Determination of Carbohydrates in Various Matrices by ...

Determination of Carbohydrates in Various Matrices by Capillary HPAE-PADTerri Christison,1 Joachim Weiss,2 Cathy Tanner,1 Frank Hoefler1 1Thermo Fisher Scientific Inc., Sunnyvale, CA, USA; 2Thermo Fisher Scientific GmbH, Dreieich, Germany

2 Determination of Carbohydrates in Various Matrices by Capillary HPAE-PAD

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Various Matrices by Capillary HPAE-PAD Terri Christison1, Joachim Weiss2, Cathy Tanner1, Frank Hoefler1 1Thermo Fisher Scientific Inc., Sunnyvale, CA, USA; 2Thermo Fisher Scientific GmbH, Dreieich, Germany

PO71293-EN 0914S

Overview Carbohydrates in beverage samples and milk products were directly determined using

high performance anion-exchange chromatography in the capillary format with pulsed amperometric detection (HPAE-PAD) on a capillary reagent-free, high-pressure system.1,2

This method eliminates the costly and labor-intensive derivitization used in other methods.

Lactose and lactulose determinations are also demonstrated on the new 4 µm high-capacity standard bore Thermo Scientific™ Dionex™ CarboPac™ SA20-4m column optimized for fast separations of mono- and di-saccharides.2

Introduction Mono- and di-saccharide determinations are important to the food industry to ensure product formulation and product quality and to report ingredients to immune- and allergy-sensitive individuals. Because carbohydrates are non-chromophoric, chemical derivitization is needed for UV detection. However, derivitization is costly, labor-intensive, and may cause changes in molecular configuration. High Performance Anion-Exchange chromatography with Pulsed Amperometric Detection (HPAE-PAD) is a proven sensitive method to directly and selectively determine carbohydrates. In HPAE-PAD, carbohydrates are ionized in strong base and separated by anion-exchange chromatography. The carbohydrates are detected by PAD with a gold working electrode using a four-potential waveform selective and sensitive for carbohydrates. This sensitivity allows carbohydrate analysis down to pmol concentrations or when sample volumes are limited. This sensitivity is moderated in beverage samples which contain g/L concentrations by minimizing the flow path combined with moderate dilution. Here we demonstrate the determinations of the different sugars used to sweeten beverages and the fast and easy determination of lactose and lactulose in milk products. This work further demonstrates the versatility of HPAE-PAD, easily optimized for low or high carbohydrate concentrations, the separations on different carbohydrate columns, high-pressure IC (HPIC), and capillary HPIC systems. Experimental Sample Preparation The beverage samples were prepared by dilution and filtration, as appropriate. The milk products (1 g/10 mL water) were treated with 200 µL each of Carrez I (potassium hexacyanoferrate(III)) and Carrez II (zinc sulfate) solutions for deproteinization according to AOAC Method 984.151 and as described in AN 2482. The samples were mixed, diluted to 100 mL, centrifuged, and the supernatant filtered and treated with a Thermo Scientific™ Dionex™ OnGuard™ IIA sample preparation cartridge to remove anionic contaminants and neutralize the sample. Method Thermo Scientific Dionex HPIC ion chromatography systems used:

Thermo Scientific™ Dionex™ ICS-5000+ HPIC™ system, standard and capillary format Dionex ICS-5000+ IC modules: EG Eluent Generator, DC Detector

Chromatography, and DP Dual Pump Thermo Scientific Dionex ICS-4000 Capillary HPIC system

Detection: Electrochemical Detector and Electrochemical cell Gold on PTFE Disposable Electrode and pH-Ag/AgCI Reference Electrode PTFE Gaskets: 0.001”, 0.002”, or 0.015”

Autosampler: Thermo Scientific Dionex AS-AP Autosampler Software: Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System Conclusion

• Capillary and analytical HPAE-PAD are direct, selective, and sensitive methods without the need for costly and labor-intensive derivitization.

• Modifying the gasket type affects sensitivity which can be advantageous when analyzing high carbohydrate concentrations.

