Application solution of galactooligosaccharides
in infant formula milk powder by ion chromatography
Qingdao Shenghan Chromatograph Technology Co., Ltd.
Foreword
Galactooligosaccharides(GOS) is a kind of functional
oligosaccharide with natural attribute.In nature,there are
trace amounts of GOS in animal milk, while human breast
milk is abundant. The establishment of bifidobacterial
florain infants depends largely on the GOS components in
breast milk.
GOS is an excellent nutrient source and effective
proliferator of beneficial bacteria such as bifidobacterium
and lactobacillus acidophilus in human intestine. It can
improve digestive and absorption function of human
intestinal tract.The digestive function of the newborn is relatively weak, so infant milk powder has
been added with galactooligosaccharides, it can not only improve the digestive function of the baby,
but also promote the absorption of calcium in the baby, thereby enhancing the baby's immunity.
Implementation standard
AOAC official method 2001.02 detection method of GOS in food,Ion exchange
chromatography
Reagents and standards
All the water used in the detection process of all the preparation solution must be 18MΩ DI
water(ultrapure water).
3.1 Phosphate buffer：0.2M，pH6.0。22.0g KH2PO4 and 6.0g K2HPO4 ・3H2O are dissolve in the water, diluted
to 1L, then sterilized in autoclave at 120℃/30min;
3.2 Hydrochloric acid solution：1M; 8.3mL concentrated HCl is diluted to 1L with water;
3.3 Sodium hydroxide solution：50%，Hydrochloride free;
3.4 Sodium hydroxide solution：1M，54mL sodium hydroxide solution is diluted to 1L with water which is
remove CO2;
3.5 β-galactosidase solution:2000U/mL, 50000U/g β-galactosidase(produced from aspergillus oryzae
fermentation) is suspended in the phosphate buffer, and the solution with final activity of 2000U/mL was
obtained.One unit hydrolyzes o-Nitrobenzene-β-D-galactose at the condition of pH4.5/25℃, then generate o-
2
Nitrophenol and D-galactose. Store the suspension in the refrigerator when it is not in use, and fully mix the
suspension before use. Use within 8 hours;
3.6 Acetonitrile：LC level；
3.7 Acetonitrile solution：20%(v/v),200mL acetonitrile is diluted to 1L with water;
3.8 Acetonitrile solution：3%(v/v),30mL acetonitrile is diluted to 1L with water;
3.9 Sodium acetate trihydrate：Sodium acetate trihydrate without water，reagent grade；
3.10 Mobile phase A：12.5mM NaOH，without carbonate;
3.11 Mobile phase B：125mM NaOH，without carbonate;
3.12 Mobile phase C：125mM NaOH(without carbonate) and 500mM Sodium acetate trihydrate;
3.13 Galactose：Without water；
3.14 Lactose：一 water(stable at 103℃);
3.15 Sugar standard storage solution: Dry galactose standard sample and lactose at 103℃ for 4 hours to
constant weight.Accurately weigh 80mg galactose (S1), put it into a volumetric flask, dilute it to scale with
water(0.8mg galactose /mL);Accurately weigh 150mg lactose monohydrate(S2′),prepare lactose solution (1.425mg
anhydrous lactose/mL ) in the same way. S2′ multiplying 0.95 obtains the weight of anhydrous lactose(S2), and
compensates the weight of lactose crystal water.
3.16 Working standard solution:Remove S1 solution and S2 solution each 5.00mL (WS1),and put them into a
1L volumetric flask and dilute them to scale;Repeat the above operation,remove the two kinds of solution for each
10.00mL(WS2),15.00mL(WS3) and 20.00mL(WS4),then dilute them to 1L solution.
Solution mLS1 mLS1 galactose +lactose
WS1 5.00 5.00 4ug/mL+7.125ug/mL
WS2 10.00 10.00 8ug/mL+14.25ug/mL
WS3 15.00 15.00 12ug/mL+21.375ug/mL
WS4 20.00 20.00 16ug/mL+28.5ug/mL
Configuration and chromatographic conditions
• Type：CIC-D120
• Infusion pump：Four element gradient pump
• IC column：PA20 sugar analysis column
• Eluent:gradient elution
• Flow rate：0.4mL/min
• Sample size：20μL
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• Detection: pulsed amperometric detector(Au working electrode，Ag/AgCl reference electrode）
Refer gradient elution procedure:
Mobile phase(%)
Time(Min) A B C
0.00 95 5 0
20.10 95 5 0
35.00 0 100 0
36.00 0 100 0
36.10 0 0 100
46.00 0 0 100
46.10 95 5 0
61.00 95 5 0
pretreatment
Before analysis,make liquid sample homogeneity.Shatter hard particles, then filter by 1mm2 sieve(No.18
sieve)
Extract
Weigh accurately to 1mg 。 Weigh 50mL plastic bottle with spiral lid(M1). Weigh a certain amount of
samples, containing GOS and lactose about 0.1-0.3g, but the sample size should not exceed 10g, then put it in
this 50mL plastic bottle(M2, test sample part).Add about 40mL hot phosphate buffer (about 80 degrees), cover
the lid and mix.Let the plastic bottle water bath at 80±2℃,after stirring for 30 minutes,then put it in ice bath
and cool it to room temperature.Measure pH value and adjust the pH value to 5.7-6.3 with 1M NaOH or 1M
HCl. Dilut with phosphoric acid buffer to about 50mL.Weigh the weight of the bottle, cap and solution, and
calculate and test the weight of the extract(M3).
