Post on 14-Jul-2018
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
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Homopolysaccharides : one type of monosaccharide
cellulose, amylose, dextran...
OHHO
Heteropolysaccharides : at least two types of different monosaccharides
galactomannan, arabinoxylan, alginate…
OHHO
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Origin Seaweeds
Animals
O
OH
CH2OH
OO
OH
CH2OH
OO
NHAcOH
CH2OH
O
NHAc
NHAc
Chitin
Plants Cellulose
Alginate, fucoidans
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Bacteria Microalgae
Fungi
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Polysaccharide
Applications
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Polysaccharide
Applications
1 - Analysis of native
polysaccharide
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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)
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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 %
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Characterization of polysaccharides
OHHO
Molecular weight
Chemical composition
Structural elucidation
Chromatography
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Chromatography
Columns
Eluent
Pump
Detector
Control and data analysis
Injector
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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
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- 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
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Carbohydrate composition
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
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Analysis
Polysaccharide
Total depolymerization
Derivatization
or not
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- 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
+
Carbohydrate composition
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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
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MeMe
or
Flame Ionization
detector
Gas Chromatography : Principle
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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
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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)
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High Perfomance Anion exchange Chromatography (HPAEC)
CarboPac column Construction
Core
Sulfonated surface
Ion-Exchange Surface
Quaternary ammonium
functionalized latex
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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
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Linkage
ose/ose
Linkages analysis
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
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
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PMAA Partially methylated alditol acetates
Linkage analysis - methylation and GC/MS
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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
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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
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Substituents
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 and position of
substituents
EA, IC, IR and NMR
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Ionic chromatography Dionex with Conductimetric detection
Substituents
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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
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O-H
C-H C=O
C-O
S=O
C-O-S
Laminarin
Sulfated
Laminarin
FT-IR
spectroscopy
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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
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1H, 13C
COSY, TOCSY
HSQC, HMBC
Analysis by Nuclear Magnetic Resonance (NMR)
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Anomeric
Substituted
H6a-6b/C6
HSQC NMR spectrum of microalgae polysaccharide (55°C, 400 MHz)
1H, 13C
COSY, TOCSY
HSQC, HMBC
Analysis by Nuclear Magnetic Resonance (NMR)
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Polysaccharide
Applications
2 - Analysis after
depolymerisation
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1. Native polysaccharide analysis
2. Partial depolymerisation
3. Fractionation
Structure
4. Oligosaccharidic fraction analysis
Partial depolymerization
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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
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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
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Separation and purification of maltodextrins from DP1 to DP10 on a Biogel P2, elution with H2O
Size Exclusion Chromatography
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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
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Oligosaccharide Analysis
Carbohydrate composition (GC)
Linkages analysis (GC/MS)
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Sequence
13
2
1D/2D NMR
Mass spectrometry (ESI-IT, MALDI-TOF)
Polysaccharide
Applications
3 - Example: Structure of a microalgae
Cell wall polysaccharide
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Purification
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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
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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
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Partial depolymerization
Analysis
Fractionation
Acidic hydrolysis
Smith degradation
Non specific degradations
Specific degradations
Depolymerization
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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
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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
Microalgae cell wall polysaccharide structure
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Mild acid hydrolysis Smith degradation
Analysis of the fractions
GC
GC/MS
2D NMR
Microalgae cell wall polysaccharide structure
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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
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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
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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
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