• An RFIC system with electrolytically generated eluent requires only adding DI water, thereby eliminating eluent preparation, increasing reliability, stability, and ease-of-use.

• Fast determinations of lactose and lactulose in milk products were demonstrated using a simple 8 min isocratic separation on the Dionex CarboPac SA10 column.

References 1. Thermo Fisher Scientific Technical Note 135: Determinations of Monosaccharides

and Disaccharides in Beverages by Capillary HPAE-PAD. Sunnyvale, CA, 2013. [Online] http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Application%20&%20Technical%20Notes/Chromatography/Ion%20Chromatography/IC%20and%20RFIC%20Systems/TN-135-Determination-Monosaccharides-Disaccharides-Beverages-TN70646-E.pdf (accessed August 6, 2014).

2. Thermo Fisher Scientific Technical Note 146: Fast Determinations of Lactose and Lactulose in Milk Products Using HPAE-PAD. Sunnyvale, CA, 2014 [Online] http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Product%20Manuals%20&%20Specifications/Chromatography/Ion%20Chromatography/TN-146-Fast-Determination-Lactose-Lactulose-Milk-TN70891-EN.pdf (accessed August 6, 2014).

Column: Dionex CarboPac PA20 with guard, 0.4 mm Column temp.: 30 C Eluent source: Dionex EGC-KOH capillary cartridge Eluent: 10 mmol/L KOH Flow rate: 0.008 mL/min Inj. volume: 0.4 L Detection: PAD, Au disposable, 4-potential carbohydrate waveform Gasket: 0.001” PTFE Ref. electrode: Ag/AgCl Sample prep.: 5000-fold dilution Peaks: 1. Void volume -- mol/L

2. Galactose 5 3. Glucose 23 4. Sucrose 7 5. Fructose 11

4 3

2

1

5

15 5 10 0 Minutes 0

70

nC

Column: Dionex CarboPac PA20 with guard, 0.4 mm Column temp.: 30 C Eluent source: Dionex EGC-KOH capillary cartridge Eluent: 10 mmol/L KOH Flow rate: 0.008 mL/min Inj. volume: 0.4 L Detection: PAD, Au disposable, 4-potential carbohydrate waveform Gasket: 0.001” PTFE Ref. electrode: Ag/AgCl Sample prep.: 5000-fold dilution, degas Peaks: 1. Glucose 98 mol/L

2. Fructose 95

2

1

15 5 10 0 Minutes 0

90

nC


2. Galactose 0.1 3. Glucose 60 4. Mannose 2 5. Sucrose 20 6. Fructose 110

4

3

6

2

1

15 5 10 0 0

70

nC

5

Column: Dionex CarboPac PA20 with guard, 0.4 mm Column temp.: 30 C Eluent source: Thermo Scientific Dionex EGC- KOH capillary cartridge Eluent: 10 mmol/L KOH Flow rate: 0.008 mL/min Inj. volume: 0.4 L Detection: PAD, Au disposable, 4-potential carbohydrate waveform Gasket: 0.001” PTFE Ref. electrode: Ag/AgCl Sample: 10 mol/L mixed standard Peaks: 1. Fucose

2. Galactosamine 3. Glucosamine 4. Galactose 5. Glucose 6. Mannose

4

3

2

1

6

15 5 10 0 Minutes

0

60

nC

5

Data & System Management High-Pressure

Non-Metallic Capillary Pump

Eluent Generator (OH– or H+)

Waste

Sample Inject (Autosampler)

H20

Separation Column

Degas

Suppressor Bypass

CRD Bypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential (V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10

FIGURE 9. Lactose and lactulose in raw unpasteurized milk.

FIGURE 8. Cane sugar in coconut water flavored beverage.

FIGURE 6. High fructose corn syrup (HFC) in a carbonated beverage.

FIGURE 5. Native sugars in apple cider.

