi na tion of psicose, fruc tose, dex trose, to tal DP 2, DP 3, and
DP 4+.
B. Ap pa ra tus
(a) Liq uid chromatograph.—With pulseless pump, re frac tive
in dex de tec tor, and re cord ing/com put ing in te gra tor.
(b) Cir cu lating bath.—45 0.005C. (Haake, Model FK,
re place ment Model D8L; Haake Buchler In stru ments, Inc., 244
Sad dle River Rd, Sad dle Brook, NJ 07662-6001, USA), or
equiv a lent.
(c) Cir cu lating bath.—78 0.05C. (Haake Model FS, or
equiv a lent. Dow Corning 200, 5cS vis cos ity sil i cone oil may be
used as bath me dium.)
(d) Sy ringe.—100 L Pre ci sion Sam pling, Se ries B, Model
01-9264, PRES SURE-LOK (Pre ci sion Sam pling Corp., 8275
West El Cajon, PO Box 15886, Ba ton Rouge, LA 70895, USA), or
equiv a lent.
(e) LC col umn.—610 7 (id) mm (24 3 8in. [od]) stain less steel
col umn packed with Aminex 50W-X4, 20–30 m, Ca resin (or,
al ter na tively, Aminex Q15-S, 19–25 m, Ca resin), and fit ted with
Wa ters As so ci ates Model 98764 col umn tem per a ture con trol block
and 3 8 in. (95 mm), 10 m frit, end fit tings.
C. Re agents
(a) Mo bile phase.—De gassed water, fil tered through 0.22 m
Millipore fil ter be fore use. Keep at ca 65C and stir slowly on hot
plate-stirrer to re move dis solved gas.
(b) Col umn pack ing.—Aminex 50W-X4, 20–30 m, or Q15-S,
19–25 m, Ca form resin. Slurry re quired amount of Aminex
50W-X4, H form, or Q15-S, Na form cat ion ex change resin
(Bio-Rad Lab o ra tories) with H 2O, trans fer to 350 mL me dium
po ros ity fritted glass fun nel, and re move H 2O with vac uum. Wash
each 10 g resin with 300 mL fil tered (Whatman No. 4 pa per, or
equiv a lent) 0.3M Ca(CH 3COO) 2 so lu tion ad justed to pH 6.2 with
CH 3COOH. Wash each 10 g resin with 200 mL H 2O. Re move H 2O
from treated resin with vac uum and store in nonactinic bot tle at
room tem per a ture.
(c) Mixed ion ex change resin.—Ex haust (2 L 1.5M NaOH) and
re gen er ate (2 L 1.5M HCl) ca 600 mL bed vol ume Duolite C-3
(Di a mond Sham rock Corp., PO Box 348, Painesville, OH 44077,
USA) cat ion resin. Re peat twice. Ex haust (2 L 1.5M HCl) and
re gen er ate (once each with 2 L 1.5M NaOH, and 2 L 1M Na 2CO 3) ca
600 mL bed vol ume Duolite ES-561 weak base an ion ex change
resin. Re peat twice. Wash fi nal re gen er ated res ins with H 2O to pH
ranges of 4–5 (C-3) and 6–7 (ES-461). Air dry res ins, com bine equal
weights of each, and mix. Avoid strong base res ins which may
pro mote al ka line isomerization of sugars.
(d) Car bo hy drate stan dards.—Glu cose; fruc tose ; malt oseH 2O;
maltotriose; acid con verted 42 Dex trose Equiv a lent (DE) corn
syrup; psicose [Carb. Res. 16, 383(1971)]; maltulose [Ce real
Sci ence To day 17, 180(1972)].
Es ti mate corn syrup com po si tion by de ter min ing level of each
saccharide shown for given DE in CRA, Inc. Crit i cal Data Ta bles,
pp 8–13 and 17. De ter mine DE as in 923.09C (see 44.1.15) and
941.14B (see 44.7.03). Pu rity of given sugar may be de ter mined by
nor mal ized LC anal y sis.
(e) Mixed sugar stan dard.—Pre pare stan dard sugar so lu tion to
con tain ca 3 g dry car bo hy drate sub stance/25 mL. Make dry ba sis
per cent of each stan dard sugar equiv a lent to level in typ i cal syrup
of given type. Use 42 DE corn syrup to rep re sent DP 4+ frac tion,
add ing dry weights of mono-, di-, and trisaccharide from 42 DE
syrup to dry weights of pure glu cose, malt ose, and maltotriose used
for stan dard prep a ra tion. Add ap pro pri ate dry weight of each
stan dard sugar to tared 50 mL beaker. Re cord weight to fifth place.
Add 20 mL H 2O, cover, and place on steam bath to dis solve.
Trans fer quan ti ta tively to 25 mL vol u met ric flask, di lute to
vol ume, and mix. Add sev eral drops to lu ene as pre ser va tive. Store
at room tem per a ture. Avoid to lu ene layer when draw ing up
so lu tion for stan dard in jec tion. Com pute dry ba sis con cen tra tion of
each sugar:
Sugar, dry ba sis, % =
weight sugar 100/(weight glu cose + a + b)
+ (weight fruc tose + c) + [(weight malt ose 0.94) + d + e]
+ [(weight maltotriose 0.94) + f + g +h] + weight DP 4+
where malt ose and maltotriose weights are mul ti plied by 0.94 to
ac count for H 2O of hydration and trace im pu ri ties; a and b = weight
glu cose from 42 DE syrup and maltulose; c = weight fruc tose from
maltulose; d and e = weights of DP 2 from 42 DE syrup and
maltulose; and f, g and h = DP 3 weights from 42 DE syrup,
maltulose, and malt ose.
