SCHAFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO.

3, 1997 603

FOOD COMPOSITION AND ADDITIVES

Determination of Sugars in Beet and Cane Final Molasses by Ion

Chromatography: Collaborative Study
KEVIN SCHAFFLER and C.MJ. DAY-LEWIS
Sugar Milling Research Institute, University of Natal, King George V Ave, Durban, 4001, South Africa
MARGARET CLARKE
Sugar Processing Research Institute, Inc., 1100 Robert E. Lee Blvd, New Orleans, LA 70124
JILL JEKOT *

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Dionex Corp., 1228 Titan Way, PO Box 3603, Sunnyvale, CA 94088-3063

Collaborators: E. Rearick; B. Tungland; G. Steinle; L. Edye; P. Morel du Boil; E. Ramphal; G. Deruy; H. Hoebregs; B. Parslow;
H. Liecker; J. Jekot

A collaborative study of an ion chromatographic tech-
nique of high-performance anion-exchange with
pulsed amperometric detection has been
completed. The procedure was used to determine
sucrose, glucose, and fructose in 8 cane molasses
samples and sucrose in 5 beet molasses samples.
Eleven laboratories took part in the study. Repeat-
ability and reproducibility values for sucrose, glu-
cose, and fructose were excellent for both types of
samples. All criteria for accepting the method for
sugars in both beet and cane molasses were met.
The ion chromatographic method for determination
of sugars in beet and cane final molasses has been
adopted first action by AOAC INTERNATIONAL.
Gas chromatography (GC) has long been used for sugar
analysis. Although results are accurate, the process involves a
time-consuming derivatization step. The Sugar Milling Re-
search Institute (2) has used HPAE-PAD to determine glucose,
fructose, and sucrose in cane molasses for many years. The
method has been modified to include sucrose in beet molasses.
Raffinose also can be determined, but revision of the procedure
to include raffinose has not been tested collaboratively.
The HPAE-PAD method has evolved over several years,
with several researchers making important contributions. In
1990, Morel du Boil and Schaffler (2) compared anion- and
cation-exchange liquid chromatography for carbohydrates.
They showed that anion-exchange columns produced more ac-
curate results than cation-exchange columns and RI detection.
In 1990, Thompson (3) described the use of lactose as an inter-
nal standard to negate errors caused by sample dilution. Detec-
tor drift was accounted for by bracketing samples with calibra-

T
he purpose of this study was to evaluate an ion chroma-
tion standards. In 1992, Day-Lewis and Schaffler (4) compared
tographic (IC) method for analysis of sugars in beet and
this method and the South African sugar factories' official GC
cane molasses by collaborative study for AOAC
method. The precision and accuracy of the HPAE-PAD results
INTERNATIONAL.
compared favorably with those of the GC method (5).
Many impurities in sugar factory liquors are concentrated in
Recently, Schaffler and Day-Lewis ran a collaborative study
final molasses, making it a difficult process stream to analyze
for determination of sugars in molasses under the auspices of
chromatographically. Some impurities co-elute with sugars and
the International Commission of Uniform Methods for Sugar
produce inflated results when samples are analyzed by cation-
Analysis (ICUMSA). The IUPAC protocol for collaborative
exchange columns with refractive index (RI) detection (1). At-
studies was officially adopted by ICUMSA in 1990 (6). Be-
tempts to remove these impurities from final molasses were not
cause all the repeatability and reproducibility criteria were met,
effective (1). High-performance anion-exchange with pulsed
the HPAE-PAD procedure was adopted as an official method
amperometric detection (HPAE-PAD) is more specific for car-
at the 1994 ICUMSA meeting (7).
bohydrates than the cation-exchange/refractive index proce-
dure. The impurities in molasses are, therefore, less important.
Collaborative Study
Submitted for publication October 13, 1995.
The recommendation was approved by the Methods Committee on Eleven laboratories analyzed 2 cane molasses samples from
Food Nutrition, and was adopted by the Official Methods Board of the Australia, 2 from Mauritius, and 4 from South Africa. Two beet
Association. See "Official Methods Board Actions" (1996) /. AOAC Int. 79,
molasses samples from Germany and 3 from the United States
42A, and "Official Methods Board Actions" (1996) The Referee, March
issue. also were dispatched by airmail to all collaborators. Instruc-
Author to whom correspondence should be addressed. tions to freeze the samples until analysis could be undertaken
604 SCHAFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, No. 3,1997

