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March 11, 2015 Michael G. Hvizd Senior Manager, CoE Laboratories

Fast, Accurate and Direct Carbohydrate Analysis Using HPAE-PAD

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Agenda

• Outline • What are Carbohydrates?

• High Performance Anion Exchange Chromatography (HPAE)

• Pulsed Amperometric Detection (PAD)

• Application Examples

• Total Solution from Thermo Fisher Scientific

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Classification of Carbohydrates

• Monosaccharide • Empirical formula (CH2O)n

• Each carbon except one contains a hydroxyl group

• The remaining carbon atom has a carbonyl oxygen • If carbonyl is at the end of the chain,

monosaccharide is called an aldose • If carbonyl is at any other position,

monosaccharide is called a ketose • Based on number of carbons (3, 4, 5, 6),

a monosaccharide is a triose, tetrose, pentose, or hexose.

Ketoses (e.g., fructose)

have a keto group, usually

at C2.

C

C OHH

C HHO

C OHH

C OHH

CH2OH

D-glucose

OH

C HHO

C OHH

C OHH

CH2OH

CH2OH

C O

D-fructose

Aldoses (e.g., glucose)

have an aldehyde group

at one end.

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glucose glucose maltose

- H2O

Classification of Carbohydrates

• Disaccharides • Monosaccharides can be joined by means of a glycosidic bond

• Formed by the reaction of carbonyl carbon of one monosaccharide with the hydroxyl group of the other monosaccharide

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OH

OH

CH2OH

O

OH

O

CH2OH

OH

OH

OHO

n

Classification of Carbohydrates

• Oligo- and Polysaccharides • Chains of monosaccharides joined by glycosidic bonds

• Since monosaccharides have multiple hydroxyl groups, oligo- and polysaccharides can be highly branched

• Oligosaccharide • Chain of 3–10 sugar molecules

• Polysaccharide • Chain of 10+ sugar molecules

• Glycosidic bonds can be broken by: • Acid digestion

• Enzyme digestion

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Challenges with Carbohydrate Analysis

• Extremely Polar, Partly Ionic • Many Similar and complex structures • Non-Chromophoric • Often present in complex matrices • Often bonded to other molecules (glycoproteins, glycolipids)

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

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High Performance Anion Exchange Chromatography

• Separates neutral and charged saccharides without derivatization

• Separates on the basis of:

• Charge, size, and composition

• Branching and linkage isomerism

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Electrochemical Methods - PAD

• Electrochemical methods: • Simple, direct detection method

• No sample derivitization • High sensitivity

• Comparable to derivitization with fluorescence detection (fmol to low pmol). • Minimal sample preparation

• Allows samples to be diluted 100 to 1000-fold, so simplifies sample prep • Compatible with high pH anion exchange column technology • Official methods (using Thermo Scientific™ Dionex™ CarboPac™ PA1

column) • Detection is linear over at least 4 orders of magnitude • PAD is a nondestructive method and can thus be used preparatively

or hyphenated to other detectors like MS

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High Performance Anion Exchange Chromatography –Pulsed Amperometry Detection (HPAE-PAD)

• Dissociation constants of some common carbohydrates

• Carbohydrate are weak acids.

• At high pH, they are at least partially ionized, and thus can be separated by anion-exchange mechanisms

• HPAE uses NaOH/KOH as eluent, sometimes with addition of Sodium Acetate to increase ionic strength.

• Pulsed-amperometric detection is an electrochemical detection method. With new waveform it provides direct, specific, highly-sensitive detection for carbohydrates.

