biofuels and food applications - analytical2013/12/05 · astm d 4806-06c1 astm d 5798-062 din en...

Biofuels and Food Applications

Joachim Weiss, Ph.D.

Thermo Fisher Scientific GmbH, Dreieich, Germany

Proprietary & Confidential 2 Dionex Confidential

BIOFUELS ANALYSIS

Proprietary & Confidential 3 Dionex Confidential Dionex Confidential Dionex Confidential

Biomass to Biofuels Conversion Pathways

Solid Biomass

wood, straw

Resources Conversion Product Market

Wet Biomass

manure

organic waste

Sugar &

starch plants

sugar cane

Oil crops

rapeseed

sunflower

Steam

Liquid (BTL)

Hydrocarbons

Bio Oil

Bio Gas

Syngas

Bio-ethanol

Biodiesel

Digestion

Microbial

Fermentation

Biochemical

Distillation

Esterification

Fischer

Tropsch

Synthesis

Combustion

Gasification

Pyrolysis

Thermochemical

Fuel

Heat

Power HTU

Hydrolysis

Extraction


What is Biodiesel?

• Alternative fuel made from any fat or vegetable oil

• Used in diesel engine with no modifications

• Petroleum blends

• 20% blend is B20

• Pure form is B100

• Renewable fuel made from plant or animal oils

• Produced via transesterification reaction


ASTM Specifications for Biofuels

Parameter Bio-ethanol Biodiesel

ASTM D 4806-06c1

ASTM D 5798-062

DIN EN 14214 ASTM D 6751

Chloride (1 ppm) ASTM D 7328

ASTM D 4806 - -

Sulfate (4 ppm) ASTM D 7318

ASTM D 7319 - -

Group I Alkali Metals Na, K (5 ppm)

- EN 14108 UOP391

Group II Alkaline-Earth Metals

Ca, Mg (5 ppm) - EN 14108 TBD

Glycerol (2 ppm) - EN 14105/14106 D6584


Plant lb. oil/acre Gallons of

biodiesel/acre

Algae 6,757 700

Coconut 2,070 285

Jatropha 1,460 201

Rapeseed 915 126

Peanut 815 112

Sunflower 720 99

Soybean 450 62

Oil Seed Crop Production for Biofuels


ASTM Determination of Sulfate and Chloride in

Bio-ethanol

0 2.5 5 7.5 10 12.5 15

10

80

µS

Minutes

1

2

Column: IonPac AS22 + guard, 4 mm

Eluent: 4.5 mmol/L Na2CO3 +

1.4 mmol/L NaHCO3

Temperature: 30 °C

Flow rate: 1.2 mL/min

Inj. volume: 200 µL

Concentrator: TAC-ULP1

Detection: Suppressed conductivity,

AutoSuppression, recycle mode

Sample: Ethanol with 5% gasoline

spiked with aqueous standards

Peaks: 1. Chloride 4 mg/L

2. Sulfate 4


Determination of Glycerol

Column: IonPac ICE-AS1

Eluent: 5 mmol/L H2SO4/

10 mmol/L mannitol

Flow rate: 1 mL/min

Detection: D.C. amperometry, Pt, 0.6 V

Inj. volume: 50 µL

Peaks: 1. Glycerol

2. Sulfite

3. Ethanol

Minutes

0 2 4 6 8 10

0

35

nA

1

2

3


Determination of Cations in Biodiesel

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.2

2.0

µS

Minutes

1

2 3

4

Peaks: 1. Sodium

2. Potassium

3. Magnesium

4. Calcium


IC Determination of Cations in Biodiesel

Analyte #

Injections

Unspiked Sample

Concentration (ppb)

Measured Spiked Sample Concentrations

(ppb)

Unspiked Sample +

Spike (ppb)

