September 2014
Benefits of Hydrogen in Liquid Chromatography.
Introducing SHARC1 HPLC Columns.
SIELC Technologies, Inc., Wheeling, IL 60090 USA
The use of hydrogen bonding interactions as a primary retention mechanism in
chromatography is usually associated with gas chromatography. However, we have recently
developed stationary phases which benefit from the presence of hydrogen in the molecule and
offer the possibility for hydrogen-bonding chromatography in the liquid phase.
Hydrogen-bonding chromatography has been neglected in favor of reversed-phase, HILIC, ionexchange, normal phase and other chromatographic modes. Stationary phases rarely achieve
retention and separation of analytes based purely on a single mechanism and SIELC
Technologies have developed many commercial stationary phases benefiting from the ability
to exploit multiple interactions in a controlled way. Hydrogen bonding, is omnipresent in each
of the techniques listed above, especially in normal phase chromatography.
The contribution of hydrogen bonding can be significant. SHARC-1 (Specific Hydrogen-bond
Adsorption Resolution Chromatography) is the first column intentionally designed to achieve
separation based entirely on the analytes ability to act as a hydrogen atom donor or acceptor.
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Unique Mobile Phase, but Common Solvents.
SHARC1 column operating conditions are unique. Solvents used for SHARC separations are acetonitrile (MeCN) as the weak
solvent and methanol (MeOH) as the strong solvent. Pure MeCN has a very insignificant degree of hydrogen bonding with the
SHARC stationary phase while MeOH interacts strongly, reducing retention of analytes capable of hydrogen interactions. By
altering the ratio of MeCN/MeOH, the optimum retention profile can be obtained for many types of molecules with high
selectivity, good peak shape, efficiency, and speed.
SHARC1 Methods and Applications
Hydrogen-bonding interaction offers unique selectivity based
on number of “interaction points” available for hydrogen
bonding. Not every hydrogen bond participates in
intermolecular hydrogen interactions with the stationary
phase. Therefore the proximity of “interaction points” to each
other within a molecule also needs to be considered since
molecules can form intramolecular hydrogen-bonds, which
compete with intermolecular interactions between analyte and
stationary phase. Intramolecular interactions reduce retention
time in hydrogen-bonding mode and such structural factors
provide unique selectivity among structural isomers,
homologs, degradation products and precursors.
N
N
O
O
O
O
N
N
N
H
N
O
O
N
N
H
Caffeine 1-Methylxanthine
1
N
N
H
O
3-Methylxanthine
0
1
2
3
4
5
min
Mobile phase: 70%
MeCN, 30% MeOH
with 0.25% Formic
acid and 0.025%
AmFm
NH2
Norephedrine
OH
H
N
NH 2
OH
OH
Phenylephrine
Norephenylephrine
3 4
1
2
Mobile phase: 95% MeCN, 5%
MeOH with 0.5% Formic acid
and 0.05% AmFm
1
4
N
H
2
N
Xanthine
3
4
Pseudoephedrine
Norephedrine
Phenylephrine
Norphenylephrine
3
Column:
SHARC 1
Size:
3.2 x 100 mm
Mobile phase: MeCN/MeOH 95/5 with
0.1% Formic acid and 0.01% AmFm
Flow:
1.0 mL/min
Detection:
UV 270 nm
2
1.
2.
3.
4.
OH
H
N
Pseudoephedrine
OH
H
N
HN
OH
SHARC 1
3.2 x 100 mm
1.0 mL/min
UV 270 nm
O
N
HN
Column:
Size:
Flow:
Detection:
1. Caffeine
2. 3- Methylxanthine
3. 1- Methylxanthine
4. Xanthine
Fig. 2. The separation of caffeine, 3-methylxanthine, xanthine, and
1-methylxanthine.
0
5
10
15
20
25 min
Fig. 1. The effect of the mobile phase composition on the separation
of pseudoephedrine, norephedrine, phenylephrine, and
norphenylephrine.