Sugar pKa Fructose 12.03 Mannose 12.08 Xylose 12.15 Glucose 12.28 Galactose 12.39 Dulcitol 13.43 Sorbitol 13.60 -Methyl glucoside 13.71

(in water at 25 °C)

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Bead Structure of Dionex CarboPac Columns

NR3+ Latex

Latex Particle ( ≤ 0.1 µm)

CORE 5 - 10 µm SO3

-

Sulfonated highly-crosslinked core structure Latex Microbeads with Anion Exchange Functionalities

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Dionex’s IC Columns for Carbohydrates Analysis

Column Applications Time (min)

Dionex CarboPac PA20 column Fast Sialic Acid

Fast separation of N-acetyl- and N-glycolylneuraminic Acids (Sialic acids) <5

Dionex CarboPac SA10 and Dionex CarboPac SA10-4μm columns

Fast and high capacity separation of mono and disaccharides in biofuels, foods, and beverages. <10

Dionex CarboPac PA20 column

High-resolution separations of mono- and disaccharides with optimized resolution of glucosamine/galactose and glucose/mannose peak pairs.

<20

Dionex CarboPac PA200 column

High resolution separations of charged and neutral oligosaccharides. Separation of neutral and sialylated N-linked oligosaccharides from glycoproteins.

<50

Dionex CarboPac PA1 column For food nutrition labeling. Replaced by SA10 or PA20 ~

Dionex CarboPac MA1 column Reduced mono- and disaccharide. alditol and galactosamine <50

Fast

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Amperometry

• Measures current or charge resulting from the oxidation or reduction of analyte on a specific electrode surface

• Oxidation – electrons go from the analyte to the electrode

• Reduction – electrons go from the electrode to the analyte

Amperometry Electron Transfer

e-

Electrode Surface

e-

Analyte

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Application Examples

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Column: Dionex CarboPac PA10 and guard Eluent: 18 mM NaOH Flow Rate: 1.5 mL/min Detection: Pulsed amperometry, gold electrode Peaks: 1. Fucose 1 nmol 2. Galactosamine 1 3. Glucosamine 1 4. Galactose 1 5. Glucose 1 6. Mannose 1

2

0 8 10

0

100

nC

1

3 4

5

6

Minutes 6 4 2

Rapid Monosaccharide Analysis with the Dionex CarboPac PA10 Column

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Monosaccharide Analysis on Dionex CarboPac PA20 Column

0

100

0 2 4 6 8

Minutes

8 mM

10 mM

12 mM

14 mM

16 mM

18 mM

20 mM

1 2 3 4 5 6

nA

Column: Dionex CarboPac PA20 Dimension: 150 mm x 3 mm ID Eluent: 8-20 mmol/L NaOH Flow rate: 0.5 mL/min Detection: Pulsed amperometry, Au electrode Peaks: 1. Fucose 2. Galactosamine 3. Glucosamine 4. Galactose 5. Glucose 6. Mannose

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Column: Dionex CarboPac MA1

Eluent: 480 mM sodium hydroxide

Flow Rate: 0.4 mL/min

Detector: PAD (gold)

Peaks: 1. Inositol 18 mg/L 2. Glycerol 9 3. Arabitol 15 4. Sorbitol 18 5. Dulcitol 18 6. Mannitol 18 7. Mannose 18 8. Glucose 18 9. Galactose 18 10. Fructose 18 11. Sucrose 34 Minutes

0 10 20

0

µC

0.2

30 40 50

1

3 4

5

6 8 9

10

11

7

2

Separation of Reduced and Reducing Carbohydrates Commonly Found in Foods and Beverages

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Column: Dionex CarboPac PA10 Eluent: Sodium hydroxide/ sodium acetate gradient Flow Rate: 1 mL/min Inj. Volume: 25 µL Detection: Pulsed amperometry, Au electrode

Maltrin is a trademark of Grain Processing Corp.