% Recovery

Sodium 3 1021 1905 1880 103

Potassium 3 5.31 13.17 12.84 104

Magnesium 3 35.95 77.08 77.87 98

Calcium 3 173.6 340.3 337.0 102


Analysis of Organic Acids by IC-MS

Mucate

Quinate

2-methyllactate

Acetate

Glycolate

Propionate

Lactate Oxalate

2-hydroxyvalerate

Isovalerate Valerate

a-ketoglutarate

Tartrate

Oxalacetate

Glutarate

Malate

trans-Aconitate cis-Aconitate

Isocitrate

Citrate

SIM

Ch

rom

ato

gra

ms O

ve

rlay

Fumarate

Maleate

Succinate

Methylmalonate

Melonate

Adipate

Gluconate

Pyruvate

Butyrate

2-keto-D-gluconate

Galacturonate

5-keto-D-gluconate


1.0 40.0

1.0 39.0

60.0 38.0

60.0 36.0

30.0 28.0

15.0 18.0

1.0 8.0

1.0 –10.0

Conc. /mmol/L Time

Hydroxide Gradient

Column: IonPac AS11-HC

Temperature: 30 °C

Eluent: KOH gradient (EG)


Inj. volume: 25 µL

Sample: 50 ng of each analyte

1st Detection: Suppressed conductivity,

AutoSuppression,

External water mode

2nd Detection: MSQ Plus

Retention Time and SIM of 33 Small Organic Acids

1 2

3,4 5,6

7

8

10 12

13

14 15

16

17,18,19

20,21

23,24

25 26

27

28

30 31

32

33

0 5 10 15 20 25 30 35 40

µS

Minutes

8

29 22

9

11

1. Quinate

2. Gluconate

3. Lactate

4. 2-methyllactate

5. Acetate

6. Glycolate

7. Propionate

8. Formate

9. Butyrate

10. 2-keto-D-gluconate

11. 2-hydroxyvalerate

12. Pyruvate

13. Isovalerate

14. Valerate

15. Galacturonate

16. 5-keto-D-gluconate

17. Glutarate

18. Adipate

19. Mucate

20. Succinate

21. Malate

22. Methylmalonate

23. Melonate

24. Tartarate

25. Maleate

26. a-Ketoglutarate

27. Oxalate

28. Fumarate

29. Oxalacetate

30. Citrate

31. Isocitrate

32. cis-aconitate

33. trans-aconitate


IC-MS Determination of Organic Acids in Fermentation Broths

0

100

0

100

0

100 0

100 10.41

10.70

11.45

0

100 18.29

18.86

SIM ms [ [email protected]]





a-HBA

Acetate

Formate

Malate

Tartrate


Carbohydrate Analysis with HPAE-PAD

• Sensitive – 0.1 to 1 pmol detection limits

• High Carbohydrate Concentration Kit permits direct injection

of 10 to 80 g/L mixtures

• 0.2 μL injection loop

• 15 mil electrochemical detector gasket

• Direct detection – no derivatization necessary

• Accurate, reproducible analysis

• High-resolution separations, excellent peak efficiency for mono-,

oligo-, and poly-saccharides


Sugars: Key Feedstock of the Future

• Sugars are emerging as a key feedstock for the future – most

abundant resource on the planet

• Sugars are microbially fermented to yield basic chemical

building blocks:

• Alcohols

• Lactic acids

• Succinic acid

• Propylene glycol

• 3-Hydroxypropionic acid

• 1,3-Propanediol

• This enables low-cost production of bulk

organic chemicals


Shift from Hydrocarbon to Carbohydrate Economy

• Environmental, manufacturing, and sustainability costs are pulling a

variety of industrial processes toward carbohydrate feedstocks:

