Application of Mixed-Mode HPLC Columns
to Samples Containing Ionic and
Hydrophobic Analytes
Jeffrey S. Rohrer, Qun Xu, Jing Chen, Ji Luo, and Lang Li, Dionex Corporation, Sunnyvale, CA, USA
Abstract
Mixed-Mode Columns
While reversed-phase chromatography with a silica-based C18 column
can successfully separate the analytes of interest in many samples, polar
and charged analytes can be challenging to retain and, if retained, can
exhibit non-ideal peak shapes that compromise their quantification and/
or resolution from other analytes. Polar-embedded phases and high-pH
stable phases can partially address these problems. Mixed-mode phases
better address these problems. These phases are constructed to contain
both reversed-phase and ion-exchange functionality. The mixed-mode
columns used in this study were specifically constructed to have a long
alkyl chain to impart reversed-phase retention and either an anionexchange or a cation-exchange group at the terminus of that chain to impart ion-exchange functionality. Careful choice of eluent ionic strength,
pH, and organic solvent content allows the retention and separation of
ionic, polar, and hydrophobic analytes in a single injection. Here, the
authors use a mixed-mode column with cation-exchange and reversedphase properties, and a mixed-mode column with anion-exchange and
reversed-phase properties to solve challenging application problems.
The anion-exchange mixed mode column was used to separate and
quantify the anti-corrosion components of engine coolants, a mixture
of acids and neutral organic compounds, and additives to carbonated
beverages, including sweeteners and preservatives. The cation-exchange
mixed mode column was used to determine melamine in milk-products
as both a primary and confirmatory method. Also, this column was
used to monitor folic acid synthesis. This presentation will discuss the
separation principles of these columns and how the above applications
were developed.
There are several methods for preparing a column with both ionexchange and reversed-phase properties (Figure 1). The Acclaim
Mixed-Mode columns are prepared with the ion-exchange group at
the terminus of the alkyl chain (Figure 1, Type IV). This allows distinct
separation of the ion-exchange and reversed-phase properties and good
control of the ion-exchange capacity. Figures 2 and 3 show diagrams of
the basic construction of the Acclaim Mixed-Mode WAX-1 and WCX-1
stationary phases.
Acclaim Mixed-Mode WAX-1
I. Blend Packaging
Silica Gel
Silica Gel
Silica Gel
II. Mixed Ligand
III. “Embedded”
IV. “Tipped”
RP–BLUE; IEX–RED
24922
Figure 1. Silica-based mixed-mode phases.
R
R R
N
R
N
R
R
N
R
R
N
R
R
N
Equipment
Features
Dionex UltiMate® 3000 Standard or Rapid Separation System with the
appropriate DAD-3000 diode array detector and WPS-3000 autosampler.
Adjustable selectivity
Selectivity complementary to RP columns
Ideal selectivity for separating anionic
molecules
All the equipment was controlled and the resulting data processed using
a Chromeleon® 7.0 Chromatography Data System software.
Multimode separation mechanism:
reversed-phase, anion-exchange,
ion-exclusion, and HILIC
Columns
Dionex Acclaim® Mixed-Mode WAX-1 and Acclaim Mixed-Mode
WCX-1 in various resin particle size and column hardware formats (see
each application).
Silica Gel
Silica:
Particle Size:
Surface Area:
Pore Size:
High-purity, porous, spherical
5 µm
300 m2/g
120 Å
Figure 2. Acclaim Mixed-Mode WAX-1 column.
24923
O–
O
C
O–
O
C
O
O–
C
O
O–
C
O
40
O–
Column:
Acclaim Mixed-Mode WAX-1
(5 µm, 4.6 × 150 mm)
45% 120 mM KH2PO4 (pH 3.0):
55% CH3CN
Temperature: 30 °C
Flow Rate: 1.5 mL/min
Inj. Volume: 5 µL
Detection: UV at 210 nm
Eluent:
1
C
6
Silica:
2
High-purity, porous,
spherical
Particle Size:
5 µm
Surface Area:
300 m2/g
Pore Size:
120 Å
mAU
7
4
3
Peaks:
1. Caffeine
4 mg/L
2. Aspartame 12
3. Sorbate
20
4. Benzoate
20
5. Citrate
120
6. Acesulfame 40
7. Saccharin
12
5
–5
Silica Gel
25008
0
1.25
2.50
3.75 5.00
Minutes
6.25
7.50
24422
Figure 4. Chromatogram of a mixed standard.
