Analytical methods and Chromatography · Ionic chromatography DIONEX with Pulsed Amperometry...

Tinaïg Le Costaouëc

Analytical methods and

Chromatography

Chromatography Service : Claire Boisset-Helbert (Scientific Supervisor)

Laurine Buon (Technical Supervisor)

Philippe Colin-Morel (Technician)

Eric Bayma (Engineer assistant)

Composition :

• Neutral residue

• Acidic residue

• Hexosamines

• Substituents : organic, inorganic

Polymer of carbohydrates, linear or branched

OHHO

Polysaccharide

2 Alage4a-b - Workshop - Grenoble 2017

Homopolysaccharides : one type of monosaccharide

cellulose, amylose, dextran...

OHHO

Heteropolysaccharides : at least two types of different monosaccharides

galactomannan, arabinoxylan, alginate…

OHHO


Origin Seaweeds

Animals

O

OH

CH2OH

OO

OH

CH2OH

OO

NHAcOH

CH2OH

O

NHAc

NHAc

Chitin

Plants Cellulose

Alginate, fucoidans


Bacteria Microalgae

Fungi


Polysaccharide

Applications


Polysaccharide

Applications

1 - Analysis of native

polysaccharide


Overall composition

Colorimetric assays

Gamme etalon (dosage protéines)

y = 0,0344x

R2 = 0,9826

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0 5 10 15 20 25

Quantité (µg)

DO

Gamme étalon (dosage sucres)

y = 0,0433x

R2 = 0,9896

0,000

0,500

1,000

1,500

2,000

2,500

0 10 20 30 40 50 60

Quantité (µg)

DO

Dosage of total carbohydrates : Dubois method (1956)

Dosage of proteins : Bradford method (1976)

Standard curve with glucose

Standard curve with Bovin Serum Abumin

Quantity (µg)

Quantity (µg)

OD

(595 n

m)

OD

(480 n

m)


Composition Mass percentage

Neutral

carbohydrates Uronic acids proteins

Sulfate groups

Polysaccharide 30 30 2.6 9.4

Carbohydrate content = 60%

Carbohydrate content : from 1% to 85 %


Characterization of polysaccharides

OHHO

Molecular weight

Chemical composition

Structural elucidation

Chromatography


Chromatography

Columns

Eluent

Pump

Detector

Control and data analysis

Injector


Molecular weight

HPSEC with double detection: - Differential refractometer

- MALLS

Chromatogram of a mixture of pullulans

eluted on SB-803OHpak and SB804OHpak shodex columns in series

0,2M sodium nitrate

212 000 Da

22 800 Da


- Polydispersity Index

The PDI reflects the heterogeneity of molecular weight

distribution of polysaccharide chainsPDI = Mw / Mn

Carbohydrate composition

4)--D-Glcp-(14)-a-D-GalpA-(14)-a-D-Galp-(13

1a-D-GlcpA

3

1-D-GlcpA

4

14,6-Pyr--D-Manp

Nature of glycosidic residues

GC, IC (HPAEC-PAD), NMR



D-glucose

D-galactose

D-mannose

L-fucose

L-rhamnose

- Neutral carbohydrates

OCH3 OH

OHOH

OH

O

CH2OH

OH

OH

OH

OH

D-glucuronic acid

D-galacturonic acid

- Uronic acids

O

COOH

OH

OH

OH

OH

- Hexosamines

N-acetyl-D-glucosamine

N-acetyl-D-galactosamine

O

CH2OH

OH

NHAc

OH

OH


Analysis

Polysaccharide

Total depolymerization

Derivatization

or not


- GC

- IC (HPAEC-PAD)

Gas Chromatography : Principle

Gas chromatograph AGILENT 6850

- with Flame Ionisation Detection (FID)

- or coupled with mass spectrometry

Total hydrolysis

+

+ +

derivatization

Analysis by GC

+



O

OHO

OHHO

OH

O

OHO

OHHO

OH

Me Me+

Methanolysis MeOH/HCl 3 N, 4 h, 100°C

O

OOH

O

O

HO

HO

OH

HO O

OH

TrimethylsilylationPyridin + Sylon BSTFA TMCS

99:1, 12 h, room Tp

O

OO

OO

O

O

OO

OO

O

Me Me

Tms

Tms

TmsTms

Tms

Tms

TmsTms +

Preparation of Trimethylsilylated derivatives (TMS) of polysaccharides

Hydrolysis TFA 2N or H2SO4 2N, 4h, 100°C

O

OOH

O

O

HO

HO

OH

HO O

OH

Reduction and

acetylation

NaBH4/pyridin, acetic anhydrid

Preparation of Alditol acetates of polysaccharides

OO

O

OO

OH

+H

H

HH

H

OAc

OO

O

OO

OH

H

H

HH

H

OAcOAc

OAc

AcOAcO

+

OAcOAc

OAc

OAc

AcOAcO

Gas Chromatography : Derivatization


MeMe

or

Flame Ionization

detector

Gas Chromatography : Principle


30 m X 0.53 mm ,

0.2 µm id

Separation of alditol acetates of monosaccharides injected on a Supelco SP-2380 column, with helium as vector gas