Xanthines are good candidates for separation by hydrogen
bonding chromatography with SHARC1 (fig. 2). In caffeine,
three of the nitrogens have substituted methyls, which
makes these groups unavailable for hydrogen bonding
interaction with the stationary phase , whereas 3-methyl-,
and 1-methylxanthine only have one substituent and thus
are retained longerand Xanthine, which has no substitution
on the nitrogen, is retained the longest.
Copyright © 2003-2014 SIELC Technologies, Inc. All rights reserved.
In another example, a mixture of 4 neurotransmitters (fig. 1) were separated based on their ability to form hydrogen bonds with
the SHARC 1 stationary phase. Pseudoephedrine and norephedrine have 2 interaction points, phenylephrine and
norphenylephrine have three. The presence of N-methyl substitution reduces hydrogen-bonding interaction of nitrogen functional
groups and reduces retention. This approach can be used for the separation of unsubstituted and N-substituted amines.
Accessibility of hydrogen-donors or hydrogen-acceptors play a critical role in how compounds are retained and eluted from
SHARC column. Longer retention is achieved with more accessible interactions and offers a wider range of mobile phase
conditions to optimize a separation.
Isomers of aminopyridine were separated by hydrogenbonding
chromatography (fig. 3). The difference in retention
NH2
Column:
SHARC 1
time was attributed to accessibility of hydrogen-bonding sites.
Mobile Phase: Acetonitrile
N
NH2
The separation of phthalic acid isomers (fig. 4) is an example
N
and MeOH (95/5) with 0.5%
which
demonstrates the effect of intramolecular bonding
3-Aminopyridine 2-Aminopyridine
FA and 0.05% AmFm
Detection:
UV 270nm
(phthalic
acid). It is less retentive compared to the other
NH2
isomers (terephthalic and isophthalic acids), which do not form
internal hydrogen bonds.
N
N
Column: 3.2 x 100 mm,
Hydrogen-bonding proved to be a good approach to retain
Pyridine 4-Aminopyridine
4 Particles: 3 m,
and separate simple organic acids (fig. 5). The mobile phase
Flow:
2.0 mL/min
for
the
separation
of
acids
typically
contains
1. Pyridine
Pressure: 155 bar
acetonitrile/methanol
and
ammonia
or
triethylamine
as an
2. 3-Aminopyridine
4
3.
4-Aminopyridine
additive.
The
mobile
phase
is
fully
MS-compatible
and
the
Column: 3.2 x 100 mm,
4. 2-Aminopyridine
Particles: 3 m,
presence of ammonia allows high sensitivity in LC/MS.
Flow:
Pressure:
12 3
1.0 mL/min
77 bar
4
Column:
Size:
Mobile phase:
H
N
1
Column: 4.6 x 150 mm,
Particles: 5 m,
Flow:
1.0 mL/min
Pressure: 37 bar
2 3
1
2
0
O
HO
Flow:
Detection:
3
5
1.
2.
3.
10
SHARC 1
3.2 x 100 mm, 3 um
MeCN/MeOH 95/5 with
10 mM Ammonia
0.5 mL/min
UV 250 nm
15
Acetaminophen
Benzoic acid
4-Acetylbenzoic acid
min
O
O
Fig. 3. The separation of aminopyridines with different column
geometry
O
1.
2.
3.
Phthalic acid
Terephthalic acid
Isophthalic acid
Column:
Size:
Flow:
Detection:
O
OH
OH
O
O
Phthalic Acid
1
Terephthalic Acid
1. Compound
XN136
(proprietary)
Column:
Size:
Flow:
Detection:
Mobile phase:
gradient
Isophthalic Acid
Mobile phase: MeCN
100% with 0.5% Formic
acid with 0.05% AmFm
1
3
8
12
Column:
Size:
Flow:
Detection:
Mobile phase:
gradient
Mobile phase:
MeCN/MeOH 98/2 with
0.2% Formic acid with
0.02% AmFm
2
4
0
2
4
6
8
min
Fig. 5 Separation of organic acids in hydrogen-bonding mode with
LC/MS conditions
3
2
CH 3
OH
HO
O
0
O
O
OH
OH
SHARC 1
3.2 x 100 mm
1.0 mL/min
UV 270 nm
OH
OH
min
Fig. 4. The effect of mobile phase composition on the separation of
phthalic, terephthalic, and isophthalic acid.