Table 996.04A. Method performance for determination of sugars in beet and cane final molasses by ion
chromatography
Molasses Sugar Sr SR ra Rb RSDr, % RSDR, %

Cane Sucrose 0.26 0.44 0.73 1.23 0.82 1.38

Glucose 0.06 0.13 0.17 0.36 1.74 3.81
Fructose 0.08 0.13 0.22 0.36 1.24 2.04
Beet Sucrose 0.29 0.85 0.81 2.38 0.57 1.69

r = 2.8 x sr.
R = 2.8 x sB

were included in each shipment. Instructions also were sent by B. Apparatus

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fax to participants.
996.04 Sugars in Cane and Beet Final Molasses,
Ion Chromatographic Method,
ICUMSA-AOAC Method
First Action 1996
(Applicable to determination of sucrose, glucose, and fruc-
tose in cane final molasses and sucrose in beet final molasses.)
Caution: See Appendix Laboratory Safety for "Safe Han-
dling of Alkalies." Dispose of waste solvents in appropriate
manner compatible with applicable environmental rules and
regulations.
Method Performance:
See Table 996.04A for method performance data.
A. Principle
Method uses pellicular anion-exchange resin column cou-
pled with highly specific and sensitive pulsed amperometric
detection. Most carbohydrates are very weak acids with pKa
values above 11. NaOH ionizes carbohydrates to anionic form
and elutes compounds of interest in approximate order of pKa.
Column selectivity can be controlled by pH of eluant. Pulsed
amperometric detector applies potential at electrode to oxidize
ionized carbohydrates and uses repeating sequence of 3 poten-
tials applied for specific durations optimized for carbohydrates
to prevent fouling of electrode surface. To drive oxidation re-
action at the working electrode of pulsed amperometric detec-
tor, pH of eluant must be at least 12.2.
Table 996.04B. Preparation of carbohydrates standard
(a) Liquid chromatographic (LC) system.—Equipped with
either isocratic or gradient pump equipped with inert polyether
ether ketone (PEEK) flow path and electrochemical detector
with amperometric cell and gold working electrode, or equiva-
lent. Fitted with injection loop capable of delivering 10-
100 |iL. Operating conditions: flow rate, 1 mL/min; tempera-
ture, ambient; detector settings, optimum parameters for
carbohydrates as provided by manufacturer. Carbohydrates re-
tention times (min): glucose, 3.90; fructose, 4.4; lactose, 7.0;
sucrose, 8.5 with resolution between peaks of 1. Do not recycle
eluant.
(b) LC column.—250 x 4 mm id; packed with polystyrene
divinylbenzene substrate (10 Lim diameter) agglomerated with
quaternary amine functionalized latex (350 nm diameter).
(c) Data collection system.— Compatible with LC.
(d) Autosampler.—Suitable for use with LC system; capa-
ble of loading a loop or doing full loop injection.
(e) Analytical balance.—Accuracy, ±0.1 mg.
(f) Volumetric flasks.—100 and 250 mL.
(g) Pyrex bottle.—For eluant preparation; fitted with screw
cap containing 3 small holes (for helium sparging tube, eluant
outflow line, and vent to ensure constant blanketing without
buildup of pressure); capable of holding 2 L.
(h) Schott screw-cap bottle.—250 mL.
(i) Beaker.—50 mL.
(j) Magnetic stirrers.
(k) Pipets.—1,5, and 25 mL.
(1) Sparger.—Vacuum can be used.
(m) Membrane filters.—0.45 u,m porosity.
Table 996.04C. Preparation of carbohydrates standard
stock solutions for cane final molasses
Carbohydrates standard stock solution

Carbohydrates standard stock solution

Carbohydrate Ci C2 c3
Carbohydrate B, B2 B3 Glucose, g 0.020 0.060 0.100
Fructose, g 0.030 0.070 0.110
Sucrose, g 0.25 0.30 0.35 Sucrose, g 0.250 0.310 0.370
Lactose internal Lactose internal
standard solution, mL 5.0 5.0 5.0 standard solution, mL 5.0 5.0 5.0
 SCHAFFLER ET A L . : JOURNAL OF AOAC INTERNATIONAL VOL. 80, No. 3, 1997 605

Table 1. Sucrose in cane molasses by HPAE-PAD, summary statistics

Sample No.

