HPAE-PAD analysis of Carbohydrates. Michael G. Hvizd Manager, Laboratory Operations Centers of Excellence Dionex Corporation Laboratory

Transcription

1 HPAE-PAD analysis of Carbohydrates Michael G. Hvizd Manager, Laboratory Operations Centers of Excellence Dionex Corporation Laboratory

2 Presentation Outline Principles of High Performance Anion Exchange Column Technologies Principles of Pulsed Amperometric Detection Applications using HPAE-PAD 226

3 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 1 to 1-fold, so simplifies sample prep Minimal Sample Preparation is necessary Unique column technology Official Methods (using CarboPac PA1)

4 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 cationexchange columns.

5 Rapid Monosaccharide Analysis with the CarboPac PA1 1 nc Column: CarboPac PA1 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 Minutes

6 Detection of Food Sugars and Sugar Alcohols 5 Column: CarboPac PA1, PA1 guard, Borate Trap Eluent: 52 mm sodium hydroxide Flow Rate: 1.5 ml/min Inj. Volume: 5 µl Detection: Pulsed amperometry, gold electrode 5 Peaks: 1. Glycerol 4. mg/ml 2. Xylitol Sorbitol Mannitol Glucose Fructose Sucrose Lactose 8. nc Potential (V) Minutes Time (s)

7 Separation of Reduced and Reducing Carbohydrates Commonly Found in Foods and Beverages.2 1 µc Minutes Column: Eluent: Flow Rate: Detector: CarboPac MA1 48 mm sodium hydroxide.4 ml/min PED (gold) Peaks: 1. Inositol 18 mg/l 2. Glycerol 9 3. Arabitol Sorbitol Dulcitol Mannitol Mannose Glucose Galactose Fructose Sucrose

8 Separation of Maltose Oligomers 7 1 Column: CarboPac PA-1 and guard Eluent: NaOH/sodium acetate gradient Flow Rate: 1. ml/min Inj. Volume: 1 µl Detection: Pulsed amperometry, Au electrode nc Peaks: 1. Glucose 2. Maltose 3. Maltotriose 4. Maltotetraose 5. Maltopentaose 6. Maltohexaose 7. Maltoheptaose 8. Maltooctaose 9. Maltononaose 1. Maltodecaose Minutes

9 Maltodextrins Maltrin M4 and M7 175 nc 175 A: Maltrin M4 B: Maltrin M7 Column: CarboPac PA1 Eluent: Sodium hydroxide/ sodium acetate gradient Flow Rate: 1 ml/min Inj. Volume: 25 µl Detection: Pulsed amperometry, Au electrode nc Maltrin is a trademark of Grain Processing Corp Minutes

10 Distribution of Chain Length of Amylopectins by HPAE-PAD 12 Rice 12 2 Corn Sweet Potato Edible Canna Retention Time (min) From: Koizumi, K., Fukuda, M., Hizukuri, S.. J. Chromatography. 1991, 585,

11 18 nc PA2 Improved Chain-Length Resolution of Inulin Polymers Columns: CarboPac PA2 (3 x 25 mm) CarboPac PA1 (4 x 25 mm) Gradient: 12- to 32-mM NaOAc in 1 mm NaOH over 4 min Flow Rate: PA2:.5 ml/min PA1: 1. ml/min Detection: Pulsed amperometry, quadruple waveform, gold electrode PA Minutes 268-1

12 Separation of N-Linked Oligosaccharides 2 na Minutes NANA Gal GlcNAc Man α2 6 Column: CarboPac PA-1 Eluent: to 25 mm NaOAc over 11 min in 1 mm NaOH Flow Rate: 1 ml/min Detector: PAD (Gold) Peaks: 1. Fucosylated Man 3 GlcNAc 2 2. Man 3 GlcNAc 2 3. Asialo, agalacto bi, core fuc 4. Asialo, agalacto bi NANA Gal GlcNAc Man α Asialo bi, core fuc 6. Asialo bi 7. Man 9 GlcNAc 2 8, 9. Disialylated tri (reduced) 1, 11. Trisialylated tri (reduced) 12, 13. Tetrasialylated tri (reduced) Peak 1 Peak 11