D. Prep a ra tion of Col umn
Place empty col umn ver ti cally, and at tach 15 in. 3 8 in. (od)
(38.1 9.5 mm [od]) stain less steel precolumn, us ing 3 8 in. (9.5 mm)
un ion. At tach end fit ting to bot tom of col umn and vac uum line to
end fit ting. At tach open-ended ves sel (ca 500 mL) to precolumn
with Tygon tub ing and se cure ves sel in same plane as col umn.
Slowly fill col umn, precolumn, and ca 1 3 of ves sel with H 2O. Slurry
ca 50 mL bed vol ume of resin and add to ves sel, us ing vac uum.
Bring col umn to 78C. Pull vac uum on col umn un til resin level in
ves sel is con stant. Dis con tinue vac uum, dis con nect ves sel from
precolumn, and pump sol vent through precolumn and col umn at
0.5 mL/min (0.7 mL/min for Q15-S resin) over night. Dis con nect
end fit ting from precolumn and precolumn from col umn. Fill end
fit ting with ca 5 mm resin and at tach to col umn. Cap end with fit tings
when stor ing col umn.
E. Op er ating Con di tions
Flow rate 0.4 mL/min (0.6 mL/min for Q15-S resin); col umn
tem per a ture 78C; de tec tor tem per a ture 45C; de tec tor at ten u a tion
2006 AOAC INTERNATIONAL

8; de tec tor out put 10 mV; re corder at ten u a tion 1, re corder range
10 mV; chart speed 2 in./h. Pro gram in te gra tor to ob tain trap e zoidal
base line cor rec tion for each peak and to pre vent er ro ne ous base line
at val ley point be tween fused peaks or on pla teau cre ated by fused
peak clus ter.
F. Stan dard iza tion
In ject 30 L (ca 3.5 mg sol ids) of stan dard so lu tion. In te grate
stan dard sugar peaks in nor mal ized mode. Sum DP 4+ frac tions
ob tained from nor mal ized print out to ob tain DP 4+ nor mal ized
re sponse. Di vide known dry ba sis con cen tra tion by nor mal ized
re sponse for each com po nent to ob tain ra tios. Di vide each
com po nent ra tio by glu cose ra tio to ob tain Sys tem I KF val ues
(cal i bra tion fac tors). Pro gram com po nent KF val ues in Sys tem I,
B(c), us ing Method I and glu cose as ref er ence (KF glu cose = 1). En ter
DP 3 KF at both DP 3 re ten tion time and at time 100 s less than DP 3
time, to pro vide KF for panose, isomaltotriose, and lin ear DP 4 when
50W-X4 resin is used. List DP 4+ KF as de fault KF for DP 5+
cal cu la tion when 50W-X4 resin is used.
G. Prep a ra tion of Test So lu tion
De ter mine ap prox i mate dry sub stance as in 945.05C (see
44.7.05). Di lute syrup by weight to ca 12% dry sub stance with H 2O.
Add ca 0.3 g mixed ex change resin to ca 6 g di luted test portion and
shake 10 min to re move pos si ble in ter fer ing ionic ma te rial which
elutes in DP 4+ re gion.
H. De ter mi na tion
Rinse sy ringe with di luted test so lu tion 4 times be fore in jec tion.
In ject 30 L di luted test so lu tion, G. Best ac cu racy is ob tained when
dry sub stance sol ids in jected for test solution and stan dard are
equiv a lent. In te grate eluted peaks, us ing Sys tem I and Method I.
Af ter in jec tion, wash sy ringe 4 times with warm tap water, al low ing
air bub bles to scrub sy ringe walls. Wash sy ringe twice with H 2O.
I. Cal cu la tions
Re sults are com puted au to mat i cally when us ing integrator. List
fruc tose, glu cose, maltulose, and other DP 2 re sults. Com bine
maltotriose, panose-isomaltotriose, and lin ear DP 4 re sults, and list
sum as DP 3-4. Sum re main ing re sults and list as DP 5+. Re port re sults
on ash-free, car bo hy drate dry sub stance ba sis. In ab sence of
com put ing in te gra tor, list ar eas for fruc tose (f), glu cose (g),
maltulose, other DP 2, sum of maltotriose, panose-isomaltotriose and
lin ear DP 4, and sum of DP 5+, and com pute re sult by equa tion:
Com po nent, % = [(area com po nent)(KF com po nent)]
100/{[(area f)(KF f)] + [(area g)(KF g)] + . . .
+ [(area 5+)(KF 5+)]}
Ref er ences: JAOAC 62, 527(1979); 69, 258(1986).
CAS-50-99-7 (dex trose)
CAS-57-48-7 (fruc tose)
CAS-50-99-7 (glu cose)
CAS-17606-72-3 (maltulose)
CAS-23140-52-5 (psicose)
2006 AOAC INTERNATIONAL