Parameter 1 2 3 4 5 6 7 8

Labs retained 11 11 11 11 9 10 10 9
Labs rejected 0 0 0 0 2 1 1 0
Mean value, % 31.18 31.11 30.69 31.20 32.48 33.71 31.64 30.77
Sr 0.26 0.34 0.16 0.28 0.22 0.24 0.26 0.29
sR 0.48 0.40 0.33 0.37 0.53 0.52 0.41 0.45
RSDr 0.85 1.11 0.53 0.89 0.68 0.72 0.84 0.95
RSDR 1.53 1.30 1.09 1.20 1.64 1.53 1.31 1.46
Horwitz value 2.38 2.38 2.39 2.38 2.37 2.36 2.38 2.39
Horwitz ratio 0.64 0.54 0.46 0.50 0.69 0.65 0.55 0.61

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r 0.75 0.97 0.46 0.79 0.63 0.68 0.75 0.83
R 1.35 1.14 0.94 1.06 1.50 1.46 1.17 1.27

C. Reagents
Note: It is essential that air (i.e., C0 2 ) be excluded at all
times. At all stages of eluant handling or preparation, degas by
using vacuum or by sparging with helium. Store NaOH solu-
tions under helium in Pyrex vessels. Do not recycle eluant.
(a) Sodium hydroxide (NaOH).—50% (w/w) aqueous
stock solution (low carbonate [<0.03%]).
If low-carbonate 50% NaOH solution is not available, pre-
pare as follows: Filter 150 mL deionized or reverse-osmosis
H20 through 0.45 u,m membrane filter into 250 mL Schott
screw-cap bottle containing magnetic stirrer. Stir and degas by
using vacuum or by sparging with helium at least 30 min.
Weigh 150 ±0.1 g low-carbonate NaOH pellets just before
use. Rapidly add pellets to degassed H 2 0 and continue degass-
ing and stirring until pellets are dissolved. Allow solution to
cool slightly. Remove stirrer and sparger or vacuum and close
bottle. Leave 50% NaOH stock solution undisturbed at least
7 days before use. Store 50% NaOH solution up to 6 months or
until carbonate precipitate is evident. Note: Always blanket so-
lution with helium when opened. Carefully pipet solution from
clear portion. Never pour from the bottle. Close bottle immedi-
ately after use.
(b) Eluant.—150 mM NaOH. Place 2 L deionized
(18 MQ.) or reverse-osmosis H 2 0 (filtered, if necessary) in Py-
rex bottle, B(g). Sparge with helium or degas ca 30 min. Pipet
15.6 mL 50% NaOH stock solution, (a), into sparged or de-
gassed H 2 0. Store eluant up to 1 week. Do not use more than
top 3 quarters of 50% NaOH solution because of possible so-
dium carbonate precipitation. Note: Never pour 50% NaOH
stock solution from the bottle. Always pipet the amount needed.
Note: Decrease in carbohydrate retention times indicate
C0 2 contamination of eluant. Prepare fresh 50% NaOH stock
solution if contamination is suspected.
(c) Carbohydrate standards.—Sucrose, glucose, fructose,
and lactose; reagent grade, desiccant dried.
(d) Lactose internal standard solution.—3.2%. Weigh
8 ± 0.01 g lactose standard into 250 mL volumetric flask, dis-
solve in H20, and then dilute to volume with H20.
D. Preparation of Carbohydrate Standard Solutions
(a) Carbohydrate standard solutions.—(1) Stock solu-
tions.—Using Tables 995.04B and/or C (depending on type of

Table 2. Glucose in cane molasses by HPAE-PAD, summary statistics

Sample No.

Parameter 1 2 3 4 5 6 7 8

Labs retained 11 11 11 11 9 9 10 9
Labs rejected 0 0 0 0 2 2 1 2
Mean value, % 4.30 4.13 4.42 4.27 1.77 2.49 4.22 3.03
sr 0.05 0.10 0.08 0.07 0.04 0.07 0.03 0.04
SR 0.15 0.16 0.16 0.18 0.08 0.11 0.11 0.11
RSDr 1.18 2.45 1.70 1.54 2.29 2.70 0.82 1.22
RSDR 3.58 3.85 3.56 4.16 4.75 4.27 2.68 3.62
Horwitz value 3.21 3.23 3.20 3.22 3.67 3.49 3.22 3.38
Horwitz ratio 1.11 1.19 1.11 1.29 1.29 1.22 0.83 1.07
r 0.14 0.29 0.21 0.19 0.11 0.19 0.10 0.11
R 0.44 0.45 0.44 0.50 0.24 0.30 0.32 0.31
606 SCHAFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, No. 3,1997

Table 3. Fructose in cane molasses by HPAE-PAD, summary statistics

Sample No.

