HPAE-PAD analysis of Carbohydrates

Michael G. HvizdManager, Laboratory OperationsCenters of ExcellenceDionex Corporation

Laboratory

Presentation Outline

Principles of High Performance Anion Exchange

Column Technologies

Principles of Pulsed Amperometric Detection

Applications using HPAE-PAD

22060

Carbohydrates -Features of HPAE-PAD

Simple, Direct Method– No derivatization is necessary

High Sensitivity– comparable to derivatization with fluorescence detection (fmol to low pmol). – Allows samples to be diluted 100 to 1000-fold, so simplifies sample prep

Minimal Sample Preparation is necessaryUnique column technologyOfficial Methods (using CarboPac PA1)

Resolving Power of HPAE-PAD

Comparison of chromatographic techniques used for the analysis of a hydrolyzed glucose syrup illustrates the remarkable resolving power of the HPAE-PAD technique. Elution order is reversed with the CarboPac® PA1 column as compared with conventional metal loaded cation-exchange columns.

Rapid Monosaccharide Analysis Rapid Monosaccharide Analysis with the CarboPacwith the CarboPac™™ PA10PA10

Column: CarboPac PA10 and guardEluent: 18 mM NaOHFlow Rate: 1.5 mL/minDetection: Pulsed amperometry,

gold electrodePeaks: 1. Fucose 1 nmol

2. Galactosamine 13. Glucosamine 14. Galactose 15. Glucose 16. Mannose 1

22

00 88 101000

100100

nCnC

11

3344 55

66

MinutesMinutes664422

1179011790--0101

Column: CarboPac™ PA10, PA10 guard,Borate Trap®

Eluent: 52 mM sodium hydroxideFlow Rate: 1.5 mL/minInj. Volume: 5 µLDetection: Pulsed amperometry, gold electrode

Detection of Food Sugars and Sugar AlcoholsPeaks: 1. Glycerol 4.0 mg/mL

2. Xylitol 2.03. Sorbitol 1.54. Mannitol 3.05. Glucose 5.06. Fructose 8.07. Sucrose 2.08. Lactose 8.0

15150-01

50

nC

4

5

6 78

12

3

0

Minutes5 10 150

–2

–1

0

1

0 0.2 0.4

Poten

tial (V

)

Time (s)

Column: CarboPac™ MA1

Eluent: 480 mM sodium hydroxide

Flow Rate: 0.4 mL/min

Detector: PED (gold)Peaks: 1. Inositol 18 mg/L

2. Glycerol 93. Arabitol 154. Sorbitol 185. Dulcitol 186. Mannitol 187. Mannose 188. Glucose 189. Galactose 18

10. Fructose 1811. Sucrose 34

Minutes0 10 20

0

µC

0.2

30 40 50

1

34

5

68 9

10

11

7

2

Separation of Reduced and Reducing CarbohydratesCommonly Found in Foods and Beverages

8460-03

Column: CarboPac™ PA-100 and guardEluent: NaOH/sodium acetate gradientFlow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: Pulsed amperometry, Au electrode

Peaks: 1. Glucose2. Maltose3. Maltotriose4. Maltotetraose5. Maltopentaose6. Maltohexaose7. Maltoheptaose8. Maltooctaose9. Maltononaose

10. Maltodecaose

Separation of Maltose Oligomers

10927-02Minutes

2

3

4

0 5 10 15 20 25

0

70

nC

1

5

67

89

10

Column: CarboPac™ PA10Eluent: Sodium hydroxide/

sodium acetate gradientFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: Pulsed amperometry,

Au electrode

Maltrin is a trademark of Grain Processing Corp.

