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8/4/2019 Structure Analysis of Polysacharides by NMR
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1November 2005
Structure analysis of polysaccharides byNMR
Lennart Kenne
Department of Chemistry, SLU, Swedish University of AgriculturalSciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden
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1. Structural information needed for carbohydrates
2. Information from NMR
3. Structure analysis by NMR
4. Modern NMR methods some applications
NMR Spectroscopy in Glycoscience
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Oligo- and polysaccharides
Structure Components
Linkages
Sequence
Conformation
Properties Interactions with solvent or other
molecules as proteins
Information on structure and properties
HO
O
H
H
HO
H
O
NHHH
OH
HO
O
H
H
OH
H
HH
H3C
O
H
O
H
HO
H
H
OHHOH
OHO
CH3
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Studies of carbohydrates
Isolated material
Carbohydrates on solids
Carbohydrates in their natural environment
When can NMR be used?
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The basic
NMR experiment
1H (100%) and
13C (1.1%)
HO
H
O
H
HO
H
H
OHH
OH
OH
Higher field higher energy more nuclei in thelower state 1 of 100,000
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FIDB0
NMR parameters
Information
Chemical shiftsChemical surrounding
Coupling constantsStereochemistry
IntensitiesNumber of atoms / molar ratio
Relaxation times T1 and T2Dynamic properties
NOE (nuclear Overhauser effect)Interatomic distances and dynamic properties
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OO
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HH H
HO
H
For most
NMR experiments
OO
O
CH2OD
DO
DO
OD
CH3
OD
OD
H
H
H
H
H
H
HHH
DO
H
Sample dissolved in D2O
Deuterated solventgives no signal andlocks the frequencies
Substituted sugars other solvents
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HO
O
H
H
HO
H
O
NHHH
OH
HO
O
H
H
OH
H
HH
H3C
O
H
O
H
HO
H
H
OHHOH
OHO
CH3
StructureComponentsLinkages
SequenceConformation
Component
Which sugar
Anomeric configuration
Absolute configuration
Substituents
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O
OH
CH2OH
HO
HO
OHH
H
H
H
H
O
OH
CH2OH
HO
HO
OHH
H
H
H
H
H-1 H-2 H-3 H-4 H-5
b-Glc 4.64 3.25 3.50 3.42 3.46
a-Glc 5.23 3.54 3.72 3.42 3.84
Difference +0.6 +0.3 +0.2 0 +0.4
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O
OH
CH2OH
HO
HO
OHH
H
H
H
H
O
OH
CH2OHHO
HO
OHH
H
H
H
H
H-1 H-2 H-3 H-4 H-5
b-Glc 4.64 3.25 3.50 3.42 3.46
b-Gal 4.53 3.45 3.59 3.89 3.65
Difference -0.1 +0.2 +0.1 +0.5 +0.2
C b h d t / NMR
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Equatorial proton +0.6 ppm(axial proton)
OO
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HHH
HO
H
d4.5 ppmd5 ppm
Chemical shifts - anomeric proton signals
Proton on a carbon linked to two oxygens
Carbohydrates / NMR
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OO
HO
CH2OH
HO
HO
OH
CH2OH
OH
OH
H
H
H
H
H
H
HHH
HO
H
A B
OH
Chemical shifts
H-1 H-2 H-3 H-4 H-5 H-6a H-6b
A 4.64 3.25 3.50 3.42 3.46 3.72 3.90B 4.89 3.95 3.66 3.60 3.38 3.75 3.91
Diff +0.2 +0.7 +0.2 +0.2 -0.1 0 0
Carbohydrate Research 188 (1989) 169-191
b-D-Glc b-D-Man
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180 degr
7-10 Hz
60 degr
1-4 Hz
O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HH
H
H
H
H
H
H
HO
1.5 Hz
7.5 Hz
Coupling constants - 3JH,H
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OO
O
CH2OH
HO
HO
OH
CH2OH
OH
OH
H
H
H
H
H
H
HH
H
HO
H
A B
Coupling constants - 3JH,H
H1,2 H2,3 H3,4 H4,5 H5,6a H5,6a H6a,b
A 7.5 10 10 10 2 5 12B 1.5 3 10 10 2 5 12
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O
OH
CH2OH
HO
HO
OHH
H
H
H
H
O
OH
CH2OH
HO
HO
OHH
H
H
H
H
C-1 C-2 C-3 C-4 C-5
b-Glc 96.8 75.2 76.8 70.7 76.8
a-Glc 93.0 72.5 73.8 70.7 72.4Difference -3.8 -2.7 -3.0 0 -4.4
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O
OH
CH2OH
HO
HO
OHH
H
H
H
H
H
H
H
H
H
a
b -gauche effect = -4 ppm
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O
OH
CH2OH
HO
HO
OHH
H
H
H
H
O
OH
CH2OHHO
HO
OHH
H
H
H
H
C-1 C-2 C-3 C-4 C-5
b-Glc 96.8 75.2 76.8 70.7 76.8
b-Gal 97.4 73.0 73.8 69.7 75.9Difference +0.6 -2.2 -3.0 -1.0 -0.9
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OO
O
CH2OH
HO
HO
OH
CH3
OCH3
OH
H
H
H
H
H
H
HHH
HO
H
+ 8 ppm
Chemical shifts
Substituted carbon +4-10 ppm
(Depending onstereochemistry around theglycosidic bond)
Anomeric carbon d 100-104 and 96-99 ppm
For mannoses almostno difference
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180 degr
7-10 Hz
60 degr
1-4 Hz
O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HH
H
H
H
H
H
H
HO
1.5 Hz
7.5 Hz
Coupling constants - 3JH,H
For manno-configuration