Parameter 1 2 3 4 5 6 7 8

Labs retained 11 9 11 9 10 9 9 8
Labs rejected 0 2 0 2 1 2 2 3
Mean value, % 6.96 6.97 7.13 6.71 5.99 5.89 6.13 6.17
sr 0.11 0.08 0.08 0.07 0.05 0.06 0.10 0.10
sR 0.13 0.08 0.12 0.06 0.27 0.19 0.09 0.08
RSDr 1.59 1.21 1.18 1.02 0.81 0.98 1.59 1.57
RSDR 1.91 1.19 1.65 0.96 4.48 3.22 1.55 1.38
Horwitz value 2.99 2.99 2.98 3.00 3.05 3.06 3.04 3.04

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Horwitz ratio 0.64 0.40 0.56 0.32 1.47 1.05 0.51 0.45
r 0.31 0.24 0.24 0.19 0.14 0.16 0.28 0.27
R 0.38 0.23 0.33 0.18 0.76 0.54 0.27 0.24

molasses tested) weigh amounts of carbohydrate standards,
C(c), into 100 mL volumetric flasks, dissolve in deionized or
reverse-osmosis H 2 0, and dilute to volume with H 2 0. (2)
Calibrating standard solutions.—Transfer 1 mL each carbohy-
drate standard stock solution, (1), into separate 100 mL volu-
metric flasks and dilute to volume with deionized or reverse-
osmosis H 2 0. Filter through 0.45 |im filter into autosampler
bottles. Prepare 2 autosampler vials of each carbohydrates cali-
brating standard solution. In addition, prepare (n + 1) autosam-
pler vials of calibrating standard solution B 2 (when testing beet
molasses) and/or C2 (when testing cane molasses), where n -
number of test samples.
Transfer standard solutions into small air-tight vials and store
frozen. Always use freshly thawed standard solution for analysis.
Before analyzing test samples, run carbohydrate calibration
standard solutions, D(a)(2) (B!-B 3 for beet molasses and Q -
C3 for cane molasses). If sucrose response is not linear, increase
dilution factors for standards and test samples.
E. Preparation of Test Samples
(a) Beet molasses.—Weigh 0.7 g test sample into beaker
and then quantitatively transfer into 100 mL volumetric flask
with deionized or reverse-osmosis H 2 0. Pipet 5 mL lactose in-
ternal standard solution, C(d), to test sample and dilute to vol-
ume with deionized or reverse-osmosis H 2 0. If 1.4 mm2 work-
ing electrode is used in analysis, place 1 mL sample-internal
standard solution into 100 mL volumetric flask and dilute to
volume with deionized or reverse-osmosis HzO. When using
3.0 mm2 working electrode, dilute 3 mL sample-internal
standard solution to 100 mL with deionized or reverse-osmosis
H 2 0. Filter diluted solution through 0.45 \im filter into
autosampler bottle. Prepare duplicate samples in separate con-
tainers starting at weighing step.
(b) Cane molasses.—Weigh 1.0 g test sample into beaker
and then quantitatively transfer into 100 mL volumetric flask
with deionized or reverse-osmosis H 2 0. Pipet 5 mL lactose in-
ternal standard solution, C(d), to test sample and dilute to vol-
ume with deionized or reverse-osmosis H 2 0. If 1.4 mm2 work-
ing electrode is used in analysis, place 1 mL sample-internal
standard solution into 100 mL volumetric flask and dilute to
volume with deionized or reverse-osmosis H 2 0. When using
3.0 mm working electrode, dilute 3 mL sample-internal
standard solution to 100 mL with deionized or reverse-osmosis
HzO. Filter solution through 0.45 jam filter into autosampler

Table 4. Sucrose in beet molasses by HPAE-PAD, summary statistics

Sample No.

