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Column Choices: A Look at
Column Choices: A Look at
Column Choices
Mark Powell
Applications Engineer
Columns and Supplies
Technical Support
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April 8, 2015
1
Overview
What to consider when choosing a column?
• Column chemistry
• Silica surface
• Bonded phase
• End-capping
• Particle size options
• Totally porous
• Superficially porous
• Method conditions
• Column lifetime
Silica Column Characteristics
Surface area
• Pore size
• Particle size CH3
Surface chemistry O Si
CH3
• Bonding
CH3
• Silanols O Si CH3
CH3
CH3
O Si
CH3
CH3
O Si CH3
CH3
CH3
O Si
CH3
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3
Pore Size
Small molecules
• 80 – 120 Å
• Maximizes loading and retention
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Pore Size
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Particle Technologies
Year(s) of Particle Size Most Popular Plates / 15cm
Acceptance Nominal Size
1950’s 100µm 200
• dp = 2.7µm
• 2 µm frit to reduce clogging 0.5um
Columns: 4.6 x 50mm, Mobile Phase: 60% ACN:40% Water Flow Rate: 2 mL/min
Efficiency Improvement
with Superficially Porous Particles
van Deemter equation:
• A term – eddy diffusion and
h A B / C flow distribution
B • particle size & packing quality
important
• narrow particle size distribution
• B term – longitudinal diffusion
• Slightly lower due to longitudinal
h
C diffusion reduction
A
A C • C term – mass transfer
• shorter diffusion paths
• better with superficially porous
Separation Speed (v) particles
• more effect on large molecules
Lower h = higher efficiency!
Agilent Confidential
4/8/2015
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Comparison of Particle Size Distributions
Totally Porous and Poroshell 120 Particles
Analyte Mass Transfer Improvements through Lower
Diffusion
Totally Porous
• Totally porous particles
• diffusion throughout particle
• Poroshell 120
• diffusion limited to outer shell
van Deemter equation:
h A B / C
Superficially Porous
• Results:
• Lower C term
• Higher efficiency
• And
• Higher flow rate with
• Minimal impact on efficiency
Pore Size and Efficiency
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Making a Poroshell Particle
Make the solid core Apply the porous shell Apply the bonded
• Smooth surface • Single coacervation step phase
• Tight particle size distribution • High yields
• Tightly packed column bed • Better reproducibility
• Higher efficiency
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Silica Particle Surface
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Typical Stationary Phase Bonding and Endcapping
Reaction
CH 3
CH 3
O Si R
OH O Si R CH 3
CH 3 CH 3 OH
OH OH CH 3 CH 3
+ Cl Si R
OH CH 3
+ Cl Si CH 3 O Si CH 3
OH
CH 3 CH 3 CH 3
OH O Si R CH 3
R = C8, C18, etc . (TMS)
CH 3 O Si R
OH HO OH OH OH
Si Si Si Si
decreasing acidity
OH
+ +
M M Si
Surface Metal Internal Metal
(activated silanol)
(most acidic)
Ion-exchange
SiO Na+ + R3NH+
_ _
SiO N+R3 + Na+
Hydrogen bonding
-SiO . . . H + . . . OOCR
_ _ _
-SiOH + RCOO
Bonded Phase Pore Size (Å) Temp. Limit (°C) pH Range Endcapped Carbon Load (%) Surface Area (m²/g)
EC-C18 120 60 2-8 Double 10 130
EC-C8 120 60 2-8 Double 5 130
SB-C18 120 90 1-8 No 8 130
SB-C8 120 80 1-8 No 5.5 130
HPH-C18 100 60 3 - 11 Double Proprietary 95
HPH-C8 100 60 3 - 11 Double Proprietary 95
Phenyl-Hexyl 120 60 2-8 Double 9 130
SB-Aq 120 80 1-8 No Proprietary 130
Bonus-RP 120 60 2-9 Triple 9.5 130
HILIC 120 60 0-8 No N/A 130
EC-CN 120 60 2-8 Double 3.5 130
PFP 120 60 2-8 Yes 5.1 130
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Why So Many Phase Chemistries?
7.00
6.00 Increasing N
2.00
1.00
0.00
Plates: 5000 10000 15000 20000 25000
Alpha: 1.10 1.35 1.60 1.85 2.1
k 2.0 4.5 7.0 9.5 12.0
Selectivity Impacts Resolution Most
• Change bonded phase Typical Method Development Parameters
• Change mobile phase
• Plates are easiest to increase
Agilent Confidential
4/8/2015
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Method Development Scheme
Other types of interactions with a bonded phase can be exploited (pi -pi
interactions etc.)
When you use Poroshell 120 columns the comparison of bonded phases
can be done quickly!!