0

175

nC

A: Maltrin M040

Minutes0 5 10 15 20 25 30

B: Maltrin M700

0

175

nC

35

Maltodextrins—Maltrin® M040 and M700

13026-01

Retention Time (min)

From: Koizumi, K., Fukuda, M., Hizukuri, S.. J. Chromatography. 1991, 585, 233–238

Distribution of Chain Length of Amylopectins by HPAE-PAD

Rice12

6

20

30 40 50

12

6

20

30 40 50 60

Corn

0 10 20 30 40

60504030

20

12

6

EdibleCanna

0 10 20 30 40

12

6 20

30 40 50 60

Sweet Potato

9661-01

Improved Chain-Length Resolution of Inulin Polymers

Columns: CarboPac™ PA200 (3 x 250 mm) CarboPac PA100 (4 x 250 mm)

Gradient: 120- to 320-mM NaOAc in 100 mM NaOH over 40 min

Flow Rate: PA200: 0.5 mL/minPA100: 1.0 mL/min

Detection: Pulsed amperometry,quadruple waveform, gold electrode

180

0 5 10 15 20 25 30 35 40 45 50 55 60–20

nC

Minutes

PA200

PA1000

20608-01

Column: CarboPac™ PA-100Eluent: 0 to 250 mM NaOAc over

110 min in 100 mM NaOHFlow Rate: 1 mL/minDetector: PAD (Gold)Peaks: 1. Fucosylated Man3GlcNAc2

2. Man3GlcNAc23. Asialo, agalacto bi, core fuc4. Asialo, agalacto bi5. Asialo bi, core fuc6. Asialo bi7. Man9GlcNAc28, 9. Disialylated tri (reduced)10, 11. Trisialylated tri (reduced)12, 13. Tetrasialylated tri (reduced)

00 1010 2020 3030 4040 5050 6060 7070

200 200

nAnA

MinutesMinutes

1122

33

4455

667

8899

10101111

12121313

NANA NANA —— Gal Gal —— GlcNAc GlcNAc —— ManMan……α2−6α2−6

Peak 10Peak 10

NANA NANA —— Gal Gal —— GlcNAc GlcNAc —— ManMan……α2−3α2−3

Peak 11Peak 11

Separation of N-Linked Oligosaccharides

Official Food Methods Using IC/PAD

AOAC Method 2000.17CarboPac PA1Low-Level Glucose and Fructose in Raw and Refined Sugar

AOAC Method 2001.02CarboPac PA1Transgalacto-oligosaccharides

AOAC Method 2000.11CarboPac PA1Polydextrose

AOAC Method 997.08CarboPac PA1Fructans in Food and Food Products

AOAC Method 995.13ISO 11292 ApprovedCarboPac PA1Carbohydrates in

Soluble Coffee

AOAC Method 996.04ICUMSA Method Approval 1994

CarboPac PA1Sugars in Molasses

Official MethodCarboPac™ ColumnAnalysis

20611-01

Column Technology

1391613916

High Performance Anion Exchange Chromatography with High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEPulsed Amperometric Detection (HPAE--PAD) PAD)

1032810328

Separates neutral and charged saccharideswithout derivatizationSeparates on the basis of:– Charge, size and composition– Branching, linkage isomerism

Dissociation Constants of Some Common Carbohydrates

13.71 α-Methyl glucoside13.60Sorbitol13.43Dulcitol12.39Galactose12.28Glucose12.15Xylose12.08Mannose12.03FructosepKaSugar

(in Water at 25 °C)

8709-03

86748674--BB

Polymeric Nonporous (“Polymeric Nonporous (“PellicularPellicular”) Packing”) Packing

Highly Highly CrosslinkedCrosslinked CoreCore

Latex Latex MicrobeadsMicrobeadswith Anion Exchange with Anion Exchange

FunctionalitiesFunctionalities

1688716887

Electrochemical Detection

1391613916

Pulsed Amperomerty Detection

Advantages of PAD for Carbohydrate Detection– No derivatization is necessary– Low detection limits: low picomole to high femtomole– Detection is linear over at least 4 orders of magnitude– PAD is a non-destructive method and can thus be used preparatively– Highly specific– Using optimized pulse settings, PAD is highly specific for carbohydrates– Versatile: isocratic or gradient elution, high pH or neutral pH (with post column base

addition)– Specific for electroactive species– Both amino acids and carbohydrates can be detected in a single run