Identification and quantification of neutral sugars and hexosamines

One peak for one type of sugar

Gas Chromatography : Alditol acetate derivatization

AGILENT 6850 with Mass Spectrometry Detection


Separation of trimethylsilylated monosaccharides of hydrolysed polysaccharide injected on a HP5-MS column, with nitrogen as

vector gas

Myo-inositol

Rh

a

Man

Glc

Gal

Ara

Glc

Gal

Gal

Man

GalN

Ac

Ara

Ara

Ara

GalN

Ac

GalN

Ac

GalN

Ac

Glu

cN

Ac

Glu

cN

Ac

?

?

?

??

Identification and quantification of neutral sugars, hexosamines and uronic acids

Between one and four peaks for one type of sugar

Gas Chromatography : Trimethylsilylation derivatization

AGILENT 6850 with Flame Ionisation Detection (FID)


High Perfomance Anion exchange Chromatography (HPAEC)

CarboPac column Construction

Core

Sulfonated surface

Ion-Exchange Surface

Quaternary ammonium

functionalized latex


High pH:

ionized

carbohydrates

NaOH

NaOAc

eluents

Ionic chromatography DIONEX with Pulsed Amperometry Detection

Separation of monosaccharides on a CarboPac PA1 column (Dionex) eluted with a NaOH/NaOAc gradient

HPAEC-PAD Analysis of carbohydrate without derivatization

4,3 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0 32,0 34,0 36,0 38,0 40,0 42,0 44,0 46,0 48,4

-6

10

20

30

40

50

60

70

80

95

ETALONNAGE-TFA-070613 #4 Etalon 4 ED_1

nC

min

2 -

6,1

67

3 -

fucose -

8,8

67

4 -

14,2

00

5 -

rham

nose -

16,2

34

6 -

arabin

ose -

18,8

84

7 -

gala

cto

se -

25,6

17

8 -

glu

cose -

28,1

00

9 -

xylo

se -

31,3

67

10 -

36,2

84

11 -

rib

ose -

38,8

17

12 -

42,1

67

13 -

42,8

34

14 -

Acid

e G

al - 4

5,2

67

15 -

Acid

e g

lc -

46,3

50

1 2 - rha... 3 - arabi... 4 - gala... 5 - glucose 6 - ma... 7 - xyl... 8 - r... 9 ...

Gala

ctu

roniq

ue a

cid


Linkage

ose/ose

Linkages analysis


1a-D-GlcpA

3

1-D-GlcpA

4

14,6-Pyr--D-Manp

Nature of glycosidic

linkages

GC/MS and NMR

Position of

substituents

GC/MS

4?

3?

4?

3?

Linkage

ose/substituent

O

2

14

3

5

6

Cyclic form of residues p or f

GC/MS


PMAA Partially methylated alditol acetates

Linkage analysis - methylation and GC/MS


GC / MS of PMAA derivatives

HDCOAc

OAc

OMe

OAc

H2COMe

161

190

Ribose linked in C2 :

1,2,4-tri-O-acetyl-1-deuterio-3,5-di-O-methyl-D-ribitol

Myo-inositol


GC

MS

Type of

monosaccharideAlditol acetates partially methylated

Primary fragments

(m/z)Corresponding

substituted residu

Fucose 1,5-di-O -acétyl-2,3,4-tri-O -méthyl-fucitol 118 131 162 175 Fucp -(1->

Rhamnose 1,5-di-O -acétyl-2,3,4-tri-O -méthyl-rhamnitol 118 131 162 175 Rhap -(1->

1,2,5-tri-O -acétyl-3,4-di-O -méthyl-rhamnitol et/ou

1,4,5-tri-O -acétyl-2,3-di-O -méthyl-rhamnitol131 190 et/ou 118 203->2) et/ou 4)-Rhap -(1->

1,3,5-tri-O -acétyl-2,4-di-O -méthyl-rhamnitol 118 131 234 247 ->3)-Rhap -(1->

1,3,4,5-tri-O -acétyl-2-O -méthyl-rhamnitol 118 275 ->3,4)-Rhap -(1->

1,2,3,5-tri-O -acétyl-4-O -méthyl-rhamnitol 131 262 ->2,3)-Rhap -(1->

Glucose 1,5-di-O -acétyl-2,3,4,6-tétra-O -méthyl-glucitol 118 161 162 205 Glcp -(1->a