13 Official Food Methods Using IC/PAD Analysis Sugars in Molasses Carbohydrates in Soluble Coffee Fructans in Food and Food Products Polydextrose Transgalactooligosaccharides Low-Level Glucose and Fructose in Raw and Refined Sugar CarboPac Column CarboPac PA1 CarboPac PA1 CarboPac PA1 CarboPac PA1 CarboPac PA1 CarboPac PA1 Official Method AOAC Method ICUMSA Method Approval 1994 AOAC Method ISO Approved AOAC Method AOAC Method 2.11 AOAC Method 21.2 AOAC Method

14 Column Technology 13916

15 High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) PAD) Separates neutral and charged saccharides without derivatization Separates on the basis of: Charge, size and composition Branching, linkage isomerism 1328

16 Dissociation Constants of Some Common Carbohydrates (in Water at 25 C) Sugar Fructose Mannose Xylose Glucose Galactose Dulcitol Sorbitol α-methyl glucoside pk a

17 Polymeric Nonporous ( Pellicular Pellicular ) Packing Highly Crosslinked Core Latex Microbeads with Anion Exchange Functionalities 8674-B

18 16887

19 Electrochemical Detection 13916

20 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

21 Principal of Pulsed Amperometric Detection (PAD) Quadruple Potential Waveform for Carbohydrates Potential (V vs. Ag/AgCl).8 Integrate Au Oxide Formation Au Oxide Reduction, Electrode Activation Time Pot. Integ Begin End Time (seconds) Electrode Cleaning 1345

22 Pulsed Amperometric Detection (PAD) Waveform and Integration Period 1 1 Potential ( V ) 1 2 Potential ( V ) Time (s) Time (s) Carbohydrate Waveform Amino Acid Waveform Integration Period 22136

23 Sugars in Sugar Cane Molasses Column: CarboPac PA1 and guard Eluent: 15 mm NaOH Flow Rate: 1. ml/min Inj. Volume: 5 µl Detection: Pulsed amperometry µa 1 Peaks: 1. Glucose 4.39% 2. Fructose Lactose (int. std.) 4. Sucrose Minutes 193-3

24 Carbohydrates in Instant Coffee Column: Eluent: Inj. Volume: Detector: CarboPac PA1 15 mm sodium hydroxide/ deionized water gradient 25 µl of 1 g/l solution Pulsed amperometry, Au electrode; postcolumn addition of.3 M NaOH na Minutes Peaks: 1. Mannitol 21 mg/l 2. Arabinose Galactose Glucose Xylose Mannose Fructose 93 Sample Preparation: Phenolics removed with OnGuard P cartridge 67-3

25 65 Sucralose in Diet Drink and Artificial Sweetener Product Red Raspberry Diet Soda Diluted 5-Fold Sucralose nc Sucralose Splenda Sweetener 1 µg/ml Column: CarboPac PA2 with guard Eluents: 4 mm sodium hydroxide/ 75 mm sodium acetate Temperature: 3 C Flow Rate:.5 ml/min, 1-g/L solution Inj. Volume: 25 µl Detection: Pulsed amperometric detection, carbohydrate waveform, disposable gold working electrode Minutes Splenda is a registered trademark of McNeil-PP 267-1

26 Oligosaccharide Profiles of Pure and Adulterated Orange Juice 5 µa 5 µa A. Pure Orange Juice B. Orange Juice Adulterated with Beet Medium Invert Sugar Column: CarboPac PA-1 Eluent: Sodium hydroxide/ sodium acetate gradient Flow Rate: 1 ml/min Conc.: 2 mg/ml Inj. Volume: 25 µl Detection: Pulsed amperometry, Au electrode Minutes

27 Apple Juice.2 µc 2 3 Column: CarboPac MA1 Eluent: 5 mm sodium hydroxide Flow Rate:.4 ml/min Detection: Pulsed amperometry Peaks: 1. Sorbitol 14 µg 2. Glucose Fructose Sucrose Minutes

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