IMPROVEMENTS IN REVERSED-PHASE HPLC COLUMNS
DESIGNED FOR POLAR COMPOUND RETENTION
Waters
Fang Xia, Paula Hong, Bonnie Alden, Cheryl Boissel, Diana Swanson, Erin Chambers, Diane Diehl,
Pamela Iraneta, Jeffrey Mazzeo, Kevin Wyndham, Nicole Lawrence, Thomas Walter
Waters Corporation, Milford, MA 01757, USA
DEWETTING
Reversed-phase LC columns designed for polar
compound retention often have low ligand densities
and/or novel ligands in order to maintain pore wetting
and enhance retention. Potential weaknesses of these
types of columns include shortened lifetimes at low pH
(e.g., TFA-containing mobile phases), poor peak
shapes above pH 5 and MS bleed. To solve these
issues, we developed an improved C18 column for
polar compound retention (Atlantis® T3) using a
trifunctional ligand and a propriety endcapping
processes. We have seen dramatic improvements in
low pH lifetimes (up to a 5-fold improvement) and high
pH lifetimes (up to 2.5-fold improvement). Minimal
dewetting is observed when the flow is stopped, then
restarted, when using 100% aqueous mobile phases.
MS response of column bleed was also measured and
compared to other RPLC columns. In addition, these
new columns exhibited similar selectivity to Atlantis®
dC18 columns for easy method transfer. Lastly, we
show additional applications that are now possible
with this new column chemistry.
LOW pH STABILITY TEST
% Change Retention Time
Low-pH Stability – Methyl Paraben Retention
-10
-30
-50
-70
-90
0
200
400
600
800
1000
1200
Exposure time (min) in 0.5% aqueous TFA at 60°C
Atlantis® dC18
Atlantis® T3
YMC-Pack™ ODS AQ™
Zorbax Eclipse Plus
SunFire™ C18
SymmetryShield® RP18
YMC-Pack™ Hydrosphere™ C18
Compounds:
1. Thiourea
2. 5-Fluorocytosine
3. Adenine
4. Guanosine-5’-monophosphate
5. Thymine
Isocratic Conditions
Mobile Phase: 10 mM
NH4COOH, pH 3.0
Flow Rate: 1.2 mL/min
Injection Volume: 7 μL
Temperature: Ambient
Detection: UV @ 254 nm
After proper equilibration, the
first set of injections are made,
the flow is stopped for 10-min,
restarted and injections made
again.
0.00
0.00
1
2
APPLICATIONS
NEW Atlantis® T3
4.6 x 100 mm, 3 µm
3
4
Water Soluble Vitamins
5
1
2
3
5
0.50
1.00
1.50
2.00
2.50
3.00
3,4
3.50
4.00
4.50
Minutes
5.00
5.50
Note enhanced
retention
6.00
< 7% Thymine Dewet
6.50
7.00
7.50
8.00
8.50
9.00
Zorbax® Eclipse Plus C18
4.6 x 100 mm, 3.5 µm
1 2
5
0.070
2,3,4
1
1.00
3
7
100% retention loss with
conventional C18
5
0.050
Thymine Dewet: ~100%
0.50
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
0.040
0.030
8
0.010
0.000
0.00
1.00
2.00
3.00
MASS SPECTROMETRY
HPLC Conditions:
Mobile Phase A: 0.1% HCOOH in water
Mobile Phase B: 0.1% HCOOH in acetonitrile
Flow Rate:
1.5 mL/min with 250 μL/min flow split to
mass spectrometer
Gradient:
10 min gradient from 5% to 100% B with
3 min initial hold
Column Temperature: 50°C
Column Dimension:
4.6 × 50 mm
Instrument:
Waters Alliance® 2695
Compound
1.
Norepinephrine
2.
Epinephrine
3.
Dopamine
4.
3,4- Dihydroxyphenylacetic acid
5.
Serotonin (5-HT)
6.
5-Hydroxy-3-indoleacetic acid
7.
