Fast, Accurate and Direct Carbohydrate Analysis Using HPAE-PAD
Transcript of Fast, Accurate and Direct Carbohydrate Analysis Using HPAE-PAD
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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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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
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0 5 10 15 20 25 30 35 40 45 50 55 60 –20
nC
Minutes
PA200
PA100 0
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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
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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
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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
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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
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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.
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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