Instrument: Dionex ICS-5000+ HPIC system Column: Dionex CarboPac SA10-4m and guard, 4 mm Column temp.: 35 C Eluent source: Dionex EGC 500 KOH cartridge Eluent: 4 mmol/L KOH Flow rate: 1.45 mL/min Inj. Volume: 10 L Detection: PAD, Au on PTFE disposable, Four-potential carbohydrate waveform Gasket: 0.002” thick PTFE Ref. electrode: pH-Ag/AgCl Sample prep.: Carrez digestion, centrifuge, filter, Dionex OnGuard IIA cartridge Sample: A: 100-fold diluted raw, unpasteurized milk B: Sample A + 0.5 mg/L lactulose C: 0.5 mg/L carbohydrate standard Peaks: A B 1. Sucrose -- -- mg/L 2. Galactose -- -- 3. Glucose -- -- 4. Lactose 3.75 3.77 5. Lactulose -- 0.48

3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4

Results Sugars in Beverages Beverages are generally sweetened with the most available and the lowest cost sugar in the respective manufacturing region. Native sugars from fruit may be used for sweetening. Manufacturers often select the sugar for their products based on availability or lowest cost. These sugars are sourced from either corn, sugar cane or sugar beets. Corn syrup, which is primarily glucose, is enzymatically hydrolyzed to create fructose for increased sweetness. Beet sugar and cane sugar, which are sucrose, are partially hydrolyzed to glucose and fructose to increase sweetness and to minimize crystallization which can cause storage problems. Figure 4 shows the separation of six carbohdyrates using capillary format HPAE-PAD, injecting 0.4 L sample onto a 0.4 mm i. d. column at a flow rate of 8 L/min.

Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose

FIGURE 1. Flow diagram for a capillary HPAE-PAD system.

FIGURE 3. Conventional and disposable gold working electrodes.

Figure 1 shows the instrument flow diagram for a capillary HPAE-PAD system. The Dionex ICS-5000+ system configured for 4 mm or 2 mm i. d. columns has the same flow path but without the CRD and suppressor bypass modules.

Figures 2 shows the four-potential waveform for the detection of carbohydrates using a gold disposable working electrode (Figure 3). This waveform is optimized to create a stable gold oxide layer resulting in highly reproducible response factors.

FIGURE 2. Four-potential waveform for carbohydrate determinations.

Conventional electrode

Disposable electrode

FIGURE 4. Six carbohydrate standard.

Figure 9 shows the 8 min separation of lactose and lactulose on the Dionex CarboPac SA10-4m column using only 4 mmol/L KOH. This method utilizes the standard bore format (4 mm) and the standard 0.002” thickness gasket. A high-pressure capable system facilitates the separation.

FIGURE 7. HFC in a carbonated beverage -- 0.015” gasket.

Column: Dionex CarboPac PA20 with guard, 0.4 mm Column temp.: 30 C Eluent source: Dionex EGC-KOH capillary cartridge Eluent: 10 mmol/L KOH Flow rate: 0.008 mL/min Inj. volume: 0.4 L Detection: PAD, Au on PTFE disposable, Four-potential carbohydrate waveform Gasket: 0.015” PTFE Ref. electrode: Ag/AgCl Sample prep.: 500-fold dilution, degas Peaks: mg/L % Ratio 1. Glucose 102 58 2. Fructose 150 42

2

1

15 5 10 0 Minutes 0

140

nC

Glucose Fructose

55%, 42%

Figure 7 shows that using a 0.015“ gasket versus the 0.001” capillary gasket reduces sensitivity, so that less dilution is required. The resulting analysis suggests the use of HFC 42 for sweetening.

Figures 5 to 7 show the analysis of beverage samples using capillary HPAE-PAD with a typical capillary gasket of 0.001” thickness. In Figure 5, the sample had a mixture of sugars, characteristic of native sugars. In Figure 6, only glucose and fructose are present indicating high fructose corn syrup (HFC) being used for sweetening.

Figure 8 shows a typical profile of a product sweetened with cane sugar (predominantly sucrose with equal parts of fructose and glucose).