Enzymolysis
Solution treatment—Weighing plastic bottle with spiral lid(M4), take 20g extract and put it into the
bottle, the net weight of sample is M5. Take 1mL β -galactosidase solution,put it into a bottle,cover
tightly with the bottle cap, and mix.
Initial test solution—Weighing plastic bottle with spiral lid(M7),take 1mL β -galactosidase solution
and 1mL phosphate buffer and make them water bath for 10minutes at 100℃, then inactivate the enzyme
and cool it.Take 20g extract, and put it into the bottle, the net weight of sample is M8,cover tightly with
the bottle cap, and mix.The above active enzyme solution and inactive enzyme solution were cultured at
60±2℃ for 30 minutes,mix it slightly and continuously,and begin to record the heating time when the
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solution temperature reaches 60℃. Avoid generating foams or bubbles during mixing.And make it cool
with ice bath.
Add 5mL 20% acetonitrile to 6.1 solution, mix, weigh the bottle (M6) with the lid tightly, add 4 mL
20% acetonitrile to 6.2 solution, mix, and weigh the bottle (M9) with the lid tightly.Centrifuge the
solution(10000*g, 10 minutes),the supernatant is filtered by 0.2 μm membrane and the filtrate
isconcentrated.The enzyme inactivation extract solution is called A1, and the hydrolytic extract solution
is called A2.
Test spectrum
Standard spectrogram:
mV
1.74
1.68
8.797' 半乳糖
9.937' 葡萄糖
1.62
1.56
1.5
1.44
20.813' 乳糖
1.38
1.32
1.26
1.2
3 6 9 12 15 18 21 24 27 min
────────────────────────────────────────
No. Retention time Name Concentration Peak area Peak separation degree
1 8.797 Galactose 10 6945 2.48
2 9.937 Glucose 10 7494 14.05
3 20.813 Lactose 20 6452 0.00
Enzymatic hydrolysis samples spectrogram:
mV
6
9.519' 葡萄糖
5.5
4.5
3.5
2.5
19.192' 乳糖
2
8.643' 半乳糖
58.823'
1.5
6 12 18 24 30 36 42 48 54 60 66 min
────────────────────────────────────────
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No. Retention time Name Concentration Peak area Peak separation degree
1 8.643 Galactose 10.4 7223 1.63
mV
2.1
2 2
9.519 Glucose 138.3 103655 11.12
3 19.192 Lactose 63.8 20584 18.45
1.9
Enzyme unhydrolyzed sample spectrogram:
1.8
1.7
1.6
1.5
1.4
19.290' 乳糖
1.3
9.361' 葡萄糖
8.293' 半乳糖
1.2
1.1
5 10 15 20 25 30 35 40 45 50 55 min
────────────────────────────────────────
No. Retention time Name Concentration Peak area Peak separation degree
1 8.293 Galactose 0.8186 569 2.53
2 9.361 Glucose 0.8734 655 13.07
3 19.290 Lactose 18.26 5892 0.00
conclusion
In this paper, Galactooligosaccharides in milk powder is determined by ion chromatography and pulsed
amperometric detection. The content of galactooligosaccharides was effectively separated and detected by
optimized gradient elution procedure. The method has high sensitivity, wide linear range, good precision and
accuracy, and can be used for the determination of galactooligosaccharides in milk powder and other samples.
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Product presentation
CIC-D120
CIC-D120 is equipped temperature-control bipolar conductivity detector which can greatly
improve the detection performance and stability of the instrument and can be compatible with
ampere detector, UV-detector, and UV and post-column derivation device and so on. Combined with
Sheng Han's leading chromatography column technology, it can analyze the anions, cations,
cyanogen, iodide, sugar, small molecular organic acids etc.and is widely applied in the fields of
environment, disease control, food, chemical industry, electronics, mining and metallurgy.
▷Temperature-control bipolar conductivity detector（CN 202033335U）
Greater detection range,better precise analysis
▷Built-in circulating 3D constant temperature technology（CN 204259917U）
Temperature stability time is less than 30 mins, ensuring the accuracy and reliability of test data
▷The world's leading full-range series of ion chromatographic columns
(CN 105126936A、CN 104788603A)
High efficiency, large capacity of the columns for detecting ions of varied compositions
▷Self-Regenerating Electrolytic Micro-membrane Suppressor（CN 102735792A）
High pressure resistance, small dead volume, highly responsive to signals
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Ion chromatographic separation column
AS the first domestic developer and manufacturer of Ion chromatographic column , now Qingdao Shenghan
have the technology of the development and production of three kinds of Ion chromatographic column
including ion exchange chromatographic column, ion exclusion chromatographic column and ion pair
chromatographic column. In addition, Sheng Han have also successfully developed and produced hydroxyl
system of Ion chromatographic column in large scale ranking the second in the world, thus broken the
monopoly of imported brands in the high-end ion chromatographic column field more than ten
years.Accordingly the application of domestic IC column can reduce the cost of operation and maintenance of
users by about 35%.