agro/wood

• Pulp/paper

• Fuel and energy

• Plastics – biochemicals/biopolymers

• Many of these processes are dependent on lignin, cellulose, and

hemicellulose carbohydrate chemistries and separation technologies


Bio-based Chemicals and Fuels from Sugars

Sugars Adipic acid

Polylactic acid

Glucaric acid

Succinic acid

Biochemicals

Food ingredients

Propylene glycol

1,3-Propanediol

Biofuels

Fuel alcohols

Biodiesel

Agricultural by-

products

Energy crops

Forestry waste

Food processing

waste

Industrial waste

Municipal waste

Chemo-Physical Treatment

and/or Enzymes

Biocatalysts

Microbial Organisms

Nutraceuticals

Drugs

Cosmetics

Pesticides

Antifreeze and deicing fluids

Paints and coatings

Nylon

Polyester

Textiles

Polymers and plasticizers

Surfactants/detergents

Ethanol

Propanol

Butanol

Dimethyl ether

Biodiesel


Analytical Needs for Biofuels Production

Challenge: to develop methods and standards for the measurement of the

hydrolysis products (sugars) of celluloses acting on lignocellulosic materials

• Published standards exist for carbohydrate analysis of biomass

• LC method E1758

• Poor peak resolution masks detection of minor sugars

• Long run times

• Anionic/cationic de-ashing columns required for desalting

• GC method E1821

• Complex, multistep derivatization

• Sugars have multiple peaks which introduces error

• HPAE-PAD methods are superior for profiling biomass carbohydrates

• Solves persistent problems of poor chromatographic fidelity and missed peaks

• CarboPac SA10 column increases throughput

• High Concentration Carbohydrate Kit solves sensitivity issue


Sources of Oligosaccharide Complexity

•Profiling of biomass carbohydrates

•Most abundant organic chemical on earth with annual biosphere production of ~90x109

metric tons

Essential Monosaccharides

• Chromatographic separations is key

• Similarity of carbohydrates (number of C, H, O) makes qualitative analysis by MS difficult

O

H

H

HO

CH3

OH H

H

OH

b-L-Fucose (Fuc)

CH2OH

HO

H

O OH

H

H OH

b-D-Galactose (Gal)

H

H

OH H

O

CH2OH

H

HO

H

H

OH

H

b-D-Mannose (Man)

OH HO

H

O

CH2OH

H

HO

H

H

OH

H

b-D-Glucose (Glc)

OH H

HO

Cellulose


Dissociation Constants of Some Common Carbohydrates

Sugar pKa

Fructose 12.03

Mannose 12.08

Xylose 12.15

Glucose 12.28

Galactose 12.39

Dulcitol 13.43

Sorbitol 13.60

α-Methyl glucoside 13.71

(in water at 25 °C)

At pH 13, most sugars deprotonate

to form anions

Even at pH 13, alditols are only weakly ionized


Separation of Biofuel Sugars on CarboPac SA10

Column: CarboPac SA10

Eluent: 1 mmol/L KOH (EG)


Inj. volume: 10 µL

Temperature: 45 °C

Detection: IPAD, Au working electrode

Sample: Biofuel sugar mix

Peaks: 1. Fucose 20 mg/L

2. Sucrose 20

3. Arabinose 20

4. Galactose 20

5. Glucose 20

6. Xylose 20

7. Mannose 20

8. Fructose 20

1

2

3

4 5

6

7

8

9 8 7 6 5 4 3 2 1 0

0

50

nC

Minutes


IC/MS Alditol Analysis of Microalgae Whole Cell Lysate

D+Arabitol

L-Arabitol

Mannitol

Algae Sample

0 10 20 30 40 50 60 70 80 90 100 110 120 130 141 –20

0

180

nC

7

6

5

4

3

2

1

Minutes

Xylitol Inositol

Dulcitol

Column: CarboPac MA1

Eluent: 500 mmol/L NaOH


Temperature: 30oC

Detection: PAD


High-Concentration Carbohydrate Analysis by HPAE-PAD

Column: CarboPac PA1, 4 x 250 mm


Flow rate: 1 mL/min

Inj. volume: 0.2 µL

Detection: IPAD, Au working electrode

Gasket: 15 mil UHMWPE cell gasket

0 1 2 3 4 5

0

5500

nC

Minutes

Glucose

Fructose

10-80 g/L Mixtures

• Reduces sensitivity

• Precludes dilution errors


FOOD & BEVERAGE ANALYSIS

Proprietary & Confidential 25 Dionex Confidential Dionex Confidential

Food & Beverage Components Analyzed by Ion Chromatography

• Inorganic anions

• Alkali- and alkaline-earth metals

• Organic acids

• Amines

• Carbohydrates

• Alcohols

• Amino acids


Food & Beverage Analytes Determined by IC

Inorganic Anions HPIC / Suppressed Conductivity, DC Amperometry, UV/Vis

IC Methodology Analyte

Alkali-/Alkaline-

Earth Metals HPIC / Suppressed Conductivity

Organic Acids HPIC / Suppressed Conductivity, HPICE / Suppressed

Conductivity

Aliphatic Amines HPIC / Suppressed Conductivity / Pulsed Amperometry

Carbohydrates HPIC / Pulsed Amperometry

Alcohols/Aldehydes HPICE / Pulsed Amperometry

Amino Acids HPIC / Pulsed Amperometry


Advantages of IC for Food & Beverage Analysis

• Sensitive Detection Samples can be diluted to decrease the concentration of

matrix components

• Specific Detection Low abundant analytes can be detected in presence of

large concentration of matrix components

• Analyte-Specific Separations The selectivity of the separator columns is tailored for the