3Thermo Scientific Poster Note • PN71293 ISC-EN 0814S




PO71293-EN 0914S



















4 3

2

1

5

15 5 10 0 Minutes 0

70

nC


2. Fructose 95

2

1

15 5 10 0 Minutes 0

90

nC



4

3

6

2

1

15 5 10 0 0

70

nC

5



4

3

2

1

6

15 5 10 0 Minutes

0

60

nC

5




Waste


H20

Separation Column

Degas

Suppressor Bypass

CRD Bypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential (V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10






3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4


Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose












2

1

15 5 10 0 Minutes 0

140

nC

Glucose Fructose

55%, 42%








PO71293-EN 0914S



















4 3

2

1

5

15 5 10 0 Minutes 0

70

nC


2. Fructose 95

2

1

15 5 10 0 Minutes 0

90

nC



4

3

6

2

1

15 5 10 0 0

70

nC

5



4

3

2

1

6

15 5 10 0 Minutes

0

60

nC

5




Waste


H20

Separation Column

Degas

Suppressor Bypass

CRD Bypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential (V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10






3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4


Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose












2

1

15 5 10 0 Minutes 0

140

nC

Glucose Fructose

55%, 42%




5Thermo Scientific Poster Note • PN71293 ISC-EN 0814S




PO71293-EN 0914S



















4 3

2

1

5

15 5 10 0 Minutes 0

70

nC


2. Fructose 95

2

1

15 5 10 0 Minutes 0

90

nC



4

3

6

2

1

15 5 10 0 0

70

nC

5



4

3

2

1

6

15 5 10 0 Minutes

0

60

nC

5




Waste


H20

Separation Column

Degas

Suppressor Bypass

CRD Bypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential (V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10






3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4


Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose












2

1

15 5 10 0 Minutes 0

140

nC

Glucose Fructose

55%, 42%








PO71293-EN 0914S



















4 3

2

1

5

15 5 10 0 Minutes 0

70

nC


2. Fructose 95

2

1

15 5 10 0 Minutes 0

90

nC



4

3

6

2

1

15 5 10 0 0

70

nC

5



4

3

2

1

6

15 5 10 0 Minutes

0

60

nC

5




Waste


H20

Separation Column

Degas

Suppressor Bypass

CRD Bypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential (V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10






3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4


Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose












2

1

15 5 10 0 Minutes 0

140

nC

Glucose Fructose

55%, 42%




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Determination of Carbohydrates in Various Matrices by Capillary HPAE-PADTerri Christison1, Joachim Weiss2, Cathy Tanner1, Frank Hoefler1

1Thermo Fisher Scientific Inc., Sunnyvale, CA, USA; 2Thermo Fisher Scientific GmbH, Dreieich, Germany

PO71293-EN 0914S




Lactose and lactulose determinations are also demonstrated on the new 4 µm high-capacity standard bore Thermo Scientific™ Dionex™ CarboPac™ SA20-4m columnoptimized for fast separations of mono- and di-saccharides.2

IntroductionMono- and di-saccharide determinations are important to the food industry to ensure product formulation and product quality and to report ingredients to immune- and allergy-sensitive individuals. Because carbohydrates are non-chromophoric, chemical derivitization is needed for UV detection. However, derivitization is costly, labor-intensive, and may cause changes in molecular configuration.

High Performance Anion-Exchange chromatography with Pulsed Amperometric Detection(HPAE-PAD) is a proven sensitive method to directly and selectively determine carbohydrates. In HPAE-PAD, carbohydrates are ionized in strong base and separated by anion-exchange chromatography. The carbohydrates are detected by PAD with a gold working electrode using a four-potential waveform selective and sensitive for carbohydrates. This sensitivity allows carbohydrate analysis down to pmol concentrations or when sample volumes are limited. This sensitivity is moderated in beverage samples which contain g/L concentrations by minimizing the flow path combined with moderate dilution.

Here we demonstrate the determinations of the different sugars used to sweeten beverages and the fast and easy determination of lactose and lactulose in milk products. This work further demonstrates the versatility of HPAE-PAD, easily optimized for low or high carbohydrate concentrations, the separations on different carbohydrate columns, high-pressure IC (HPIC), and capillary HPIC systems.

ExperimentalSample PreparationThe beverage samples were prepared by dilution and filtration, as appropriate.