Parameter 1 2 3 4 5

Labs retained 9 10 10 10 10
Labs rejected 2 1 1 1 1
Mean value, % 51.39 51.83 51.66 50.49 47.60
Sr 0.25 0.33 0.34 0.27 0.26
sR 0.78 1.02 0.67 0.90 0.88
RSDr 0.49 0.63 0.65 0.53 0.54
RSDR 1.52 1.97 1.30 1.79 1.85
Horwitz value 2.21 2.21 2.21 2.22 2.24
Horwitz ratio 0.69 0.89 0.59 0.81 0.83
r 0.71 0.93 0.95 0.76 0.73
R 2.21 2.89 1.90 2.55 2.49
 SCHAFFLER ET A L . : JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 3, 1997 607

m c o c D c D f - c n ' ^ j - o c o o c o bottle. Prepare duplicate samples in separate containers starting

d i - :
T - :
o d o d ' t c o c ) T ^ at weighing step.
COCOCOCOCOCOCOCOCMCOCO

« i i o N T - T - n « i ' t * o o F. Preparation ofLC Column

0 ) O C O N I I I « ) ( I I * S I O T -
o i ^ ^ r d d d ^ i r i c i d Check condition of LC column by running one of the carbo-
CMCOCOCOCOCOCOCOCMCOCO
hydrate calibrating standard solutions, D(a)(2). If retention
times have decreased and/or resolution is unacceptable, clean
r ^ i - r ^ c D o c N o c o c o c O ' q -
C M i n c o i o c o c o c o c n c n ' t c o or regenerate column (see manufacturer's instruction). De-
^ T ^ N i ^ r ^ l l i ^ d ^ O J
cococococococococococo crease in retention times or unacceptable resolution can be
caused by carbonate coming from eluant buildup on LC col-
i ^ t - - * u n c o o c D i n c \ i m c n
T-iotOT-oiSMrinnto umn. If C0 2 contamination is suspected, prepare fresh eluant,
•r^T-^CJT^i^T^inCMr-^-i-^T-^
cococococococococococo
C(b). Wash column 15 min with fresh eluant. Rerun carbohy-
drate calibrating standard solution to check retention times.

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If retention times still are not acceptable, LC column may
T- in n n •* w s (D r S S
O O CD CM -<t O r^ need to be regenerated as follows: Wash column extensively
n T t T t ^ c o f c o c om i ni n n co
c oco
COCOCOCOCOCOCOCOCMCOCO with H 2 0. Disconnect and bypass electrochemical cell. Flush
c o o o o c n m c n m c n c o c D c n
column 1 h with 1.0M NaOH. Reconnect electrochemical cell
T - c o f - c o c M c n - ^ c q c o i n o
c o - ^ ^ c o c o c o c o c o c o c o c o and reequilibrate column with eluant, C(b). Rerun carbohy-
cocOcOcOcOcocOcocOcoc O
drates calibrating standard solutions, D(a)(2), (B!-B 3 for beet
molasses and Q-C3 for cane molasses) to check retention
times and resolution. If LC column is still not delivering ac-
t - c o c D o j c n c n c M i n r-
m c o N t s o i o q cn
ceptable retention times and resolutions, acid wash may be neces-
cT^ o cCMo cCOo cCMo cCM
o c To^ c oc
CM ocMcoco
cri C M C M sary. Follow manufacturer's instructions in troubleshooting guide.
c o i - c n i - r ^ r v c o o o c n c D c o
ooT-:'itT-:cocMCDr^cnocM G. LC Determination
i-^COCOCMCMCMCMCOCnCMCM
COCOCOCOCOCOCOCOCMCOCO
Before analyzing test samples, run carbohydrate calibration
standard solutions, D(a)(2) (B!-B 3 for beet molasses, and Q -
O t t ) o i s o ) i - c o n o < f n C3 for cane molasses) to establish system's linearity. Run du-
l O T - c o i o c o ^ u i c o o i T - i n
plicate vials from each test sample and carbohydrate calibrating
cococococococococococo
standard solution, either B 2 or C2 (depending on type of molas-
( o « ] 0 ) M O * * s o > c « n ses), so test vials are "sandwiched" by standard solution. Con-
< O T t i n ( D O U ) S * N " f O
cococococococococococo tinue until all test samples have been analyzed.
H. Calculations
cncMcococnTtcMinT-incD
c n c n c o - i - c o c o - t c q i o c D c n
For calibration, use internal standard technique using peak
c r i d o T ^ d d d d d d d heights or peak areas. Use average response factors from car-
CMCOCOCOCOCOCOCOCOCOCO
bohydrate calibrating standard solution B 2 or C2 bracketing test
c D o o T - o c o m c n o o o o c n
c n c o ^ o c o m: c o o o o i n: c o
c r i d - ' - T - o d o d i - d o
samples to calculate sugar concentrations for each test sample
CMCOCOCOCOCOCOCOCOCOCO (e.g., for beet molasses: B2, sample la, sample lb, B2, sample
2a, sample 2b, B2> etc.).
Calculate relative response factor (RRF) for each sugar in
S O l O T t T - O O O B l t O )
L o r ~ - o t c o o r - - ' * T f r - . o o each carbohydrate calibration standard solution that is run. For
d d c M T ^ i - ^ T ^ i - ^ i - ^ T - ^ d d
cococococococococococo example, calculate RRF for sucrose (RRFSUC) as follows:
• > - T - m i n c n c D i n - * c \ i o j c n
TfCOCO-i-OCMr^-COCO'^CO ^*suc-std Mac-std
d d i - ^ i ^ T - ^ - i - ^ d T ^ d T ^ d RRF„,C = X
COCOCOCOCOCOCOCOCOCOCO P
1
M,lac-std
suc-std