0

175

nC

A: Maltrin M040

Minutes 0 5 10 15 20 25 30

B: Maltrin M700

0

175

nC

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Maltodextrins—Used to Make Natural Soda

HPAE-PAD is the only way to develop a quantitative analysis of the polysaccharide chain link

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Improved Chain-Length Resolution of Inulin Polymers

Columns: Dionex CarboPac PA200 (3 × 250 mm) CarboPac PA100 (4 × 250 mm) Gradient: 120 to 320 mM NaOAc in 100 mM NaOH over 40 min Flow Rate: Dionex CarboPac PA200: 0.5 mL/min Dionex CarboPac PA100: 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

PA100 0

20

Sialic Acid Analysis of Bovine Fetuin on Dionex CarboPac PA20 Column

Column: Dionex CarboPac PA20 (150 mm x 3 mm ID) Gradient: 20-200 mmol/L NaOAc in 0.1 mol/L NaOH over 10 min Flow rate: 0.5 mL/min Detection: Integrated pulsed amperometry, disposable Au electrode

-150

0

250

Minutes 0 2 4 6 8 10 12 14 16 18 20

nC

NANA NGNA

A

B

NANA

NGNA

B. NANA & NGNA standard

A. 0.1 mol/L HCl fetuin hydrolysate

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Fetuin N-Oligosaccharide Profiles: CarboPac PA200 vs. PA100

Column: Dionex CarboPac PA200 (3 x 250 mm) Dionex CarboPac PA100 (4 x 250 mm) Eluent: Sodium acetate gradient in 100 mM sodium hydroxide Flow Rate: Dionex CarboPac PA200: 0.5 mL/min Dionex CarboPac PA100: 1 mL/min Detection: Pulsed amperometry, Quadruple waveform, Au electrode Peaks: 1. Disialylated, triantennary 2. Disalylated, triantennary 3. Trisialyated, triantennary 4. Trisialyated, triantennary 5. Tetrasialylated, triantennary 6. Tetrasialylated, triantennary

0 10 20 30 40 50 60 70 Minutes

2

1

2

3 4

5 6

1 2

3 4

5 6

20

nC

160

Fetuin N-Oligosaccharide Profiles: Dionex CarboPac PA200 Column vs. Dionex CarboPac PA100 Colmun

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Data and System Management

High-Pressure Non-Metallic Pump

Eluent (OH– )Generator

Waste

Sample Inject (Autosampler)

EGC

H20

CR-TC

ED

Separation Column

HPAE-PAD System Flow Diagram with RFIC

Recycle Mode

Electrochemical Detector

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Fast Separation of Food Sugars Using Dionex CarboPac SA10 Column

-10

100

nC

0 2 4 6 8 10 Minutes

1

2

3 4 5

6

Column: Dionex CarboPac SA10-4µm and guard, 4 mm Eluent Source: Thermo Scientific Dionex EGC 500 KOH Cartridge Eluent: 1 mM KOH Flow Rate: 1.5 mL/min Inj. Volume: 10 µL Column Temp.: 40 °C Detection: PAD, Au on PTFE disposable, Four-potential Carbohydrate waveform Ref. Electrode: pH-Ag/AgCl Sample: Food Sugars Standard, 5mg/L Peaks:

1. Sucrose 2. Glucose 3. Fructose 4. Lactose 5. Cellobiose 6. Maltose

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Column: Dionex CarboPac SA10 (4.0 × 250 mm) Temp: 45 °C Eluent: 1 mM KOH (EG/CR-TC) Flow Rate: 1.5 mL/min Inj. Volume: 10.0 µL Det. Met.: PAD (carbohydrate quadruple waveform) Electrode: Au Peaks: 1. Mannitol 2. Arabinose 3. Galactose 4. Glucose 5. Xylose 6. Mannose 7. Fructose

Separation of Coffee Sugars on the Dionex CarboPac SA10 Column

0 1 2 3 4 5 6 7 8 9

20

80

nC

Minutes

1

2

3 4 5

6

7

25

Carbohydrates in Instant Coffee

Column: Dionex CarboPac PA1 Eluent: 150 mM sodium hydroxide/ deionized water gradient Inj. Volume: 25 µL of 10 g/L solution Detector: Pulsed amperometry, Au electrode; postcolumn addition of 0.3 M NaOH Peaks: 1. Mannitol 21 mg/L 2. Arabinose 140 3. Galactose 76 4. Glucose 44 5. Xylose 26 6. Mannose 51 7. Fructose 93 Sample Preparation: Phenolics removed with Thermo Scientific™ Dionex™ OnGuard™ P cartridge