specific requirements of the various analyte classes


• Meat and meat products

Simultaneous analysis of analytes in meat products:

Column: IonPac AS11 with AG11


Detection: UV, 225 nm

• Milk and milk products

Simultaneous analysis of analytes in milk products:


Eluent: 40 or 50 mmol/L KCl and 5 mmol/L Tris, pH 7.5


IC Methods for Nitrite/Nitrate Analysis


• 10.0 g of sample in 100 mL of water

• Homogenize for 1 min

• Heat sample between 70°C and 80°C for 15 min

• Centrifuge at 5000 × g for 10 min

• Filter the supernatant

• Collect the filtrate for ion chromatography

Extraction of Nitrate and Nitrite from Meat Products


• Dissolve 1.0 g of milk powder into 20 mL of water

• Filter through a 1.2 µm filter

• Centrifuge the filtrate in a Centricon 3 filter at 5000 × g for 30 min

• Pass the Centricon 3 filtrate through OnGuard-RP cartridge

• Collect the filtrate for HPLC analysis

Extraction of Nitrate and Nitrite from Milk Products


Determination of Nitrate and Nitrite in a Meat Extract



Flow rate: 1 mL/min

Inj. volume: 25 µL


Peaks: 1. Nitrite 1.16 mg/L

2. Nitrate 0.54

0 5 10

0.00

0.02

AU

Minutes

1

2


Sulfite in Food

• Sulfiting agents added to foods as antioxidants, preservatives,

and whiteners

• Added as sulfur dioxide, sodium sulfite, sodium and

potassium bisulfite, and sodium and potassium metabisulfite

• Many people suffer allergic reaction to ingestion of sulfites

• FDA requires warning labels on foods and beverages

containing 10 ppm of SO2

• AOAC contains several methods for determination of sulfites:

• Modified Monier-Williams method

• Ion exclusion-DC amperometric method (Kim & Kim)


Column: IonPac ICE-AS1

Eluent: 5 mmol/L sulfuric acid


Detection: DC amperometry,

Pt electrode, 0.6V

Inj.-volume: 50 µL

Peaks: 1. Glycerol

2. Sulfite 1.5 mg/L

3. Ethanol

Sample diluted 1:10. Sulfite concentration

was 15 mg/L in the undiluted sample

Separation of Sulfite, Glycerol, and Ethanol in White Wine

Minutes

0 2 4 6 8 10

0

35

nA

1

2

3


• Ion chromatography with suppressed conductivity or

UV detection

• Reversed-phase chromatography with DC amperometry

(silver electrode) (IDF/AOAC Method)

• Ion chromatography with DC amperometry

(platinum electrode)

• Ion chromatography with DC amperometry

(silver electrode)

Chromatographic Methods for Iodide Analysis


Iodide in Baby Formula

0 2 4 6 8 10

Minutes

Iodide

73 µg/L

Iodide

45µg/L

10

nA

-10

10

nA

-10

A.

B.

Column: IonPac AG11 with AS11

Eluent: 5 mmol/L HNO3


Inj. volume: 100 µL

Detection: DC amperometry, 0.05 V

Ag electrode

Samples: A. Milk-based baby formula

B. Soy-based baby formula


Colum: IonPac AS11 (2 mm)


Inj. volume: 20 µL

Detection: Suppressed conductivity

Peaks: 1. PO4

2. P2O7

3. P3O9

4. P3O10

5. P4O12

6. P4O13

Polyphosphate Analysis

Minutes

30

0

µS

0 10 5 15 25 45 40 35 20 30

1

3

2

4 6

5

Courtesy of E. S. Baluyot (Rhone-Poulenc Inc.)

