Chromatography of Carbohydrates
 Modes
M d off Ch
Chromatography
t
h
 Affinity (AC)
 Ion Exchange (IX)
• Anion
A
Exchange
E h
o Strong Anion Exchange (SAX)
o Weak Anion Exchange (WAX)
• Cation Exchange
 Hydrophilic Interaction (HI)
 Reverse Phase (RP)
• Reverse Phase – Ion Pairing (RPIP)
 Size Exclusion Chromatography
 Electrophoresis
• Direct Detection
• Indirect Detection
 Modes of Detection
 Absorption or Emission Spectroscopy
• Intrinsic Chromo-/fluoro- phore
• Tagged Chromo-/fluoro- phore
 Pulsed Amperometry
 Mass Spectrometry
1
Modes of Chromatography
Affi i (AC)
Affinity (AC)
Sample
Matrix
Eluent
mixture; varying affinity
= mixture; varying affinity
= ligand‐coated
= aqueous w/ competitor
Si E l i (SEC)
Size Exclusion (SEC)
= mixture; varying sizes
mixture; varying sizes
= porous
= aqueous I E h
Ion Exchange (IXC)
(IXC)
mixture; varying charges
= mixture; varying charges
= anionic or cationic
2
= salt‐containing
Modes of Chromatography
H d hili I
Hydrophilic Interaction (HI)
i
(HI)
Sample
Matrix
Eluent
Polar/hydrophilic
= Polar/hydrophilic
= polar, normal phase‐like
= aq. organic R
Reverse Phase (RP)
Ph
(RP)
= non‐polar/hydrophobic
non polar/hydrophobic
= non‐polar capped
= aq. organic H d h bi I
Hydrophobic Interaction
i
non polar/polar
= non‐polar/polar
= non‐polar capped
3
= salt‐containing
Modes of Detection
 Challenges
Ch ll
 Chromophore?
 Complexity arising from similarity of structures
 Highly polar and water soluble nature
 Detection Approaches
 Absorption or Emission Spectroscopy
• Intrinsic Chromo-/fluoroChromo /fluoro phore
• Tagged Chromo-/fluoro- phore
4
Modes of Detection
 Modes
M d
 Pulsed Amperometry
o pulsed … cycle
o amperometry … application of voltage to induce current
o application of a short voltage oxidizes an analyte, which generates a current
o further oxidation cleans the surface of electrode for further cycling
o duration of less than 1 sec
o underivatized carbohydrates can be analyzed … no need to label
o pH >12 … -OH groups ionized … can be made to interact with hydrophilic
columns or charged columns … resolution
 Mass Spectrometry
o Direct ionization and structure elucidation
o MS/MS pparticularlyy useful
f
5
Affinity Chromatography
+
+
+
carbodiimide
carbodiimide
coupling
reduction
coupling
+
reduction
+
+
Carbohydrate-relevant affinity columns

Concanavalin A (ConA)
member of the lectin family of proteins; binds specifically to sugars containing 
D-mannosyl and -D-glucosyl units; elution with -methyl-mannoside

Many other lectins
(see Freeze, H. Curr. Protoc. Prot. Sci. Ch. 9; May 2001)

H
Heparin
i
member of glycosaminoglycan family of sugars; binds numerous electropositive6
proteins; elution with varying salt concentration
Size Exclusion Chromatography

Usually good for large molecules or complexes; covalent or non-covalent
non covalent

Also referred to as GPC or GFC (gel permeation/filtration chromatography); if sample
permeates through the matrix (GPC, non-aqueous); if sample does not permeate
(filters GFC,
(filters,
GFC aqueous)

SEC separates molecules according to their size; more accurately the hydrodynamic
volume of a molecule. This correlates with the elution volume

May correlate
l with
i h apparent molecular
l l weight
i h (M
( R);
) appropriate
i standards
d d are
necessary

Provides the size distribution for a polydisperse sample; particularly good for
carbohydrate polymers. Forms an essential method of polymer characterization
including number-average (MN) and weight-average molecular weights (MW), and
polydispersity (P)

Principle: A molecule that penetrates pores of the stationary phase takes longer path to
elute in comparison to that molecule which does not penetrate matrix pores, or is
excluded. This results in the separation. Typically small molecules penetrate pores
more than
th large
l
molecules.
l l Thus,
Th larger
l
molecules
l l elute
l t earlier
li than
th smaller
ll
molecules.