where Msuc_std = mass of sucrose in carbohydrate calibrating

incMi-cnincor~--*-^-cMCM
t o o i o i i O T - N o ^ q c o standard solution, g; Psuc-Std - P ea k height of sucrose in cali-
d r d r O N ^ r r r ^ d
cococococococococococo
brating standard solution; Mlac_std = mass of lactose in calibrat-
c o o o c M r - c M o o c n ' ^ - c o i - ing standard solution, g; P^-s^ = peak height for lactose in
i n o N t i o s t o o t o i o f
d - r ^ T - ^ - ^ d i ^ d d T ^ T ^ T - : calibrating standard solution.
cococococococococococo
Calculate concentration of sugars (e.g., sucrose) in test sam-
ples as follows:
(1) Determine average RRF value of sucrose (/^?Fave_suc)
from RRF of calibrating standard solutions immediately before
T - w n - j i n ^ s c o o i o ^ and after injecting final molasses sample:
608 SCHAFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, No. 3,1997

Table 6. Raw data for glucose in cane molasses by HPIC, 1993 ICUMSA collaborative study

Sample

1 2 3 4 5 6 7 8

Laboratory A B A B A B A B A B A B A B A B

1 4.10 4.07 3.95 3.95 4.36 4.32 4.16 4.13 1.72 1.75 2.41 2.42 4.11 4.13 2.96 2.97
2 4.18 4.23 4.06 4.06 4.48 4.48 4.21 4.16 1.76 1.69 2.41 2.44 4.06 4.05 2.96 3.00
3 4.14 4.21 3.81 4.20 4.19 4.05 4.10 3.88 1.69 1.79 2.41 2.64 4.12 4.35 2.94 2.98
4 4.39 4.35 4.39 4.19 4.43 4.42 4.32 4.33 1.79 1.74 2.54 2.52 4.27 4.29 3.24 3.16
5 4.23 4.25 4.15 4.12 4.54 4.61 4.28 4.25 1.78 1.73 2.45 2.52 4.14 4.21 3.09 3.14
6 4.25 4.33 4.06 4.12 4.39 4.34 4.18 4.23 1.71 1.73 2.43 2.46 4.17 4.22 3.02 3.00
7 4.43 4.57 4.28 4.28 4.32 4.57 4.55 4.67 1.87 1.87 5.94 5.97 4.33 4.29 3.03 3.08

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8 4.30 4.31 4.02 4.10 4.46 4.32 4.28 4.28 2.24 2.26 2.51 2.45 4.21 4.28 3.89 3.86
9 4.57 4.43 4.17 4.30 4.60 4.58 4.38 4.41 2.92 2.91 4.49 8.25 4.37 4.31 2.78 3.15
10 4.50 4.49 4.38 4.31 4.58 4.62 4.47 4.40 1.92 1.93 2.71 2.69 4.38 4.39 3.17 3.15
11 4.15 4.13 3.95 3.93 4.30 4.20 4.17 4.03 1.72 1.63 2.44 2.31 4.13 4.06 2.92 2.82

Table 7. Raw data for fructose in cane molasses by HPIC, 1993 ICUMSA collaborative study

