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Merlin K. L. Bicking, Ph.D.
ACCTA, Inc.
Richard A. Henry, Ph.D.
Consultant Minnesota Chromatography Forum 2014 Session: 2D-LC and Novel LC Phases
1
Is A Second Retention Mechanisms in LC a
Curse or Cure?
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2
Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN USA 55125 Email: [email protected]
Richard A. Henry, Ph.D. Consultant State College, PA USA 16801 Email: [email protected]
“Analytical Solutions to Analytical Problems”
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Curse: The Effect of Silica Purity on Selectivity
Zorbax XDB C18 Moderate Purity Silica (fully endcapped)
ACE C18 High Purity Silica (fully endcapped)
Zorbax SB C18 Moderate Purity Silica (non-endcapped)
Zorbax ODS Low Purity Silica (fully endcapped)
9,700 pl/m
23,100 pl/m
63,400 pl/m
Decreasing silica purity can change selectivity, but poor chromatography and reproducibility may result from surface interactions.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
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A second retention mechanism from the silica affected the chromatography
Column: 250 x 4.6mm, 5m Mobile phase: 80:20 MeOH/25mM KH2PO4 (pH6.0) Flow: 1.00ml/min Components; 1: Norephedrine, 2: Nortriptyline, 3: Toluene, 4: Imipramine, 5: Amitriptyline
1, 2, 4 and 5 not eluted
Reproduced with kind
permission of Mac-Mod Analytical 3
min 0 0.5 1 1.5 2 2.5 3
mAU
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120
min 0 0.5 1 1.5 2 2.5 3
mAU
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20
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60
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100
120
Cure: Change Some Retention Times, Not All • Decreasing the acid content causes an increase in the retention of the
basic analytes (ionic effects). Since the acetonitrile content has not changed, the hydrophobic retention region does not change.
1 Creatinine 2 Procainamide 3 Protriptyline
Retention Window for Hydrophobic Components
1 2 3
1 2
3 0.10% Sulfuric Acid/ 80% Acetonitrile
0.01% Sulfuric Acid/ 80% Acetonitrile
Column: 4.6 X 50 mm PFPP
4
• When the presence of a second retention mechanism is part of the phase design, maybe we can use both mechanisms to improve the separation
• This is possible only if,
– We understand what the mechanisms are
– We understand how they behave
– We know how to control their behavior
So, is it a curse or cure?
5
Experimental Details • Equipment
– Agilent 1100 with autosampler, column compartment, and diode array
– OpenLab ChemStation software
• Mobile Phase Components – Acetonitrile (HPLC Grade) – Ammonium Formate
• 10, 20, and 30 mM
• Operating Conditions – Flow: 1.75 mL/min – Injection: 2 uL – Temperature: 35 C – Detection: 220 nm
Manufacturer Phase Dimensions Phase
Supelco Ascentis Express C18 4.6X50 mm, 5 um* Hydrophobic
Supelco Ascentis Express F5 4.6X50 mm, 5 um* Pentafluorophenylpropyl
Supelco Ascentis Express OH5 4.6X50 mm, 5 um* OH/Diol
Zirchrom Zirchrom-PBD 4.6X50 mm, 3 um Hydrophobic with adsorbed phosphate
Imtakt Scherzo SM-C18 4.6X50 mm, 3 um Hydrophobic + Cation Exchange + Anion Exchange
* Fused core design. Equivalent to 3 um particles. 6
• Test two mobile phase parameters that may affect retention
– Acetonitrile Content
– Buffer concentration
• Test high and low levels of each parameter
– Acetonitrile: 50% and 90%
– Buffer Concentration: 10 mM and 30 mM
22 Factorial Screening Design
7
• Why choose this design? – Factorials evaluate two
or more variables with a minimum number of experiments
• Why choose these conditions? – The choice is arbitrary
– Ideal for general LC and LC-MS applications
22 Factorial Screening Design
mM
Bu
ffer
% Acetonitrile
30mM
10mM
50% 90%
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Procedure: Measure retention Calculate k Calculate Main Effects
• The Main Effect of a parameter is the difference between the results at high and low settings.
• Larger values mean a larger change in retention over the range studied.
• Small values mean that this variable does not produce a significant change in retention.
Calculating Main Effects
mM
Bu
ffer
% Acetonitrile
30mM
10mM
50% 90%
Ace
ton
itri
le H
igh
Ace
ton
itri
le L
ow
Buffer Low
Buffer High
mM
Bu
ffer
% Acetonitrile
30mM
10mM
50% 90%
High Average – Low Average
Hig
h A
vera
ge –
Lo
w A
vera
ge
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0.407 (k=0.55)
0.452 (k = 0.72)
2.742 (k=9.34)
3.076 (k=10.6)
Example Calculations
mM
Bu
ffer
% Acetonitrile
30mM
10mM
50% 90%
Nicotinic Acid (OH5)
Main Effect for Acetonitrile
Main Effect for Buffer
(9.34+10.6) 2
(0.55+0.72) 2
- = +9.3
(0.55+9.34) 2
(0.72+10.6) 2
- = -0.7
• There is a significant increase in retention with an increase in acetonitrile.
• There is a small decrease in retention with an increase in buffer conc.
10
What Do Main Effects Mean?
Sign of the Main Effect
Negative Near Zero Positive
Acetonitrile Reversed Phase Trend
No effect Normal Phase Trend
Buffer Conc. Ion Exchange Trend
No effect Salting Out Trend
Important Note: the existence of a trend suggests, but does not prove, that the stated mechanism is responsible for the effect.