0
2
4
6
8
SHARC 1
3.2 x 100 mm
1.0 mL/min
UV 270 nm
ACN/MeOH
Generic C18
3.2 x 100 mm
0.5 mL/min
UV 270 nm
ACN/H2O
min
Fig. 6. The effect of the column and mobile phase composition on
analysis of water-sensitive compound
Copyright © 2003-2014 SIELC Technologies, Inc. All rights reserved.
The separation of phthalic acid isomers (fig. 5) is another example of the separation of compounds with identical numbers
of interaction sites. This example also demonstrates that when intramolecular bonding exists (phthalic acid) compounds are
less retentive compared to the other isomers (terephthalic and isophthalic acids).
Hydrogen bonding separation can be demonstrated with Triton X100, where degree of retention is correlated to the amount
of oxygen in the molecule. This approach is suitable for other surfactants with different alkyl chain length substituents. The
Hydrogen-bonding approach can also be successfully used when analytes are water sensitive. Employing non-aqueous
mobile phases keep water-sensitive compounds intact (fig. 6). When developing a method using SHARC conditions, reactivity
of analytes with alcohols must be considered.
Additional benefits
Fast Analysis due to Low Back Pressure
MeCN/MeOH mixtures have 2-3 times lower
viscosity than water/MeOH or water/MeCN
mixtures (fig. 7). As a result, smaller particles for
column packing can be used without an increase
of working pressure compared to conventional
HPLC. Separation UPLC-like conditions can be
easily obtained on standard HPLC instruments
with a 2-3 times higher eluent linear velocity using
columns with smaller particle size. Increases in
analysis speed of up to 5 times are routinely
achieved using this combination (fig. 3). Use of
UPLC (RRLC, UHPLC) equipment allows a
further increase in analysis speed of 5-6 times.
2
2
1.8
1.6
1.8
MeOH/water
1.4
1.2
1.4
MeCN/water
1
1.2
1
0.8
0.6
1.6
0.8
MeCN/MeOH
0.6
0.4
0.4
0.2
0.2
0
0
0
10
20
30
40
50
60
70
80
90 % 100
Solubility of Analytes
MeOH is one of the most universal solvents for
organic compounds. Combinations of MeOH with
Fig. 7 Viscosity profile of mixtures MeOH/water, MeCN/water, and MeCN/MeOH
MeCN will dissolve almost any molecules with
very high or very low polarity. Highly hydrophobic
molecules such as surfactants, lipids and oil soluble vitamins are easily soluble in this solvent combination. Very polar
molecules such as sugars, diols, salts of amino compounds and carboxylic acids also can also be efficiently dissolved in
these solvent systems. Other organic solvents can be used as diluents without affecting the separation.
LC/MS and Preparative Chromatography Compatibility
MeCN/MeOH mixtures have a low boiling point and are much easier to evaporate than water. As result, this solvent system
is much more convenient for preparative chromatography. Additionally, the benefit of the low viscosity eluent systems allows
preparative separations with higher throughput/flow rate. These eluents are MS friendly, which enables mass directed
preparative strategies. In most cases isocratic methods can be used due to the high selectivity of the column which permits
eluent recycling - minimizing solvent consumption. High organic content of the mobile phase guarantees high sensitivity in
MS applications.
Alternative Selectivity
Since hydrogen bond formation is very specific in terms of interaction energy and strongly depends on molecule geometry as
well as the number and position of functional groups, the separation of similar molecules such as isomers or oxidation or
reduction products can be achieved in SHARC with high selectivity. A wide range of molecules can be analyzed with the
SHARC technique. Practically any molecule with functional groups containing oxygen and nitrogen can be retained and
separated from similar compounds using this technology.
Contact SIELC for additional information on column performance, method development,
ordering, and availability. Phone 847-229-2629; Fax 847-655-6079; Email: [email protected]
Copyright © 2003-2014 SIELC Technologies, Inc. All rights reserved.