The milk products (1 g/10 mL water) were treated with 200 µL each of Carrez I(potassium hexacyanoferrate(III)) and Carrez II (zinc sulfate) solutions for deproteinization according to AOAC Method 984.151 and as described in AN 2482. Thesamples were mixed, diluted to 100 mL, centrifuged, and the supernatant filtered andtreated with a Thermo Scientific™ Dionex™ OnGuard™ IIA sample preparation cartridge to remove anionic contaminants and neutralize the sample.MethodThermo Scientific Dionex HPIC ion chromatography systems used:




Autosampler: Thermo Scientific Dionex AS-AP AutosamplerSoftware: Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System

Conclusion • Capillary and analytical HPAE-PAD are direct, selective, and sensitive methods without

the need for costly and labor-intensive derivitization.

• Modifying the gasket type affects sensitivity which can be advantageous whenanalyzing high carbohydrate concentrations.

• An RFIC system with electrolytically generated eluent requires only adding DI water,thereby eliminating eluent preparation, increasing reliability, stability, and ease-of-use.

• Fast determinations of lactose and lactulose in milk products were demonstrated usinga simple 8 min isocratic separation on the Dionex CarboPac SA10 column.


and Disaccharides in Beverages by Capillary HPAE-PAD. Sunnyvale, CA, 2013.[Online]http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Application%20&%20Technical%20Notes/Chromatography/Ion%20Chromatography/IC%20and%20RFIC%20Systems/TN-135-Determination-Monosaccharides-Disaccharides-Beverages-TN70646-E.pdf (accessed August 6, 2014).

2. Thermo Fisher Scientific Technical Note 146: Fast Determinations of Lactose andLactulose in Milk Products Using HPAE-PAD. Sunnyvale, CA, 2014 [Online]http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Product%20Manuals%20&%20Specifications/Chromatography/Ion%20Chromatography/TN-146-Fast-Determination-Lactose-Lactulose-Milk-TN70891-EN.pdf (accessedAugust 6, 2014).

Column: Dionex CarboPac PA20 withguard, 0.4 mm

Column temp.: 30 C Eluent source: Dionex EGC-KOH capillary

cartridgeEluent: 10 mmol/L KOHFlow rate: 0.008 mL/minInj. volume: 0.4 LDetection: PAD, Au disposable, 4-potential

carbohydrate waveform Gasket: 0.001” PTFERef. electrode: Ag/AgClSample prep.: 5000-fold dilution

Peaks: 1. Void volume -- mol/L2. Galactose 53. Glucose 234. Sucrose 7 5. Fructose 11

43

2

1

5

155 100 Minutes0

70

nC


Column temp.: 30 CEluent source: Dionex EGC-KOH capillary

cartridgeEluent: 10 mmol/L KOHFlow rate: 0.008 mL/minInj. volume: 0.4 L

Detection: PAD, Au disposable, 4-potentialcarbohydrate waveform

Gasket: 0.001” PTFERef. electrode: Ag/AgClSample prep.: 5000-fold dilution, degas

Peaks: 1. Glucose 98 mol/L2. Fructose 95

2

1

155 100 Minutes0

90

nC


Column temp.: 30 C Eluent source: Dionex EGC-KOH capillary

cartridgeEluent: 10 mmol/L KOHFlow rate: 0.008 mL/minInj. volume: 0.4 LDetection: PAD, Au disposable, 4-potential

carbohydrate waveform Gasket: 0.001” PTFERef. electrode: Ag/AgClSample prep.: 5000-fold dilution

Peaks: 1. Void volume -- mol/L2. Galactose 0.13. Glucose 604. Mannose 25. Sucrose 206. Fructose 110

4

3

6

2

1

155 1000

70

nC

5


Column temp.: 30 C Eluent source: Thermo Scientific Dionex EGC-

KOH capillary cartridgeEluent: 10 mmol/L KOHFlow rate: 0.008 mL/minInj. volume: 0.4 LDetection: PAD, Au disposable, 4-potential

carbohydrate waveform Gasket: 0.001” PTFERef. electrode: Ag/AgClSample: 10 mol/L mixed standard