Sample

1 2 3 4 5 6 7 8

Laboratory A B A B A B A B A B A B A B A B

1 6.82 6.86 6.85 6.96 6.99 7.02 6.75 6.76 5.93 5.91 5.96 5.94 6.15 6.22 6.08 6.24
2 6.82 6.93 6.97 6.95 7.13 7.11 6.80 6.71 5.87 5.83 5.87 5.87 6.02 6.01 6.09 6.21
3 7.18 6.89 7.50 7.55 7.33 7.30 7.08 7.11 6.33 6.20 6.25 6.38 6.55 6.72 6.65 6.58
4 6.94 6.98 6.91 6.87 6.94 7.02 6.69 6.69 5.77 5.73 5.80 5.83 6.12 6.11 6.18 6.11
5 6.78 6.77 6.99 6.90 7.10 7.18 6.70 6.61 5.77 5.70 5.70 5.88 6.01 6.11 6.17 6.24
6 6.93 6.95 6.99 7.07 7.12 7.05 6.67 6.71 5.84 5.86 5.84 5.80 6.13 6.21 6.15 6.13
7 7.02 7.35 7.28 7.29 7.20 7.37 7.05 7.26 6.09 6.16 8.14 8.37 6.43 6.26 6.22 6.25
8 6.97 6.97 7.08 7.04 7.09 7.19 6.83 6.70 6.50 6.60 5.84 5.83 6.11 6.28 6.83 6.89
9 7.14 6.90 6.82 7.08 7.09 7.05 6.66 6.69 6.16 6.09 6.89 6.66 6.13 6.01 5.23 5.99
10 6.98 6.97 7.04 7.00 7.10 7.14 6.76 6.67 5.76 5.79 5.97 5.94 6.09 6.19 6.22 6.27
11 7.00 6.95 7.07 6.90 7.32 7.02 6.76 6.56 6.07 5.73 5.60 5.68 6.34 6.03 6.26 5.95

Table 8. Raw data for sucrose in beet molasses by HPIC, 1993 ICUMSA collaborative study

Sample

1 2 3 4 5

Laboratory A B A B A B A B A B

51.24 51.26 51.26 51.57 51.60 51.65 50.74 50.50 48.05 48.33
52.56 52.23 50.99 51.60 50.99 51.03 50.64 50.02 47.12 46.80
52.84 52.41 52.74 52.86 52.09 52.47 50.67 50.19 48.16 48.16
50.20 49.81 50.86 50.54 51.88 50.92 49.18 49.51 46.82 46.12
51.27 51.14 51.41 51.30 51.22 51.19 50.19 49.98 47.50 47.32
51.13 51.39 51.86 52.25 51.17 51.86 50.79 50.72 46.70 47.31
53.68 55.61 53.77 52.79 52.80 52.88 51.87 51.67 49.38 49.17
51.24 51.33 51.06 50.59 51.40 51.43 49.57 50.20 47.07 47.07
51.36 50.84 51.42 51.00 50.92 50.98 49.56 49.61 47.15 47.03
51.12 51.68 53.32 53.49 51.94 52.78 51.89 52.31 48.57 48.14
 SCHAFFLER E T AL.: JOURNAL OF A O A C INTERNATIONAL VOL. 8 0 , NO. 3 , 1997 609

Table 9. HPIC conditions used by participants

Collaborator
Variable Rearick Tunglanc1 Steinle Edye duBoil Ramphal

Column & guard CarboPac PA1 CarboPac CarboPac CarboPac CarboPac CarboPac
Column temp. Ambient 23°C Ambient Ambient 26°C Ambient
Flow, mL/min 1.0 1.0 0.6 1.0 1.0 1.0
Column pressure,
psi 1350 850 1600 1500 1400 1250
Volume injected,
HL 20 20 20 20 20 20
Pump Dionex Dionex Dionex Dionex Waters M-45 SP IsoChrom
Detector PAD-2 PED-2 PAD-2 PED-2 PAD-2 PAD-2

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Range 10K(nA) 10 uC 10K(nA) 30 uC 10K(nA) 10K(nA)
Integrator HP 3396 (II) Dionex AI-450 Apex Chrom Dionex AI-450 HP 3396 HP 3396
Autosampler Waters 717 Dionex GPM-II Micro-meritics SP 8800 SGE LS3200 SP 8800
Integration mode Area Height Height Height Height Height

Collaborator

Details Deruy Hoebregs Parslow Lieker Jekot

Column & guard CarboPac CarboPac CarboPAc CarboPac CarboPac

Column temp. 25°C 40°C 25°C 20°C Ambient
Flow, mL/min 1.0 1.0 1.0 1.0 1.0
Column pressure, psi 908 1100 1490 1520 1100
Volume injected, uL 50 100 50 20 50
Pump Dionex Dionex GPM-2 Dionex Dionex GMP Dionex
Detector PAD-2 PED-1 PAD-2 PAD-2 PAD-2
Range 3K(nA) 10 uC 10K(nA) 1K(nA) 30K(nA)
Integrator Shimadzu ICR1B Shimadzu CR-4AX Drew Chrom Data Sys Merck D-2000 Dionex Al 450
Autosampler Gilson 231 Dionex Dionex SP 8800 Dionex
Integration mode Height Height Height Height Height