Caveats: analytes should not interact with stationary phase; each analyte should be
l d d simultaneously;
loaded
i lt
l column
l
should
h ld be
b uniformly
if
l packed
k d andd nott contain
t i channels
h
l

See Anal. Bioanal. Chem. 2011, 399, 1413-1423.
7
SEC of LMW Heparins
F i i
Fraxiparin
Fragmin
Enoxaparin








Two columns in series: TSK2000W and TSK3000W
Sample: Underivatized LMWHs; Abs@206 nm
Eluent: 250 mM sodium sulfate, pH 4.5
Calibration using partially depolymerized heparins
Blue dextran (Mr = 2,000,000) and NaN3 for V0 and Vt
Slice into 100 – 1000 peaks; tabulate Ai and Ti
Convert all Ti into Mi using calibration equation
MN = Ai×Mi / Ai; MN = Ai×Mi2 / Ai×Mi ; P = MW / MN
Logiparin
gp
Oligo Mix
Taken from Desai et al. Carbohydr. Res. 1994, 255, 193-212.
8
Ion Exchange (IX) Chromatography

Usually good for molecules that are charged (positive or negative) at a defined pH;
practically all molecules have charged groups; technique useful for a large range of
molecules

Separation of ions is based on their nature and number of charges

Ions can be cations or anions; Positively charged matrix will carry negatively charged
ions that can be exchanged for negatively charged analyte = anion exchange
chromatography and vice versa
Anion Exchange Resin
Cation Exchange Resin

IX can be strong or weak depending upon the pKA characteristics of the ionizing
group; strong anion exchange (SAX) implies the use of quaternary amines that are
positively charged independent of the pH (e.g., matrix–NR3+); weak anion exchange
utilizes matrices with ggroups
p such as –NH3+, –NH2R+, –NHR2+, etc;; strongg cation
exchange (SCX) uses groups such as –SO3-, while weak cation exchange (WCX) uses
–COO- groups

Strongg and weak terms do not refer to the strength
g of matrix – analyte
y interaction!
9
Ion Exchange (IX) Chromatography

Principle: A molecule with a charge opposite to that present on the matrix will be more
retained in comparison to the molecule with fewer charges of the same type or the
molecule with charges of the opposite type. This results in the separation.

Elution: Typically performed using a linear salt or pH gradient.
gradient

Caveats: analytes should primarily utilize ionic interactions; should be stable under the
pH conditions, if drastic; and should be easy to separate from salt

See J. Chromatogr.
h
A 2006, 1118, 168-179.
Less sulfated species
Highly sulfated species
10
Ion Exchange (IX) Chromatography

WAX example … separation of 13 neutral oligos … reported for the first time in 1988
by Hardy and Townsend (Nature vol. 335, pg. 279-380)

Another example … separation of underivatized sialylated N-glycans from 1-acid
glycoprotein …. note the importance of number of neuraminic acid residues and local
structure! … Taken from Methods Mol. Biol. 2008, 446, 239-254
11
Hydrophilic Interaction Liquid Chromatography (HILIC)

Usually good for polar molecules; practically all carbohydrates are polar; even the
neutral N- or O-glycans are polar; technique useful for a large range of molecules

Principle: Polar molecules will interact with a polar stationary phase through
hydrogen bonding … greater the strength of this interaction
interaction, greater will be the
retention … resolution is highly structure dependent …

Is a form of liquid-liquid partition chromatography … is not adsorption based because
water is one of the eluents and molecules interact with polar groups of the matrix that
are present in the water layer surrounding the matrix

Is exactly opposite reversed-phase chromatography … hence elution gradient is
opposite too

Elution: polar solvent will disrupt this interaction … proportion of water increases

Organic solvents typically useful in this include acetonitrile, dioxane, tetrahydrofuran,
butanol, …. all water miscible

Matrix: amine-bonded; silanol groups containing; carboxylic acid terminated; …
12
Hydrophilic Interaction Liquid Chromatography (HILIC)

Example … separation of fetuin 2-AB
2 AB derivatized NN and O-glycans
O glycans using amine
aminebonded column (2-AB = 2-amino benzamide)
13
Hydrophilic Interaction Liquid Chromatography (HILIC)

HPLC v/s UPLC of 22-AB
AB labeled human IgG N-glycans
N glycans
BEH glycan column
amide column

UPLC of 2AB-labeled dextran ladder
14
Reversed-Phase HPLC

Usually good for non
non-polar
polar molecules; technique not very useful for carbohydrates

Principle: Non-polar molecules will interact with a non-polar stationary phase through
van der Waals interactions … greater the strength of this interaction, greater will be the
retention … resolution is dependent on the presence of number and type of
hydrophobic groups …

Is exactly opposite normal-phase chromatography … hence elution gradient is
opposite too

Elution: non-polar solvent will disrupt interaction … proportion of non-polar solvent
increases

Organic solvents typically useful in this include acetonitrile, dioxane, tetrahydrofuran,
ethanol, ….