0 10 20 30 40

1 2

3 4

5 6 7

1000

0

nA

Minutes

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Six Carbohydrates in 10 min Using Capillary Dionex CarboPac PA20 Column

Column: Dionex CarboPac PA20, 0.4 mm Eluent Source: Dionex EGC-KOH capillary with capillary Thermo Scientific Dionex CR-ATC Continuously Regenerated Anion Trap Column Eluent: 10 mM KOH Flow Rate: 0.008 mL/min Inj. Volume: 0.4 µL Column Temp.: 30 °C Detection: PAD, Au disposable, 4-Potential Carbohydrate waveform Gasket: 0.001” PTFE Ref. Electrode: Ag/AgCl Samples: 10 µM 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 0

60

nC

5

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Lactose and Lactulose in Raw Unpasteurized Milk Column: Dionex CarboPac SA10-4µm and guard, 4 mm Eluent Source: Dionex EGC 500 KOH cartridge Eluent: 4 mM KOH Flow Rate: 1.45 mL/min Inj. Volume: 10 µL Column Temp.: 35 °C 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

TN 146, Thermo Scientific, 2013

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Biofuel: Corn Stover Hydrolysate

0 2 4 6 8 0

180

nC

Minutes

1

2

3

4

5

Column: Dionex CarboPac SA10, Guard and Analytical , 2mm Eluent Source: Dionex EGC 500 KOH cartridge Eluent: 1 mM KOH Flow Rate: 0.38 mL/min Inj. Volume: 2.5 µL Column Temp.: 45°C Detection: PAD, Au on PTFE disposable, Four-potential Carbohydrate waveform Gasket: 15 mil thick PTFE Ref. Electrode: pH-Ag/AgCl Sample: Corn Stover Hydrolysate (150g/L, 1:200 Dilution) Peaks:

1. Arabinose 2. Galactose 3. Glucose 4. Xylose 5. Mannose

AN 282, Thermo Scientific, 2013 Dionex CarboPac SA10 Column Data sheet, Thermo Scientific, 2013

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Native Sugars in Apple Cider

Column: Dionex CarboPac PA20, 0.4 mm Eluent Source: Dionex EGC-KOH capillary cartridge with capillary Dionex CR-ATC column Eluent: 10 mM KOH Flow Rate: 0.008 mL/min Inj. Volume: 0.4 µL Column Temp.:30 °C 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 -- µM

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

Minutes

TN 135, Thermo Scientific, 2013

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Carbohydrates of Interest for Urine Analysis

Column: Dionex CarboPac PA20, 0.4 mm Eluent Source: Dionex EGC-KOH capillary cartridge with capillary Dionex CR-ATC column Gradient: 10 mM KOH (-7 to 1 min), 10–30 mM KOH (1–9 min), 30–35 mM from (9–16 min) Flow Rate: 0.008 mL/min Inj. Volume: 0.4 µL Column Temp.: 30 °C Detection: PAD, Au disposable, 4-Potential Carbohydrate waveform Gasket: 0.001” PTFE Ref. Electrode: Ag/AgCl Peaks: 1. Mannitol

2. 3-O-Methylglucose 3. Rhamnose 4. Galactose 5. Glucose 6. Xylose 7. Sucrose 8. Ribose 9. Lactose