RRFt + RRF2 Results and Discussion

/</</ < a v e _ s u c —
where RRF^^s^ - average RRF for sucrose; RRFi = RRF
of sucrose before injecting molasses sample; RRF2 = RRF of
sucrose after injecting molasses sample.
(2) Determine percentage of sucrose in final molasses sam-
ple as follows:
v
Sucrose, % = f - x RRFme_suc x —
* !ac-smp
lar-«mn
where P,sue—smp = peak height of sucrose in test sample;
Miac-smp = mass of lactose internal standard in test sample, g;
Plac_smp = peak height of lactose in test sample; Mmol_smp =
mass of test sample, g.
Relative standard deviation (RSDr) should be no more than
2% for duplicates of sucrose and no more than 3% for dupli-
cates of glucose and fructose. Otherwise reinject test sample
onto LC column as in G, or prepare fresh test samples as in E
and proceed with analysis.
Ref.: J. AOAC Int. 80, 603-610(1997)
No participant reported any serious problem with the proce-
dure. The collaborative study was truly international, with par-
ticipants from 8 countries. Statistical summaries for all sugars
in cane and beet molasses can be found in Tables 1-4. Sum-
mary data for the collaborative study are presented in Tables 5 -
8. The experimental conditions used by participants are shown
in Table 9.
100 The recommended procedure for testing outliers was fol-
lowed. The number of outliers never exceeded the limit of 2 in
^ " mol-smp
i
9 laboratories. The HPAE-PAD technique produced excellent
within-laboratory precision for all sugars. Average RSDr values
for sucrose, glucose, and fructose were 0.8,1.7, and 1.2%, respec-
tively. Average RSDr for sucrose in beet molasses was 0.6%.
The procedure also produced most acceptable laboratory-to-
laboratory scatter. Average RSDR for sucrose, glucose, and
fructose in cane molasses were 1.4,3.8, and 2.0%, respectively.
The average RSDR for sucrose in beet molasses was excellent
(1.7%). The Horwitz ratios were well below 2 for all cases,
confirming that the variation between laboratories (RSDR) was
excellent. These same samples were also analyzed by GC in
610 SCHAFFLER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, No. 3, 1997
5 laboratories (7). The agreement between techniques was
excellent.
Raffinose is the major oligosaccharide in beet products. A
preliminary investigation into measuring raffinose was briefly
looked at during the study. Because raffinose is present in beet
molasses at fairly low levels, (0.3-1.0%) and because of the
high dilution factor required for optimal sucrose analysis, a re-
liable estimation of raffinose could not be made. An accurate
determination of the raffinose content can be made by injecting
the initial solution without dilution. The raffinose content
should not be greater than 100 mg/L. Above this concentration,
the detector's response is less linear.
Recommendation
On the basis of the results of this study, it is recommended
that the ion chromatographic method for determination of sug-
ars in cane and beet final molasses be adopted first action.
Acknowledgments
We thank Erik Halbert of CSR Australia; Lyn Wong Sak Hoi
of Mauritius Sugar Industry Research Institute; Danielle de
Gaye of SMRI, South Africa; George Steinle of Siidzucker,
Germany; and Brian Tungland of Holly Sugar, Colorado
Springs, CO, for preparing and distributing the molasses samples.
The following analysts took part in the collaborative study:
Eugene Rearick, Amalgamated Sugar, Twin Falls, ID
Brian Tungland, Holly Sugar, Colorado Springs, CO
George Steinle, Siidzucker AG Mannheim/Ochsenfurt,
Obrigheim, Germany
Les Edye, SPRI, New Orleans, LA
Pamela Morel du Boil and Eshara Ramphal, SMRI, Univer-
sity of Natal, Dalbridge, South Africa
Genevieve Deruy, IRIS, Villaneuve, D'ascq Cedex, France
Hubert Hoebregs, Raffinerie Tirlemontoise, Tienen, Belgium
Bob Parslow, British Sugar, Norwich, United Kingdom
H. Liecker, Braunschweig Technical University, Braunschweig,
Germany
Jill Jekot, Dionex Corp., Sunnyvale, CA
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