^


Polyphosphates in Cheese

Column: IonPac AG11 / AS11 (2 mm),

Eluent: NaOH gradient


Inj. volume: 10 µL


AutoSuppression, recycle mode

Peaks: 1. PO4

2. P2O7

3. P3O9

4. P3O10

5. P4O12

6. P4O13

A: Bad Quality

B: Good Quality

E. Baluyot and C.G. Hartford, J. Chromatogr. A 739 (1996) 217-222


Column: IonPac AS19 (0.4 × 250 mm)

and AG19 (0.4 × 50 mm)

Eluent: KOH (EG)

Gradient: 7 min 15 mmol/L isocratic,

in 18 min to 60 mmol/L

Temperature: 30 °C

Flow rate: 10 µL/min


Detection: Suppressed conductivity

Sample: English breakfast tea

0 10 20 30

–10

90

µS

Minutes

Ch

lorid

e

Nitra

te

Su

lfa

te

Ma

late

Ort

ho

ph

osph

ate

Citra

te

Anions in Tea


Fast Analysis of Orthophosphate and Citrate in Cola Drinks

0 2 4 6 -5

35

1 2

3

Minutes

µS

4

Column: IonPac Fast Anion IIIA with guard


Temperature: 30 °C

Flow rate: 1 mL/min



AutoSuppression, Recycle Mode

Peaks: 1. Unknown — mg/L

2. Unknown —

3. Orthophosphate 456

4. Citrate 52


Gradient Elution of Anions in Diet Coke

0 4 6 8

-5

40

Minutes

µS

2

1

2 3

4

5

6

Column: IonPac Fast Anion III with guard

Eluent: KOH (EG)

Gradient: 5 mmol/L from 0-2 min,

5-30 mmol/L from 2-5 min

Temperature: 30 °C

Flow rate: 1 mL/min



AutoSuppression, Recycle Mode

Peaks: 1. Unknown — mg/L

2. Chloride 20

3. Benzoate 78

4. Sulfate 102

5. Orthophosphate 227

6. Citrate 145


LC Methods for Organic Acid Analysis

• Ion-Exclusion Chromatography

• Anion Exchange Chromatography

• Reversed-Phase Chromatography

• Ion suppression techniques

• Mixed-Mode Chromatography

• Ion-exchange, reversed-phase

• HILIC


Column: Acclaim Mixed-Mode WAX-1, 5 µm

Dimensions: 150 mm × 4.6 mm i. d.

Temperature: 30°C

Eluent: 25 mol/L phosphate buffer, pH 6


Inj. volume: 10 µL


Peaks:

1. Quinic acid

2. Shikimic acid

3. Glycolic acid

4. Lactic acid

5. Acetic acid

6. Formic acid

7. Ascorbic acid (Vitamin C)

8. Iso-ascorbic acid

9. Propionic acid

Separation of Monovalent Carboxylic Acids

AU

Minutes 0 5 10 15

1

2

3 6

7

9

8 4

5


Analysis of Organics in Soft Drink Products

Column: Acclaim Mixed-Mode WAX-1, 5 µm

Dimension: 150 mm × 4.6 mm i. d.

Temperature: 30°C

Eluent: 57/43 v/v MeCN / 120 mmol/L

phosphate buffer, pH 2.9

Flow rate: 2 mL/min



Sample: Direct injection of degassed sample

Peaks: 1. Caffeine

2. Aspartame

3. Sorbate

4. Benzoate

5. Citrate

6. Acesulfam K

0 1.5 3

Minutes

AU

Standard

Diet COKE

Diet PEPSI

2

3 6 4

1

5

2

4

1

5

2

4

1

5


HILIC – Hydrophilic Interaction Chromatography

• HILIC is a variation of normal-phase chromatography

without the disadvantages of using solvents that are not

miscible with water

• Also called “reverse reversed-phase” or “aqueous normal

phase” chromatography

• HILIC is used to retain very polar compounds

• Stationary phase is a polar material such as silica, cyano,

amino, diol, etc.

• Mobile phase is highly organic (>80 %) with a small

amount of polar solvent (e.g, H2O)


What are the Benefits of HILIC?