Peaks: 1. Fucose2. Galactosamine3. Glucosamine4. Galactose5. Glucose6. Mannose

4

3

2

1

6

155 100Minutes

0

60

nC

5

Data & System ManagementHigh-Pressure


EluentGenerator(OH– or H+)

Waste

Sample Inject(Autosampler)

H20

Separation Column

Degas

SuppressorBypass

CRDBypass

Regen flow in back

ED

CR-TC

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Pote

ntia

l (V

vs. A

g/Ag

Cl)

Time (Seconds)

Time (s)

Potential(V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10





Instrument: Dionex ICS-5000+ HPIC system Column: Dionex CarboPac SA10-4m

and guard, 4 mm Column temp.: 35 C Eluent source: Dionex EGC 500 KOH cartridge Eluent: 4 mmol/L KOH Flow rate: 1.45 mL/min Inj. Volume: 10 L Detection: PAD, Au on PTFE disposable,

Four-potential carbohydrate waveform Gasket: 0.002” thick PTFE Ref. electrode: pH-Ag/AgCl Sample prep.: Carrez digestion, centrifuge, filter,

Dionex OnGuard IIA cartridge Sample: A: 100-fold diluted raw, unpasteurized

milk B: Sample A + 0.5 mg/L lactulose C: 0.5 mg/L carbohydrate standard

Peaks: A B 1. Sucrose -- -- mg/L 2. Galactose -- -- 3. Glucose -- -- 4. Lactose 3.75 3.77 5. Lactulose -- 0.48

3 2 5

Minutes

1

A

B

C

8 2 6 4 0 30

50

nC

4

ResultsSugars in BeveragesBeverages are generally sweetened with the most available and the lowest cost sugar in the respective manufacturing region. Native sugars from fruit may be used for sweetening. Manufacturers often select the sugar for their products based on availability or lowest cost. These sugars are sourced from either corn, sugar cane or sugar beets. Corn syrup, which is primarily glucose, is enzymatically hydrolyzed to create fructose for increased sweetness. Beet sugar and cane sugar, which are sucrose, are partially hydrolyzed to glucose and fructose to increase sweetness and to minimize crystallization which can cause storage problems.

Figure 4 shows the separation of six carbohdyrates using capillary format HPAE-PAD, injecting 0.4 L sample onto a 0.4 mm i. d. column at a flow rate of 8 L/min.

Working electrode

Au Contact pad

Glucose Fructose

55%, 42%

Glucose

Sucrose

Fructose

Glucose

Sucrose

Fructose



Figure 1 shows the instrument flow diagram for a capillary HPAE-PAD system. TheDionex ICS-5000+ system configured for 4 mm or 2 mm i. d. columns has the same flow path but without the CRD and suppressor bypass modules.




Disposableelectrode


Figure 9 shows the 8 min separation of lactose and lactulose on the Dionex CarboPacSA10-4m column using only 4 mmol/L KOH. This method utilizes the standard boreformat (4 mm) and the standard 0.002” thickness gasket. A high-pressure capable system facilitates the separation.



Column temp.: 30 CEluent source: Dionex EGC-KOH capillary

cartridgeEluent: 10 mmol/L KOHFlow rate: 0.008 mL/minInj. volume: 0.4 LDetection: PAD, Au on PTFE disposable,

Four-potential carbohydratewaveform

Gasket: 0.015” PTFERef. electrode: Ag/AgClSample prep.: 500-fold dilution, degas

Peaks: mg/L % Ratio1. Glucose 102 582. Fructose 150 42

2

1

155 100 Minutes0

140

nC

Glucose Fructose

55%, 42%




http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Application%20&%20Technical%20Notes/Chromatography/Ion%20Chromatography/IC%20and%20RFIC%20Systems/TN-135-Determination-Monosaccharides-Disaccharides-Beverages-TN70646-E.pdf

http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Product%20Manuals%20&%20Specifications/Chromatography/Ion%20Chromatography/TN-146-Fast-Determination-Lactose-Lactulose-Milk-TN70891-EN.pdf

Date post:	23-Feb-2023
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