1-2 Hz
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1JC,H = 170 Hz
Equatorial proton
O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
H H
H
H
HO
Coupling constants - 1JC,H - (Anomeric configuration)
1JC,H = 160 Hz
Axial proton
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HO
O
H
H
HO
H
O
NHHH
OH
HO
O
H
H
OH
H
HH
H3C
O
H
O
H
HO
H
H
OHHOH
OHO
CH3
StructureComponentsLinkages
Sequence
Linkage position
S b tit ti li k
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Substitution - linkages
O
OH
CH2OHHO
O
OHH
H
H
H
HO
H
CH2OH
HO
HO
OHH
H
H
H
O
OH
CH2OHHO
O
OHH
H
H
H
HO
H
HOH3C
H
H
H
HOH
HO
Glycosylation shifts
Dd-values
Substitution of a carbon = a b
+9 +9 -2.5
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HO
O
H
H
HO
H
O
NHHH
OH
HO
O
H
H
OH
H
H
H
H3C
O
H
O
H
HO
H
H
OHHOH
OHO
CH3
StructureComponentsLinkages
Sequence
Sequence of sugar residues
A
B
C
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O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
H
H
H
H
HO
Dipolar interactions - NOE - (Sequence information - interresidue
Connects the residues
NOESY or ROESY
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O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
H
H
H
HO
H
Dipolar interactions - NOE - (anomeric protons -intraresidue)
Through space short distances
NOESY or ROESY
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O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
H
H
H
HO
H
O O
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
H
H
HO
H
Three-bond coupling3JC,H - (Sequence information)
HMBC
Also 4-bond H,H-coupling
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1D-NMR of polysaccharides
Viscous solution = broad signals
Complex spectrum many overlapping signals
Following an enzymatic hydrolysis of three disaccharides1D-NMR
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Fuca12GalbOMe Fuca16GalbOMeFuca13GalbOMe
T = 0
T = 12 H
T = 24 H
a-L-Fucose
b-L-Fucose
Following an enzymatic hydrolysis of three disaccharidesand an a-L-fucosidase
1D NMR
Polysaccharide from an Plesiomonas LPS1D-NMR
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HO
O
H
HHN
H
HO
H
H3C
O
O
H
O
HHO
H
HNH
H
CH3
OH3C
OH
HO
H
H
HH
H
NHO
O
HO
HH
OCH3
Polysaccharide from an PlesiomonasLPS
corePS
1D NMR
T di i l NMR
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Two-dimensional NMR
COSY H-1/H-2 H-2/H-3 H-3/H-4 H-4/H-5 H-5/H-6a,6b
OO
O
CH2OH
HO
HO
OH
CH2OH
OH
OH
H
H
H
H
H
H
HH
H
HO
H
A B
H-1 H-2
T di i l NMR
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Two-dimensional NMR
COSY H-1 H-2
TOCSY H-1 H-2 H-3 H-4 H-5 .....NOESY through space
HMQC C-H
HMBC C-C-H and/or C-X-C-H (2 or 3 bonds)
OO
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HHH
HO
H
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TOCSY
HO
O
H
H
HN
H
H
OH
H3C
OO
H
O
H
HO
H
H
NHH
CH3
O
H3C
OH
HOH
HH
HH
NH
O
O
HO
HH
O
CH3
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HMQC H,C-correlated
HO
O
H
H
HN
H
H
OH
H3C
O
O
H
O
H
HO
H
H
NHH
CH3
O
H3C
OH
HOH
HH
HH
NH
O
O
HO
HH
O
CH3
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NOESY
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HO
O
H
H
HN
H
H
OH
H3C
OO
H
O
H
HO
H
H
NHH
CH3
O
H3C
OH
HOH
HH
HH
NH
O
O
HO
HH
O
CH3
NOESY
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NMR spectroscopy Carbohydrates
Some available methods
1D NMR
2D NMRLC-NMR
HR-MAS
Saturation Transfer Difference NMR Spectroscopy
NMR imagingSolid-state NMR
LC-NMR Problems for
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HPLC 1
NMR
LC NMR Problems forstructural analysis
Solvent LC - NMRDifferent amountsTime for each compound1D 2D experiments
Structureinformation
LC SPE - NMR
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HPLC 1 SPE
SPE
SPE
SPE
SPE
LC SPE NMR
NMR
MS Advantages
Change solvent
Remove water
Several runs accumulate
Handling of compounds
Different scales
Manual or automation
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NMR analysis
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1D and 2D
0.1 - 1.5 mg
Multivariate data analysis structure analysis
Studies of carbohydrates by NMR
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Studies of carbohydrates by NMR
Carbohydrates in their natural environment The role of the hydroxyl groups
O
OH
H
HO
H
H
OHH
O
HO
H
O
H
HO
H
OOH
H
H
HO
Normally 1H NMR inD2O fast exchange
of hydroxyl protons
= not observed
but in 85% H2O /15% aceton-d6 theOH protons can beobserved
Sample preparationRemove ions that can
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Remove ions that canincrease the exchange
Expanded region of the 2D DQF-COSY spectrum (85% H2O/15% (CD3)2CO, -10 C) of maltose,
showing the scalar connectivities between OH and CH protons.