4-Hydroxy-3-methoxyphenyl
acetic acid (HVA)
% Initial Ace N 5 sigma
%Initial Acenaphthene k
C18
Abundance
1.40E+09
Symmetry
C18
Conventional
C18
®
Atlantis
dC18dC18
Atlantis
8.00E+08
6.00E+08
®
Atlantis
T3
Atlantis
T3 prototype
No
NoColumn
column
2
4
6
8
10
12
14
16
SELECTIVITY COMPARISON
2
3
13.00
14.00
15.00
UPS Tailing
1.37
1.34
1.29
1.08
1.49
1.18
Sample
Concentration
25 (µg/mL)
25 (µg/mL)
10 (µg/mL)
25 (µg/mL)
30 (µg/mL)
25 (µg/mL)
0.9
25 (µg/mL)
3
2
0.015
7
0.005
0.00
2.00
4.00
6.00
Atlantis T3
60
50
2
3
4
5
Atlantis® dC18
Atlantis® T3
Atlantis® dC18
0.0
1.0
2.0
3.0
10.00
12.00
14.00
16.00
Minutes
18.00
20.00
22.00
24.00
26.00
28.00
30.00
CONCLUSIONS
®
70
1
8.00
Figure 7: Separation of catecholamines and metabolites
Column:
Atlantis® T3 4.6 x 150 mm, 5 μm
Mobile Phase A: 10 mM NH4COOCH3, pH 5.0
Mobile Phase B: Acetonitrile
Flow Rate:
1.0 mL/min
Isocratic:
98/2 (A/B)
Injection Volume: 10.0 μL
Sample:
As indicated in the chromatogram above
Column Temp:
30 °C
Detection:
UV @ 280 nm
Instrument:
Waters Alliance® 2695 with 2996 PDA
5
5
10
15
Hours in 50mM TEA at 50°C
5
4
4
80
Figure 2: Comparison of stability of C18 stationary phases
during exposure to 50 mM TEA pH 10 mobile phases at
50°C. (2a): plate count; (2b): capacity factor
Analyte: acenapthene
Test mobile phase: 65:35 (MeOH:20 mM K2HPO4, pH 7) at
50°C
12.00
0.000
1.00E+09
(2a)
(2b)
11.00
0.010
1.20E+09
1
AU
SymmetryShield
RP18
Embedded Polar
Figure 4: Comparison of column bleed in mass spectrometry.
SunFire™ C18
10.00
0.020
Time (min)
0
9.00
6
0.025
1.80E+09
0
SunFire™ C18
8.00
Figure 6: Separation of water soluble vitamins
Column:
Atlantis® T3 4.6 x 150 mm, 5 μm
Mobile Phase A: 0.1% CF3COOH in water
Mobile Phase B: Acetonitrile
Flow Rate:
1.4 mL/min
Gradient:
Time
%A
%B
0
100
0
4
97
3
6
85
15
15
80
20
24
80
20
Injection Volume: 10.0 μL
Sample:
As indicated in the chromatogram
Detection:
UV @ 260 nm
Low-pH Gradient ES+ TIC (50-1000 m/z)
0.00E+00
90
7.00
Catecholamines and Metabolites
2.00E+08
Atlantis® dC18
6.00
1
4.00E+08
100
5.00
Minutes
MS Conditions:
Ion Source: ES+
Capillary: 3.5 kV
Cone:
33.0 V
Extractor: 3.0 V
RF Lens:
0.3 V
Source Temp:
150 °C
Desolvation Temp: 300 °C
Cone Gas: 60 L/hr
Desolvation Gas: 509 L/hr
Ion Energy: 0.5
Instrument:
Waters Micromass® ZQ™
HIGH pH STABILITY TEST
Atlantis® T3
4.00
Walter, T. H. et al J. Chromatogr. A 1075 (2005) 177-183
Trammell, B. C. et al J. Chromatogr. A, 1060 (2004) 153-163.
110
5
4
0.020
1.60E+09
5
10
15
Hours in 50mM TEA at 50°C
2
1
0.030
0
Sample
Concentrations
25 (µg/mL)
12.5 (µg/mL)
25 (µg/mL)
50 (µg/mL)
50 (µg/mL)
40 (µg/mL)
12.5 (µg/mL)
6 (µg/mL)
USP Tailing
1.05
1.23
1.22
1.19
1.28
1.17
1.11
1.11
0.060
Figure 3: Results of the dewetting study on Atlantis® T3 and a
conventional C18 column.
Figure 1: Comparison of efficiency loss for C18 stationary
phases during exposure to 0.5% TFA mobile phases at 60°C.
110
100
90
80
70
60
50
40
30
Compound
L-ascorbic acid
Nicotinic Acid
Thiamine (vitamin B1)
Pyridoxal
Pyridoxine
Folic Acid (vitamin B11)
Caffeine
Riboflavin (vitamin B2)
6
1.
2.
3.
4.
5.
6.
7.
8.
4
AU
OVERVIEW
4.0
Minutes
5.0
6.0
7.0
8.0
9.0
Figure 5: Comparison of column selectivity.
Column: 4.6 x 150 mm, 5 μm
Flow Rate: 1.0 mL/min
Analytes: (1) thiourea, (2) 5-fluorocytosine, (3)adenine, (4)
guanosine-5’-monophosphate, (5) thymine
Test mobile phase: 10 mM NH4COOH, pH 3 (v/v) at 30°C
Atlantis® T3 columns provide:
• Enhanced retention for polar analytes
• Superior stability under low and high pH conditions
• Minimal dewetting when the flow is stopped, then
restarted, when using 100% aqueous mobile phases
• Low MS bleed
®
• Comparable selectivity to Atlantis dC18 columns
• Good peak shapes
• Available in a variety of dimensions and particle
sizes
Wyndham, K.D. et al Anal. Chem. 2003 (75) 6781-6788.
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