11
• How do we use this information? – We want to separate the polar compounds from the less polar 1-
ring aromatics. – Change buffer concentration to move the polar compounds, with
little change in retention for the aromatics. – Change the acetonitrile content to move the polar compounds in
the opposite direction from the aromatics.
Significant Main Effects for F5 (PFP) Compound Class Main Effects Trend
Acetonitrile Buffer
BTEX Aromatics* Non-Polar, neutral -2.9 -0.1 RP, No ionic
Benzyl Amine Polar, strong base +3.6 -2.6 NP, Ion Exchange
Phenethyl Amine Polar, strong base, more hydrophobic than Benzyl Amine
+4.2 -3.4 NP, Ion Exchange
BTMA** Polar, cation +6.0 -5.3 NP, Ion Exchange
12 *Benzene, Toluene, Ethyl Benzene, Xylenes **Benzyltrimethylammonium chloride
Improving the Separation on F5 (PFP)
min 0 0.5 1 1.5 2
mAU
-10
-5
0
5
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15
20
25
1
2 3
90%, 30 mM
1 2
3
50%, 10 mM
min 0 0.5 1 1.5 2
mAU
-5
0
5
10
15
1 2
3
50%, 30 mM
min 0 0.5 1 1.5 2
mAU
-20
-10
0
10
20
Main Effects
Acn Buffer
BTEX -2.9 -0.1
Benzyl Amine (1) +3.6 -2.6
Phenethyl Amine (2) +4.2 -3.4
BTMA (3) +6.0 -5.3
13
90%, 10 mM
1
2
3
min 0 1 2 3
mAU
-2
0
2
4
6
8
10
12
14
30mM
10mM
50% 70%
Improving the Separation on PBD
1 2
3
50%, 10 mM
min 0 0.5 1 1.5 2 2.5 3
mAU
-15
-10
-5
0
5
10
15
20
1 2
3
50%, 30 mM
min 0 0.5 1 1.5 2 2.5 3
mAU
-20
-10
0
10
20
30
70%, 10 mM
1 2
3
min 0 0.5 1 1.5 2 2.5 3
mAU
0
5
10
15
20
25
1 2
3
70%, 30 mM
min 0.5 1 1.5 2 2.5 3 3.5
mAU
0
5
10
15
20
25
30
35
Main Effects
Acn Buffer
BTEX -1.6 -0.1
Benzyl Amine (1) +6.4 -8.0
Phenethyl Amine (2) +6.3 -8.8
BTMA (3) -0.1 -1.4
14
30mM
10mM
50% 70%
Probably not a linear
trend.
Improving the Separation on SM-C18 Main Effects
Acn Buffer
BTEX -10.0 -0.2
Creatinine (1) +0.30 0.0
Benzyl Amine (2) +2.1 -0.9
Phenethyl Amine (3) +0.5 -0.5
Nicotinic Acid (4) +2.4 -1.1
Benzene Sulfonate (5) 0.0 -1.1
1
2 3
50%, 30 mM
min 0 0.25 0.5 0.75 1 1.25 1.5 1.75
mAU
-20
0
20
40
60
80
100
4
5
1 2 3
50%, 10 mM
min 0 0.25 0.5 0.75 1 1.25 1.5 1.75
mAU
0
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30
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50
60
70
80
4
5
min 0 0.25 0.5 0.75 1 1.25 1.5 1.75
mAU
0
5
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30
35
40
90%, 10 mM
1
2 3
4
5
1
2 3
90%, 30 mM
min 0 0.25 0.5 0.75 1 1.25 1.5 1.75
mAU
0
10
20
30
40
50 4
5
15
30mM
10mM
50% 90%
Improving the Separation on OH5 Main Effects
Acn Buffer
BTEX 0.0 0.0
Creatinine (1) +2.1 0.0
Benzyl Amine (2) +2.3 -1.1
Phenethyl Amine (3) +2.3 -0.1
Nicotinic Acid (4) +9.3 -0.7
Phenylalanine (5) +9.6 +0.1
1,2,3
50%, 30 mM
4
5
min 0 0.5 1 1.5 2 2.5 3
mAU
0
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40
60
80
100
1,2,3
50%, 10 mM
4
5
min 0 0.5 1 1.5 2 2.5 3
mAU
0
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40
60
80
100
1
2
3
90%, 30 mM
4
5
min 0 0.5 1 1.5 2 2.5 3
mAU
0
5
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15
20
90%, 10 mM
1
2,3
5,4
min 1 2 3 4
mAU
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30
35
16
30mM
10mM
50% 90%
Main Effects Comparisons
17
BTEX Main Effect: ACN
SM-C18 -10.0
F5 -2.9
PBD -1.6
OH5 0.0
Benzyl Amine
Main Effect: ACN
F5 +6.4
PBD +6.4
OH5 +2.3
SM-C18 +2.1
Benzyl Amine
Main Effect: Buffer
PBD -8.0
F5 -2.6
OH5 -1.1
SM-C18 -0.9
• The 22 factorial method can be used to easily identify general retention trends.
• Each of the four columns studied demonstrated a unique combination of multiple retention mechanisms.
• When two mechanisms are present, it is possible to separately move classes of compounds in different directions by adjusting the mobile phase composition. – On a single mode column, this would only be possible by
changing the chemistry of the mobile phase components (e.g., change from acetonitrile to methanol).
– On a multi-mode column, the retention time selectivity can be changed by adjusting the composition of the mobile phase, without changing the chemistry.
Summary
18
Acknowledgements
Columns, reagents, and other resources were kindly provided by:
For more information, contact: Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN 55125 Email: [email protected] Internet: www.accta.com
19
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