Matrix: alkyl chain modified silanol groups; C18, C12, C6-alkyl chains …

Particularly interesting exploitation of this technique for carbohydrates is reversedphase ion-pairing LC (RPIP-LC) … especially for glycosaminoglycans

+
+
Hep-OSO
p
p
y ggroup,
p,
3 Na is converted in situ into Hep-OSO
3 NH3R , where R is an alkyl
e.g., butyl, pentyl, hexyl, … enables interaction with C18 columns … more the sulfate
groups, greater the interaction, better the resolution … elution performed with aqueous
– organic co-solvent
15
Reversed-Phase Ion-Pairing HPLC
Taken from Jones et al. Anal. Chem. 2011, 83, 6762-6769.
16
Reversed-Phase Ion-Pairing HPLC
Taken from Xue et al. J. Mass Spectrom. 2011, 46, 689-695.
17
Reversed-Phase Ion-Pairing HPLC
Taken from Xue et al. J. Mass Spectrom. 2011, 46, 689-695.
18
Capillary Electrophoresis

Useful for charged molecules; especially useful for acidic carbohydrates,
carbohydrates e.g.,
eg
glycosaminoglycans and sialylated glycans

Principle: Charged molecules are attracted to opposite electrodes when placed in an
electric field … attraction is a function of the charge density on the molecule

A small diameter capillary enhances the resolution dramatically because heat
dissipation is very high … very sharp peaks are observed

Severall modes
d off capillary
ill
electrophoresis
l
h
i have
h
been
b
developed
d l d including
i l di zone
electrophoresis, micellar electrokinetic electrophoresis, affinity electrophoresis, gel
electrophoresis ….

Useful for separation, kinetics, structure identification, affinity measurements …

See major reviews including: Biomed. Chromatogr. 2011, 55, 775-801; Electrophoresis
2011, 32, 3467-3481; Anal. Bioanal. Chem. 2011, 399, 541-547; Electrophoresis 2008,
29, 2508-2515; Electrophoresis 2008, 29, 3095-3106; and many others
19
Capillary Electrophoresis
 Principle
Pi i l
 Application of a high voltage across electrodes dipped in electrolysis buffer results
in a unidirectional flow of current in a microcapillary. An electroosmotic flow
( O ) is also
(EOF)
l generatedd under
d certain conditions
d
 Charged and neutral molecules are resolved according to their electric mobilities,
which corresponds to their charge to mass ratios
- - - - - - - - - - - - - - - - - - - Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+
1Na+
1-
0
Na+
Na+
1PO4-
0
Na+
Na+
1Na+
1PO4
1-
PO4-
0
Na+
PO4-
PO4-
1+
1+
0
0
Na+
PO4-
Na+
0
Na+
Na+
Na+
PO4-
1+
Na+
0
11-
PO4-
1+
Na+
1-
Na+
0
Na+
0
PO4-
1+ 1+
Na+ PO4
1Na+
Na+
1-
0
PO4-
Na+
1+
1+
1+
Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+
- - - - - - - - - - - - - - - - - - - 20
Capillary Electrophoresis
+
-
EOF migration
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O-
Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+
Na+ Na+
1-
Na+
Na+
1-
Na+
Na+
Na+
0
Na+
1-
Na+
11
Na+
Na+
1+
Na+
Na+
1-
Na+
Na+
Pi--
Pi-Na+
Na+
Na+
Pi--
1+
Na+
Pi--
Pi--
Na+
3
PO43-
Na+
0
Na+
Pi--
Na+
Pi--
Pi--
1+
Na+
Na+
0
Na+
Pi--
Na+
Pi-Na+
Pi--
Stern layer
Compact
layer
Pi-Na+
Na+
0
Na+
Pi--
Na+
Pi--
Na+
Pi--
Na+
Pi--
Na+
Na+
PO43-
Na+
1+
Pi-Na+
Pi--
Pi--
Pi--
Na+
0
Na+
Pi-Na+
Na+
Na+
1+
D
I
F
F
U
S
E
L
A
Y
E
R
Na+
Pi--
Pi--
Na+
Na+
Na+
Na+
Na+
Na+
Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+
O- O- O- O- O- O- O- O- O- OSi Si Si Si Si Si Si Si Si Si
-
O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- OSi Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
+
Electrostatic Migration
Si Si Si Si Si Si Si Si Si Si
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
O
H
O
H
O O O
H H H
Na+
Na+
O
H
O
H
Na+
O
H
O
H
Na+
Na+
1
1-
Pi--
Na+
Pi--
Na+
1-
2-
Pi-Na+
Pi--
1-
Na+
Na+
O O
H H
Na+
Na+
H H H H H H H H H H
O O O O O O O O O O
Si Si Si Si Si Si Si Si Si Si
O O O
H H H
Na+
Na+
Pi--
Na+
Pi--
2Na+
2-
Na+
O O O O O
H H H H H
Na+
3-
Na+
3-
Na+
Pi--
Pi--
3-
Na+
Na+
Na+
Na+
Na+
Pi--
Na+
Na+
Pi-Na+
Pi--
Na+
3-
Na+
Pi-Pi--
O O
H H
Na+
2-
Na+
Na+
O O
H H
Na+
Pi--
Pi--
O
H
2-
Na+
Na+
Pi--
O
H
Na+
1-
1-
Na+
O
H
Pi--
Na+
3-
Pi-Na+
H H H H H H H H H H H H H H H H
O O O O O O O O O O O O O O O O
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
21
MECC of Heparin Oligosaccharides
Taken from Desai et al. Anal. Biochem. 1993, 213, 120-127.
22