Figure 3. Column chemistry of Acclaim Mixed-Mode WCX-1 column.
Column:
Acclaim Mixed-Mode WAX-1
(5 µm, 4.6 × 150 mm)
Eluent:
45% 120 mM KH2PO4 (pH 3.0):
55% CH3CN
Temperature: 30 °C
Flow Rate: 1.5 mL/min
Inj. Volume: 5 µL
Detection:
UV at 210 nm
400
1
Analyte retention times on these mixed-mode phases can be altered by
adjusting three mobile phase properties.
1. Organic solvent content: increasing it will decrease retention of
analytes bound by hydrophobic interactions.
2. pH: for some ionic analytes this can change ionization and
therefore binding to the ion-exchange group (either increase or
decrease depending on the pH and the analyte.)
3. Ionic strength: increasing ionic strength will reduce retention of
analytes bound by ion-exchange.
mAU
5
3
B
Peaks:
4
2
6
7
A
–100
0
1.25
2.50
3.75
5.00
Minutes
6.25
7.50
1.
2.
3.
4.
5.
6.
7.
Caffeine
Aspartame
Sorbate
Benzoate
Citrate
Acesulfame
Saccharin
3.3 mg/L
10
16.7
16.7
100
33.3
10
24427
For this reason, these mixed-mode columns have adjustable selectivity.
Figure 5. Overlay of chromatograms of carbonated beverage diet cola #2, diluted
three-fold (chromatogram A) and the same sample spiked (chromatogram B).
Application 1: Determination of Additives in
Carbonated Beverages
Application 2: Determination of Additives in
Engine Coolants
Carbonated beverages such as colas and other soft drinks contain
preservatives, sweeteners, and other additives (e.g., caffeine). One
beverage can contain ionic, polar, and neutral compounds that must
be quantified but are difficult to determine in a single traditional (C18)
reversed-phase separation. Figure 4 shows an isocratic separation
of seven compounds often found in carbonated beverages using the
Acclaim Mixed-Mode WAX-1 column. These include three low calorie
sweeteners (aspartame, saccharin, acesulfame), three preservatives
(benzoate, citrate, sorbate), and caffeine. Figure 5 shows five of these
seven compounds in a diet cola sample. This method was accurate for a
variety of carbonated beverages.1
Aqueous solutions of ethylene glycol are commonly used as engine coolants. With engine use, the ethylene glycol degrades to glycolic acid which
can corrode the engine. For this reason, corrosion inhibitors are added to
engine coolants. As no single corrosion inhibitor is 100% effective, most
engine coolants contain more than one. These inhibitors generally are
from three compound classes; azoles, inorganic salts, and organic acids,
and therefore represent both organic and anionic compounds. This makes
a mixed-mode WAX column ideal for a simultaneous determination of
glycolate and the inhibitors in an engine coolant. Figure 6 shows glycolate
and four inhibitors separated using a C18 column (chromatogram A), two
polar-embedded columns (B,C), and the Acclaim Mixed-Mode WAX-1.
While the C18 column can separate all five compounds, the retention of
glycolate is poor. The mixed-mode column is better suited for this analysis. With inclusion of benzoate in the mixture, the adjustable selectivity
of this column was investigated by varying the pH, ionic strength, and
organic content of the mobile phase (Figure 7). Figure 8 shows analysis
of six engine coolant samples (a seventh sample, sample #4 is shown in
reference 2 with sample #6) have little or no inhibitors. As these coolants
were not used, no glycolate was detected.