• Retain highly polar compounds that would not be retained by

reversed-phase chromatography

• Complementary selectivity to reversed-phase columns

• Enhanced sensitivity in mass spectrometry

• Utilizing high organic mobile phases (> 80 %) enhances ESI-MS

response

• Shortens sample preparation procedure

• Final SPE or liquid/liquid extraction step usually uses an organic

solvent (e.g., acetonitrile, isopropanol, etc.)

• Then organic solvent must be evaporated to dryness and

reconstituted in mobile phase before injecting onto a reversed-phase

HPLC column.

• Elimination of evaporation/reconstitution step by directly injecting the

sample


HILIC Types

• “Neutral” surface

• Diol group (simple retention mechanism, fast equilibration,

demonstrated applications)

• Cyano group

• “Ion-Exchange” surface

• Silanol group

• Amino group

• “Zwitterionic” surface

Silica

CN

Silica

NH

2

Silica

Silica

OH

OH

O

HO

HO

Silic

a

N SO3

Me

Me

+ -

Proprietary & Confidential 47

Acrylic Acid and Oligomers

Column: Acclaim HILIC-10, 3 µm

Dimensions: 150 mm × 4.6 mm i. d.

Eluent: 90/10 v/v MeCN/10 mmol/L (total) NH4OAc, pH5

Flow rate: 1 mL/min

Temperature: 30 °C

Inj. volume: 2 µL


Peaks: (5 mg/mL in eluent)

1. Acrylic acid (monomer)

2. Dimer

3. Trimer

4. Tetramer

5. Pentamer

6. Hexamer

0 6 12 9 3 15

Minutes

mA

U

0

30

1

2

3

4

5 6


Surface Chemistry of Acclaim Mixed-Mode HILIC-1

Silica

OH

OH

OH

OH

OH

OH

OH

OH

Hydrophilic portion

Hydrophobic portion


Analysis of Fructooligosaccharides in Strawberry Joghurt

1 2

3

4

5

6 7

nC

40

-5

0 10 20 30

Minutes

Column: CarboPac PA1

Eluent: NaOH / NaOAc gradient

Flow rate: 1 mL/min

Inj. volume: 20 µL

Detection: Pulsed amperometry,

Au electrode

Sample

preparation: Carrez 1 & 2

Peaks: 1. 1-Kestose

2. Maltose

3. Nystose

4. Maltotriose

5. Inulotriose

6. Fructosylnystose

7. Maltotetraose


Column: CarboPac MA1

Eluent: 150 mmol/L NaOAc,

0.2 % (v/v) acetic acid, pH 5.5


Inj. volume: 25 µL

Detection: Integrated pulsed amperometry

Peaks: 1. 4-Cl-Galactose 7.8 mg/L

2. 1,6-di-Cl-Fructose 2.4

3. Sucralose

Analysis of Sucralose

nA

780

0

1

2

0

Minutes

30 40 50 60 10 20

3


Monosaccharides in Soluble Coffee

0 5 10 15 20 25 30 35 40 45 50

20

35

nC

1

Standard (1-9 µg/mL)

2 3

4 5

6

7

8 9 10

11 Column: CarboPac PA10 with guard


Flow rate: 1 mL/min

Inj. volume: 5 µL

Detection: Pulsed amperometry,

Au electrode

Peaks :

1. Mannitol 7. Sucrose

2. Fucose 8. Xylose

3. Arabinose 9. Mannose

4. Rhamnose 10. Fructose

5. Galactose 11. Ribose

6. Glucose

0 5 10 15 20 25 30 35 40 45 50

Minutes

20

35

nC

Instant Coffee

(OnGuard RP Treatment)

1

2

4

3

5

6 7 8

9

10 11


Conclusions

• IC is a powerful tool for the automated analysis of foods

and beverages

• Simple, direct methods with minimal sample

preparation can often be developed by using specific,

high sensitivity detection combined with column

selectivities tailored to the analytes

• Sample preparation for beverages can often be

reduced to only a dilution step

53 Proprietary & Confidential

Thank you for your kind attention!

biofuels and food applications - analytical2013/12/05 · astm d 4806-06c1 astm d 5798-062 din en...

Documents