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O
O
O
OH
OH
O
HOOH
OH
O
1'
4
OH
2'H
HH
H
g p
Detection of hydrogen bond interaction
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O
O
OMe
O
O
OH
OH
OH
OH
O
HOHO
OH
Me
OHHO
O
O
OMe
O
HOOH
OH
OHHO
HO
O
OH
OH
O
OH
HO
a-D-Galp
a-D-Glcp
a-D-Glcp
a-D-Galp
a-D-Glcp b-L-Fucp
5"
5"
2'
2'
Dd=- 0.852 ppm3JH,OH = 10.3 Hz
dd / dT = 4.8 ppb/K
Dd=- 1.438 ppm3JH,OH = 3.5 Hz
dd / dT = 5.5 ppb/K
Average values for other hydroxy protonsDd = 0.2 | ppm3JH,OH = 5.5 Hz
dd / dT =10 ppb/K
Hydrogen bondingChemical exchange
Chemical shift of the hydroxy proton of methanol as a function of the mole fraction of
methanol in water (), diethyl ether (), tetrahydrofuran () and dioxane ().
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3.1
3.3
3.5
3.7
3.9
4.1
4.3
4.5
4.7
4.9
5.1
5.3
5.5
0.0 0.2 0.4 0.6 0.8 1.0
Mole Fraction of Methanol
ChemicalShift(ppm)
HO
OH
H
HO
H
H
OHH
O
HO
CH2OH
O
OH
OH
H
H
H
H
H
H
H-O-Me
O(3)H of Me a-D-Galp
O(3)H of Me a-D-Galp
Structure analysis with two-dimensional NMR
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y
COSY H-1 H-2
TOCSY H-1 H-2 H-3 H-4 H-5 .....
NOESY through space
HMQC C-H
HMBC C-C-H and/or C-X-C-H (2 or 3 bonds)
OO
O
CH2OH
HO
HO
OH
CH3
OH
OH
H
H
H
H
H
H
HHH
HO
H
Molecules in dilute solutionsB0
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Results in high-resolution NMR spectra
Molecules in dilute solutionscan tumble and thus averageout several negative effects
as chemical shift anisotropyand dipolar couplings
HO
O
H
H
HO
H
H
OHH
OH
HO
5.5 5.0 4.5
B
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HO
OH
H
HO
H
H
OHH
OH
HO
B0
Too broad to be seen
BDifferent shielding effects
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HO
O
H
H
HO
H
H
OHH
OH
HO
B0
q
gdepending on thedifferent surroundings ofthe molecules
Chemical shift anisotropy
NO TUMBLING causes dipolar couplings and CSA
Very broad lines
Tilting the sample at 54.7 o and spinning at high speedovercome these problems (1-3cos2q)
HR-MAS NMR -High-Resolution Magic-Angle-Spinning NMR
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- Analysis of small molecules or biopolymers that
are mobile in the cells or in a semi-solid systems.
air
Q = 54.7 ("magic angle")
Q
spinning2-15 kHz
sample in D2O( 10-30 ml)
rotor
sealing screw
rotor spacer
rotor capB0 Spinning at magic angle
removes effects of dipolarinteractions and chemicalshift anisotropy
improved linewidths
T2-filter CPMG pulse sequence - (t-180-t)n
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t =387 ms
n=1
n=500
Artefacts generated by the filterT2-filter
( 180 )
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Artefacts generated by the filter(t-180-t)n
multipletsIntensity differences
DQF-COSY
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DQF-COSY
5000 Hz
Ca 1 mg alga
(dry weight)
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Relay-COSY
5000 Hz
Ca 1 mg alga(dry weight)
TOCSY
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TOCSY
5000 Hz
Ca 1 mg alga
(dry weight)
HMQC
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HMQC
14600 Hz
Ca 1 mg alga
(dry weight)
Pichia anomala
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5000 Hz
Studies of the metabolism
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Pichia anomalainhibit the growth of mold in
stored cereals.