2
Application of Mixed-Mode HPLC Columns to Samples Containing Ionic and Hydrophobic Analytes
Column:
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Sample:
A: Acclaim C18 (4.6 × 150 mm)
B: Acclaim PA (4.6 × 150 mm)
C: Acclaim PA2 (4.6 × 150 mm)
D: Acclaim Mixed-Mode WAX-1
(4.6 × 150 mm)
Columns A-C
A: 0.5% H3PO4 in water
B: Acetonitrile gradient
Column D
A: 100 mM KH2PO4 - H3PO4 buffer
B: Acetonitrile, isocratic (A-42%, B-58%)
Eluents:
120
1
Peaks:
1
E
mAU D
2
5
2
5
Chromatograms:
A:
B:
C:
D:
E:
F:
7
C
5
A
0
4
3
1
4
B
4
3
2
mAU C
3
Acclaim Mixed-Mode WAX-1
(4.6 × 150 mm)
Eluent:
A: 100 mM KH2PO4 –
H3PO4 buffer (pH 3.07)
B: Acetonitrile gradient
Flow Rate: 1.0 mL/min
Temperature: 30 °C
Inj. Volume: 10 µL
Detection:
UV at 220 nm
Sample:
100 µg/L mixture of standards
F
1. Glycolate
2. Tolyl-triazole (TT)
3. Sebacate
4. 2-Mercaptobenzothiazole (MBT)
5. 2-Ethyl hexanoate
2
D
Column:
30 °C
1.0 mL/min
5 µL
UV at 210 nm
Mixture of 5 compounds
2
4
Minutes
6
8
11
Peaks:
5
2,3
2.
3.
4.
5.
7.
4,5
B
1
–140
3
0
1
2
Benzotriazole (BT)
Tolyl-triazole (TT)
Benzoate
Sebacate
2-Ethyl hexanoate
25493
4
2
A
Sample 1 (fourfold dilution)
Sample 2 (fourfold dilution)
Sample 3 (fourfold dilution)
Sample 5 (tenfold dilution)
Sample 6 (tenfold dilution)
Sample 7 (tenfold dilution)
3
Figure 8. Chromatograms of samples 1-3 and 5-7.
5
Minutes
4
5
6
7.5
25482
Figure 6. Chromatograms of the corrosion inhibitors and glycolate using
different Acclaim columns.
Acclaim Mixed-Mode WAX-1(4.6 × 150 mm)
A: 100 mM KH2PO4 – H3PO4 buffer with different
pH values
B: Acetonitrile, isocratic (A-60%, B-40%)
Temperature: 30 °C
Flow Rate: 1.0 mL/min
Inj. Volume: 10 µL
Detection: UV at 214 nm
Acclaim Mixed-Mode WAX-1(4.6 × 150 mm)
A: 100 mM KH2PO4 – H3PO4 buffer with
different pH values
B: Acetonitrile, isocratic (A-60%, B-40%)
Temperature: 30 °C
Flow Rate:
1.0 mL/min
Inj. Volume: 10 µL
Detection:
UV at 214 nm
Column:
Eluent:
A
14
glycolate
TT
benzoate
sebacate
TT
B
glycolate
16
benzoate
10
MBT
sebacate
TT
C
benzoate
sebacate
14
MBT
MBT
2-ethyl hexanoate
12
2-ethyl hexanoate
10
8
6
4
6
2-ethyl hexanoate
12
8
Retention time (min)
Retention time (min)
glycolate
Retention time (min)
16
Column:
Eluent:
10
8
6
4
4
2
2
0
2
2.16
2.87
3.25 3.77
pH value
4.12
25
50
75
Buffer concentration (mM)
100
0
45
50
55
60
65
70
Buffer proportion (%)
25483, 25484, 25485
Figure 7. Effect of phosphate buffer pH (A), phosphate buffer concentration (B), and proportion of phosphate buffer in the mobile phase (C) on analyte retention.
3
Application 3: Determination of Melamine in Milk
Guard Column: Acclaim Mixed-Mode WCX-1
5 µm, 4.3 × 10 mm
Anal. Column: Acclaim Mixed-Mode WCX-1
5 µm, 4.6 × 250 mm
Eluents:
HAc-NH4Ac buffer
450
A
(10 mM, pH 4.3)
CH3CN (8 : 2, v/v)
mAU
400
B
Column Temp.:
Flow Rate:
Inj. Volume:
Detection:
Peaks:
mAU
1
30 °C
1.0 mL/min
20 µL
UV at 240 nm
1. Melamine
0
2
4
6
8
Minutes
10
12
Reduction
HCI
NH2
N
NH2
N
H2N
H2SO4
2,4-Diamino-5-isonitroso
2,4,5-Triamino
-6-hydroxypyrimidine
-6-hydroxypyrimidine
H2SO4
NH2
N
O
O
HN
N-(4-Aminobenzoyl)-L-glutamic acid
N
HO
N
N
H
OH
O
N
N
NH2
Folic Acid
26762
Figure 10. A synthethic process for folic acid.