Multiple column choices available with different high speed technologies make this easy
Change in Retention with pH for Ionizable
Compounds is Compound-Dependent
More retention for non-charged analytes (i.e. acids at low pH and bases at high pH)
12
0
pH 2.5 pH 6.5 pH 8 pH 11.5
Mobile Phase: 45% MeOH, 55% 20 mM Phosphate Buffer
Change in Retention with pH for Ionizable
Compounds is Key to Method Development
• Non-charged analytes have better retention (i.e. acids at low pH and
bases at high pH)
100
Poroshell 120 EC-C18 2,5 6
50
0
0 2 4 6 8 10 12
3 4 7 8
mAU
1
150
100
Poroshell 120 SB-C18 2,5,6
50
0
0 2 4 6 8 10 12
mAU
3 4 7 8
150
100
Poroshell 120 Phenyl Hexyl
1 2 56
50
0
0 2 4 6 8 10 12
3,2 6,4 7,8
mAU
150
1
100 Poroshell 120 Bonus RP 5
50
0
0 2 4 6 8 10 12
Agilent Confidential
4/8/2015
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Separation of 8 Steroids with Methanol Gradient
30
Beta Blockers with Methanol Gradient
Best Resolution of all analytes with Poroshell Bonus-RP
mAU
300
2
1 3 5 6 7
200
-100
0 2 4 6 8 10 12 min
mAU
2
300
1 6 7
3 5
200
100
4 Poroshell 120 SB-C18
0
-100
0 2 4 6 8 10 12 min
mAU
300
2
5
Poroshell 120 Phenyl Hexyl
200
1 3 4 6
100 7
0
-100
0 2 4 6 8 10 12 min
mAU
2
Poroshell 120 Bonus RP
300
5
200 1 3 4 7 6
100
-100
0 2 4 6 8 10 12 min
Agilent Confidential
4/8/2015
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Beta Blockers with Methanol Gradient
4/8/2015
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ZORBAX SB-Aq Phase
5991-1992EN
Page 33
New Phase on Poroshell 120
Poroshell 120 PFP
• USP L43
• pentafluorophenyl bonded phase
• Excellent choice for polar analytes
• Unique pi-pi interactions
• The ring system electron deficient,
making it a Lewis acid
• Allows for electronic interactions with
electron-donating Lewis bases
• Alternative phase chemistry orthogonal
to C18 chemistries
• Recommended operating range
- pH 2-8
- Maximum temp: 60°C
Agilent Confidential
4/8/2015
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Positional isomers on Poroshell 120 phases
Poroshell 120,
4.6 × 50 mm,
70:30 water:MeOH,
1.5 mL/min, 40 °C,
254 nm
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NSAID Separation Poroshell 120 with a Methanol
Gradient
Best Resolution of all analytes with Poroshell 120 PFP
mAU 1 Poroshell 120 PFP
2
300 5
200
4 9
3 6 7 8
100
0
0 1 2 3 4 5 min
1 5,7 9
mAU
2 4 Poroshell 120 EC-C18
300
200 3
6 8
100
0
0 1 2 3 4 5 min
Time % Organic
1 5 Poroshell 120 Bonus RP
mAU 0 8 2
300 6 100
7 100 4 9
200
8 8 3
100
67 8
2mL/min 254 nm
0
0 1 2 3 4 5 min
1 Poroshell 120 Phenyl Hexyl
mAU 2 5,6
300
4 9
A) 20 mM NH4HCO2, pH 3.0; B) methanol, 40 °C
200 3 7 8
100
0
0 1 2 3 4 5 min
1. APAP, 2. Phenacetin, 3. Piroxicam, 4. Tolmetin, 5. Ketoprofen, 6. Naproxen, 7. Sulindac, 8. Diclofenac, 9. Diflunisal
Agilent Confidential
4/8/2015
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NSAID Separation Poroshell 120 with a Methanol
Gradient
4/8/2015
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Method Development at High pH
From Mid pH
Agilent Confidential
4/8/2015
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Column Lifetime with High pH Methods
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4/8/2015
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Column Lifetime with High pH Methods
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4/8/2015
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Column Lifetime with High pH Methods
Agilent Confidential
4/8/2015
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HILIC
Hydrophilic Interaction Chromatography
• HILIC offers more retention than reversed-phase for very polar bases
Page 43
HILIC – comparison with C18
Page 44
HILIC Separation of Catecholamines Poroshell 120
2.1 x 100, 2.7 micron
DAD1 A, Sig=280,8 Ref=360,100 (CATECHOLAMINE2 \MIX1000025.D)
A: 100 mM NH4HCO2
3.404
mAU
B: Acetonitrile
Poroshell 120 HILIC
175 2.1 x 100, 2.7 micron 168 Bar
3.970
125
5 97
3.635
6.67 97
100
75 1. Dopamine
2. Epinephrine
3. Norepinephrine
50
1.659
0.5 µl injection
2 4 6 8 min
Poroshell 120 Options
Agilent Confidential
4/8/2015
46
Method Development Kits
ZORBAX RRHD Aqueous SB-Aq, Bonus-RP, Eclipse Plus Phenyl-Hexyl 2.1 x 50 mm 5190-6154
Agilent Confidential
4/8/2015
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Poroshell 120 Options
Agilent Confidential
4/8/2015
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Benefits of 4 µm Poroshell 120
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Consistent Selectivity Across Particle Sizes
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Lot Reproducibility
Batch-to-batch reproducibility of Poroshell 120 columns
mAU
2010 100 B10015
50
0
1 2 3 4 min
mAU
100 B10018
50
0
1 2 3 4 min
mAU
150
100
B11041
50
0
1 2 3 4 min
mAU
150
100
B11256
50
0
1 2 3 4 min
mAU
150
100
B12041
2012 50
0
1 2 3 4 min
Beverage Additives
Method Validation Kits
Thank you!
LC-column-support@agilent.com
AdvanceBio RP-mAb
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4/8/2015
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