Disadvantages of PAD for Carbohydrate Detection ***– Each sugar gives a different signal, thus calibration curves must be run for quantitative

results

– *** True for every quantitative analysis

13450

Principal of Pulsed Amperometric Detection (PAD)Quadruple Potential Waveform for Carbohydrates

–2

0

0.8

0.0 0.1 0.2 0.3 0.4 0.5Time (seconds)

Poten

tial (V

vs. A

g/AgC

l)

Electrode Cleaning

Au Oxide Reduction,Electrode Activation

Au Oxide Formation Time Pot. Integ.0.00 +0.10.20 +0.1 Begin0.40 +0.1 End0.41 -2.00.42 -2.00.43 +0.60.44 -0.10.50 -0.1

Integrate

Pulsed Amperometric Detection (PAD)Waveform and Integration Period

Carbohydrate Waveform

– 3

– 2

– 1

0

1

0 0.2 0.4 0.6 0.8 1.0Time (s)

Poten

tial (

V )

Amino Acid Waveform

0 0.2 0.4 0.6 0.8 1.0Time (s)

Poten

tial (

V )

– 3

– 2

– 1

0

1

Integration Period

22136

Column: CarboPac™ PA1 and guardEluent: 150 mM NaOHFlow Rate: 1.0 mL/minInj. Volume: 50 µLDetection: Pulsed amperometry

Peaks: 1. Glucose 4.39%2. Fructose 6.673. Lactose (int. std.) –4. Sucrose 30.8

Sugars in Sugar Cane Molasses

2 4 6 8Minutes

2

1

3

4

µA

3

8

010930-03

Carbohydrates in Instant CoffeeColumn: CarboPac™ PA1 Eluent: 150 mM sodium hydroxide/

deionized water gradientInj. Volume: 25 µL of 10 g/L solutionDetector: Pulsed amperometry,

Au electrode; postcolumn addition of 0.3 M NaOH

Peaks: 1. Mannitol 21 mg/L 2. Arabinose 1403. Galactose 764. Glucose 445. Xylose 266. Mannose 517. Fructose 93

Sample Preparation: Phenolics removed with

OnGuard® P cartridge0 10 20 30 40

12

34

5 6 7

1000

0

nA

Minutes

6007-03

Sucralose in Diet Drink and Artificial Sweetener Product

Column: CarboPac™ PA20 with guard Eluents: 40 mM sodium hydroxide/

75 mM sodium acetateTemperature: 30 °CFlow Rate: 0.5 mL/min, 10-g/L solutionInj. Volume: 25 µLDetection: Pulsed amperometric

detection, carbohydrate waveform, disposable gold working electrode

0 1 2 3 4 5 6 7 8 930

65

Sucralose

Red Raspberry Diet SodaDiluted 50-Fold

0Minutes

2 4 6 8 10 12

Sucralose

20

nC

16

Splenda® Sweetener100 µg/mL

Splenda is a registered trademark of McNeil-PP

20607-01

Column: CarboPac PA-100Eluent: Sodium hydroxide/

sodium acetate gradientFlow Rate: 1 mL/minConc.: 2 mg/mLInj. Volume: 25 µLDetection: Pulsed amperometry,

Au electrode

5

0

µA

A. Pure Orange Juice

5

0

µA

0 10 20 30Minutes

B. Orange Juice Adulterated with Beet Medium Invert Sugar

Oligosaccharide Profiles of Pure and Adulterated Orange Juice

11399-01

Apple Juice

Column: CarboPac™ MA1Eluent: 500 mM sodium hydroxideFlow Rate: 0.4 mL/minDetection: Pulsed amperometryPeaks: 1. Sorbitol 14 µg

2. Glucose 1303. Fructose 364. Sucrose 0.2

0.2

µC

0

0 30 60Minutes

1

2

3

4

2010 50408588-03