1,4,5-tri-O -acétyl-2,3,6-tri-O -méthyl-glucitol 118 162 233 277 ->4)-Glcp -(1->

Galactose 1,5-di-O -acétyl-2,3,4,6-tétra-O -méthyl-galactitol 118 161 162 205 Galp -(1->

1,3,5-tri-O -acétyl-2,4,6-tri-O -méthyl-galactitol 118 161 162 233 ->3)-Galp -(1->

1,4,5-tri-O -acétyl-2,3,6-tri-O -méthyl-galactitol 118 162 233 277 ->4)-Galp -(1->b

1,3,5,6-tétra-O -acétyl-2,4-di-O -méthyl-galactitol 118 189 234 305 ->3,6)-Galp-(1->

Mannose 1,5-di-O -acétyl-2,3,4,6-tétra-O -méthyl-mannitol 118 161 162 205 Manp -(1->a

1,4,5-tri-O -acétyl-2,3,6-tri-O -méthyl-mannitol 118 162 233 277 ->4)-Manp -(1->b

1,2,3,5-tétra-O -acétyl-4,6-di-O -méthyl-mannitol 161 262 ->2,3)-Manp -(1->

Glucuronic acid 1,5,6-tri-O -acétyl-2,3,4-tri-O -méthyl-6-d 2-glucitol 118 162 191 235 GlcpA -(1->

1,3,5,6-tétra-O -acétyl-2,4-di-O -méthyl-6-d 2-glucitol 118 191 234 307 ->3)-Glcp A-(1->

1,4,5,6-tétra-O -acétyl-2,3-di-O -méthyl-6-d 2-glucitol 118 263 ->4)-Glcp A-(1->

Galacturonic acid 1,3,4,5,6-tétra-O -acétyl-2-di-O -méthyl-6-d 2-galactitol 118 335 ->3,4)-Galp A-(1->

Ramifications and/or Substituents

Corresponding substituted residue


Substituents


1a-D-GlcpA

3

1-D-GlcpA

4

14,6-Pyr--D-Manp

Nature and position of

substituents

EA, IC, IR and NMR


Ionic chromatography Dionex with Conductimetric detection

Substituents


Separation of organic acids and inorganic groups on IonPac AS11 column (Dionex)

eluent: NaOH gradient

Elemental analysis: Central Analysis Service of Lyon

Inorganic substituents :

Sulfate, phosphate contentSO3

-

1H NMR spectra of native bacterial EPS (70°C, 500 MHz)

ppm

Pyr

Lac

Lactate

Pyruvate

1D/2D NMR

Organic substituents :

Lactate, pyruvate, succinate


O-H

C-H C=O

C-O

S=O

C-O-S

Laminarin

Sulfated

Laminarin

FT-IR

spectroscopy


Full structure / Sequence

Analysis by Nuclear Magnetic Resonance (NMR)

4)--D-Glcp-(14)-a-D-GalpA-(14)-a-D-Galp-(1 3

1a-D-GlcpA

3

1-D-GlcpA

4

14,6-Pyr--D-Manp

Linkages position

Presence and position of

substituents

Anomeric configuration

a /

Sequence

43

OH

OH

13

2


1H, 13C

COSY, TOCSY

HSQC, HMBC



Anomeric

Substituted

H6a-6b/C6

HSQC NMR spectrum of microalgae polysaccharide (55°C, 400 MHz)

1H, 13C

COSY, TOCSY

HSQC, HMBC



Polysaccharide

Applications

2 - Analysis after

depolymerisation


1. Native polysaccharide analysis

2. Partial depolymerisation

3. Fractionation

Structure

4. Oligosaccharidic fraction analysis

Partial depolymerization


Chemical hydrolysis

Depolymerization of polysaccharides

Methanolysis (MeOH/HCl)

Acidic hydrolysis (HCl, H2SO4, TFA …)

Radical Hydrolysis (H2O2)

Smith degradation

-Elimination

Lithium degradation …

Non specific degradations Specific degradations

Enzyme hydrolysis Specific degradations


Purification of oligosaccharides

Size Exclusion Chromatography

(SEC) Largest molecules

Excluded

Separated

smallest molecules

Exclusion

Gel granule

Pores in

the granule gel

Late elution

Elution with

Vo


Separation and purification of maltodextrins from DP1 to DP10 on a Biogel P2, elution with H2O

Size Exclusion Chromatography


HPAEC-PAD for Oligosaccharides separation

Ionic chromatography DIONEX with Pulsed Amperometry Detection

14,3 16,3 17,5 18,8 20,0 21,3 22,5 23,8 25,0 26,3 27,5 28,8 30,0 31,3 32,5 33,8 35,0 36,3 37,5 38,8 40,0 41,3 43,0

-24

0

25

50

75

100

125

150

175

200

225

250

291

maltoD (1à7)-150610 #1 [modified by Administrateur] maltoD DP:1à7 ED_1

nC

min

1 -

maltose -

24,5

00

2 -

maltotr

iose -

27,9

50

3 -

28,5

00

4 -

maltoté

traose -

30,6

17

5 -

30,8

67

6 -

maltopenta

ose -

32,7

67

7 -

maltohexaose -

34,6

84

8 -

maltohepta

ose -

36,6

67

... ... ... ... ... ...