10. Lactulose

nC

4

3

2

5

1

6

0 Minutes

10 16 4 12 8

50

7

8 9 10

TN146, Thermo Scientific, 2013

31

Minutes 1 2 3

19

31

0

nC A

B

C

2

1

Column: Dionex CarboPac PA20 Fast Sialic Acid, 3 × 30 mm Eluent: 70-300 mM acetate in 100 mM NaOH from 0−2.5 min, 300 mM acetate in 100 mM NaOH from 2.5−2.9 min, 300−70 mM acetate from 2.9−3.0 min; 1.5 min of equilibration at 70 mM acetate in 100 mM NaOH Flow Rate: 0.5 mL/min Inj. Volume: 4.514 µL (full loop) Column Temp.: 30 °C Detection: PAD, Au on PTFE, 2 mil gasket Ref. Electrode: pH-Ag/AgCl Samples: A) h. α1-acid glycoprotein, 1:100 dilution (23 ng protein) B) fetuin hydrolyzate, 1:100 dilution (18 ng protein) C) s. α1-acid glycoprotein, 1:100 dilution (7.9 ng protein) Peaks: (pmol) A B C

1. Neu5Ac 13 5.6 6.1 2. Neu5Gc — 0.20 1.2

N-acetylneuraminic acid (Neu5Ac) N-glycolylneuraminic acid (Neu5Gc).

AU 181, Thermo Scientific, 2011

Sialic Acids (Neu5Ac and Neu5Gc) in Glycoproteins

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0 2 4 6 8 10 12 14 16 18 20 21 35

70

Minutes

nC

1

2

3

4 5

6

Column: Dionex CarboPac PA20, and Thermo Scientific™ Dionex™ AminoTrap™ column Eluent Source: Dionex EGC-KOH cartridge with Dionex CR-ATC column Eluent: 10 mM KOH from 0−13 min 100 mM KOH from 13−16 min 10 mM KOH from 16−21 min Flow Rate: 0.5 mL/min Inj. Volume: 10 µL Column Temp.: 30 °C Detection: PAD, Au disposable, 4-Potential Carbohydrate waveform Ref. Electrode: Ag/AgCl Samples: 10 µM Mixed Standard Peaks: 1. Fucose

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

TN 40, Thermo Scientific, 2012

Glycoprotein Monosaccharide Analysis

Column Regen.

33

0 5 10 15 21

30

130

Minutes

nC

30

120

nC

B

1

2

3

4

1

2

3 4

Column: Dionex CarboPac PA20, and Dionex AminoTrap Eluent Source: Dionex EGC (III)-KOH cargtridge with Dionex CR-ATC column Eluent: 10 mM KOH from 0−13 min 100 mM KOH from 13−16 min 10 mM KOH from 16−21 min Flow Rate: 0.5 mL/min Inj. Volume: 10 µL Column Temp.: 30 °C Detection: PAD, Au disposable, 4-Potential Carbohydrate waveform Ref. Electrode: Ag/AgCl Samples: A: Fetuin HCl hydrolysate B: Fetuin TFA hydrolysate Peaks: 1. Galactosamine 2. Glucosamine 3. Galactose 4. Mannose

A

TN 40, Thermo Scientific, 2012

Monosaccharides in Digested Glycoprotein Samples

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High-Pressure Ion Chromatography (HPIC) System

Thermo Scientific™ Dionex™ ICS-5000+ both analytical and capillary systems

HPIC - High Resolution, Fast Analyses

Thermo Scientific™ Dionex™ ICS-4000 Capillary system

HPIC: A metal-free system which operates up to 5000 psi

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Conclusions

• HPAE-PAD is a fast carbohydrate analysis method • Directly quantify nonderivatized carbohydrates with high sensitivity and

selectivity

• HPAE-PAD is the one system for monosaccharides, sialic and other sugar acids, sugar phosphates, sugar alcohols, sulfate sugars, aminoglycoside antibiotics, oligosaccharides (charged and neutral), and small polysaccharides

• With innovations, such as HPIC and CarboPac columns, carbohydrates are analyzed in as little as 5 minutes