35
Sample #4
–90
0
2
4
6
8
Minutes
10
A
Temperature:
Flow Rate:
Inj. Volume:
Detection:
Samples:
30 °C
1.0 mL/min
5 µL
UV at 254 nm
Stock solutions diluted in water
THP
DNHP
Folic Acid
mAU
12
25830
Figure 9. Chromatograms of (A) milk powder samples #1– #3, and (B) liquid
milk samples #4 and #5.
a
–5
35
B
DNHP
THP
Folic Acid
mAU
Application 4: Determination of Folic Acid and
Related Compounds
Folic acid is an important B vitamin, especially for the developing fetus,
and therefore is a popular dietary supplement. Figure 10 shows one
synthetic route for folic acid. The precursors are polar and not well retained under standard reversed-phase conditions. As these compounds
can be cationic, they are therefore good candidates for separation using
the Acclaim Mixed-Mode WCX-1 column. Figure 11 shows that the two
precursors are well retained and separated from folic acid on the WCX-1
column. This figure also demonstrates the adjustable selectivity of the
mixed-mode column which allows an unknown peak on the tail of the
folic acid peak to be moved to a location that does not interfere with folic
acid quantification. Addition separations can be found in reference 5.
4
H2N
NH2
N
NH2
N
Sample #5
Sample #1
–100
OH
Column: Acclaim Mixed-Mode WCX-1, 5 µm,
4.6 × 150 mm
Eluents: (A) CH3CN - 20 mM acetate buffer
(mixture of 600 mL of 20 mM HAc and 200 mL
of 20 mM NH4Ac without pH adjustment)
(5:95, v/v)
(B) CH3CN - 30 mM acetate buffer (mixture of
600 mL of 30 mM HAc and 200 mL of 30 mM
NH4Ac without pH adjustment) (5:95, v/v)
1
Sample #3
Sample #2
N
N
H2N
OH
OH
O
In 2008, milk in China was deliberately contaminated with melamine to
increase its apparent protein content. The Chinese government issued
a HPLC method using a C18 column with an ion-pairing reagent to
determine melamine in dried (powdered) milk,3 but the method was
unable to determine melamine in liquid milk due to interferences.
Melamine is a perfect candidate for a mixed-mode separation because
it can be cationic. The adjustable selectivity of the Acclaim MixedMode WCX-1 column allows the analyst to move melamine and other
peaks so that melamine can be quantified without interference. Figure 9
shows the analysis of three powdered milk samples and two liquid milk
samples. Melamine was found in all samples except for powdered milk
sample #1. Melamine quantification in these samples was reproducible
and accurate.4 The mobile phase makes this method compatible with
MS detection.
a
–5
0
2
4
Minutes
6
8
10
26764
Figure 11. Chromatograms of a stock solution mixture diluted with water using
(A) 20 mM NH4Ac-HAc (~pH 4.3) and (B) 30 mM NH4Ac-HAc (~pH 4.0) buffer in
the mobile phase.
Application of Mixed-Mode HPLC Columns to Samples Containing Ionic and Hydrophobic Analytes
Conclusions
References
• Acclaim mixed-mode columns allow the simultaneous analysis of
neutral, polar, and charged analytes.
• Acclaim mixed-mode columns have adjustable selectivity that
allows separations to be easily altered to separate the analyte(s) of
interest from interferences.
1. Dionex Application Note 93, LPN 1886, 5/07.
2. Dionex Application Note 204, LPN 2074-01, 3/10.
3. Dionex Application Note 224, LPN 2184, 3/07.
4. Dionex Application Note 221, LPN 2181, 3/09.
5. Dionex Application Update 171, LPN 2383, 1/10.
Acclaim, Chromeleon, and UltiMate are registered trademarks of Dionex Corporation.
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5
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