Separation of maltodextrins from DP 2 to DP 7 on a CarboPac PA1 column (Dionex)

eluted with NaOH/NaOAc gradient


Oligosaccharide Analysis

Carbohydrate composition (GC)

Linkages analysis (GC/MS)


Sequence

13

2

1D/2D NMR

Mass spectrometry (ESI-IT, MALDI-TOF)

Polysaccharide

Applications

3 - Example: Structure of a microalgae

Cell wall polysaccharide


Purification


Microalgae powder

Residue

Soxhlet extractionEthanol, then acetone

Oven 60°C overnight

Storage polysaccharide

(β-Glucan)

Residue

Chlorite (80°C, 3h) /NaOH 1% (RT,1h)

Residue

Cell wall polysaccharide

Filtration 0.45µmlyophilisation

Concentration, dialysis 1kDa, filtration 0.45 µm, lyophilisation

Residue

Water-80°C-2h X2

X3

NaOH 4% (100°C, 45 min)

66 % w/w

58 % w/w

3-5 % w/w

12 % w/w

Native cell wall polysaccharide analysis

Colorimetric assays

Total

sugar %

w/w

Proteins

% w/w

Sulfate

groups

% w/w

Mw

g/mol

Cell wall

Polysacch

aride

51,3 ± 4,7 7,0 ± 1,5 5,0 130 000

- Total sugar: Dubois

- Proteins: Bradford assay

- Sulfate groups: elemental analysis (EA)

Molecular weight

HPSEC/MALLS


Native cell wall polysaccharide

Linkage analysis: GC/MS

Partially methylated alditol acetates

preparation (Hakomori, 1964)

Carbohydrate composition: GC

Trimethylsilyl derivatives preparation

Man / GlcA

4,5 / 1

Residue type % of total

residues

Manp-(1→ 2,0

→3)-Manp-(1→ 60,5

→2,3)-Manp-(1→ 31,7


Partial depolymerization

Analysis

Fractionation

Acidic hydrolysis

Smith degradation

Non specific degradations

Specific degradations

Depolymerization


Smith degradation / Fractionation

NaIO4

NaBH4

H3O+

H3O+

Microalgae cell wall polysaccharide structure

Toyopearl HW-40 Gel 16/100,

NH4OAc 0.1M, 0.4 ml/min

50 mM NaIO4 oxidation, hydrolysis

1,5% Acetic acid, 2h, 100°C


Mild acid hydrolysis / Fractionation

Hydrolysis 0.2 N TFA, 2h, 100°C

1st purification:

Superdex 30

HiLoad 26/600,

Ammonium

carbonate 0.1M,

1.5 ml/min

2nd purification: 3

Superdex 30

HiLoad 26/600,

Ammonium

carbonate 0.1M,

1.5 ml/min



Mild acid hydrolysis Smith degradation

Analysis of the fractions

GC

GC/MS

2D NMR



1H NMR spectra of (A) DP2, (B) DP3 and (C) DP5 fractions recovered after mild acid

hydrolysis and size-exclusion purification of the microalgae cell wall polysaccharide

(D2O, 25°C, 400 MHz)

Partial 1H-13C HMBC spectra of (A) polysaccharide after

Smith Degradation (D2O, 50°C, 500 MHz, with cryoprobe), (B)

trisaccharide and (C) pentasaccharide, (D2O, 25°C, 400 MHz)

NMR Fractions after mild acid hydrolysis


1H spectra

1H-13C HMBC spectra

3

2

2

3

DP2

DP3

33

2

3

DP5

1H NMR spectra of native and SD polysaccharide, D2O, 80°C, 500 MHz

NMR Fractions after Smith degradation


Native polysaccharide

SD Fraction

1H spectra

… → 3)α-Man(1→3)α-Man(1→3)α-Man(1→3)α-Man(1→3)α-Man(1→3)α-Man(1→…

2)

↑

SO3

2)

↑

β-GlcA

Structure hypothesis

Many tools for polysaccharide structure analysis !

Nature of glycosidic residues

GC, NMRNature of glycosidic linkages

GC/MS and NMR

Sequence of

glycosidic residues

2D NMRNature and position of

substituents

GC/MS, EA and NMR

Anomeric configuration

of glycosidic linkages

NMR


Analytical methods and Chromatography · Ionic chromatography DIONEX with Pulsed Amperometry...

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Transcript of Analytical methods and Chromatography · Ionic chromatography DIONEX with Pulsed Amperometry...