- How will oxygen limitation influence the
metabolism?
- Extract or analyse intact cells?
- NMR needs normally a homogenious sample in
solution.
Pichia anomala living cells
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g
Control
O2-limitations
TrehaloseArabitol
GlnGlu
Ethanol
Glycerol
Trehalose
ArabitolGlycerol
Exo- and intra-cellular metabolites compared by GC and HR-MAS NMR
HR MAS a non destructive method?
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HR-MAS - a non-destructive method?
HO
O
HOOH
HO
O
O
HOOH
HO
O
O
HOOH
HO
HO
O
HOO
HO
HO
O
HOOH
HO
O
O
HOOH
HO
O
O
CH2OH
OH
microthecin
Increased amounts ofmicrothecin after 16 hof spinning of alga(>5000 Hz)
Gracilariopsis lemaneiformis
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5-15 kHz, over night
1 mg alga (dry weight)
Characterization of Ligand Binding by SaturationT f Diff NMR S t
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Transfer Difference NMR Spectroscopy
Angew. Chem. Int. Ed. 1999, 38, No. 12 1784-1788
The difference between a saturation transfer spectrum and a normal NMRspectrum provides a new and fast method (saturation transfer difference (STD)NMR spectroscopy) to screen compound libraries for binding activity to proteins.STD NMR spectroscopy of mixtures of potential ligands with as little as 1 nmol of
protein yields 1D and 2D NMR spectra that exclusively show signals frommolecules with binding affinity. In addition, the ligands binding epitope is easilyidentified because ligand residues in direct contact to the protein show muchstronger STD signals.
Moriz Mayer and Bernd Meyer
B
Normal Irradiation
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a-L-fucosidaseHN OH
OH
CH2OHOH
1-Deoxymannojirimycin (DMJ) - inhibitor
Irradiation/saturationB0
Normal spectrum
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a-L-fucosidaseHN OH
OH
H2C
OH
OH
HN OH
OH
H2C
OH
OH
Saturation
1-Deoxymannojirimycin (DMJ)
Difference spectrum
NMR imaging
http://images.google.se/imgres?imgurl=www.physics.brocku.ca/~edik/MRI/pax1018.gif&imgrefurl=http://www.physics.brocku.ca/~edik/MRI/fruits.html&h=384&w=381&sz=106&tbnid=oAxdreAMgOEJ:&tbnh=118&tbnw=118&prev=/images%3Fq%3DNMR%2Bimaging%26start%3D20%26hl%3Dsv%26lr%3D%26ie%3DUTF-8%26sa%3DN8/4/2019 Structure Analysis of Polysacharides by NMR
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http://images.google.se/imgres?imgurl=www.physics.brocku.ca/~edik/MRI/pax1018.gif&imgrefurl=http://www.physics.brocku.ca/~edik/MRI/fruits.html&h=384&w=381&sz=106&tbnid=oAxdreAMgOEJ:&tbnh=118&tbnw=118&prev=/images%3Fq%3DNMR%2Bimaging%26start%3D20%26hl%3Dsv%26lr%3D%26ie%3DUTF-8%26sa%3DNhttp://www.chemistry.mcmaster.ca/facilities/nmr/images/600-1a-512ds.jpg8/4/2019 Structure Analysis of Polysacharides by NMR
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Bruker Daltonics ESI-Ion Trap MS (esquire3000plus and esquire2000),
APEX Fourier Transform Mass Spectrometer (FTMS),BioTOF II Time-of-Flight mass spectrometer.
http://www.chemistry.mcmaster.ca/facilities/nmr/images/700b-512.jpghttp://www.chemistry.mcmaster.ca/facilities/nmr/images/600-1a-512ds.jpg8/4/2019 Structure Analysis of Polysacharides by NMR
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World's Largest, Most PowerfulNMR Spectrometer
8/4/2019 Structure Analysis of Polysacharides by NMR
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The Department of Energy's PacificNorthwest National Laboratory
celebrated the arrival of the world'slargest, highest-performance nuclearmagnetic resonance spectrometerafirst-of-its-kind 900 megahertz (MHz)wide-bore system developed byOxford Instruments and Varian Inc.
A powerful magnet developed for chemical, biological and materials research was lifted by acrane into DOE Office of Science's William R. Wiley Environmental Molecular SciencesLaboratory