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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Pore Size

Small molecules
• 80 – 120 Å
• Maximizes loading and retention

Peptides, proteins, other large biomolecules

• 120 Å (Peptides)
• 300 Å to 450 Å (or higher)
• Maintain high efficiency

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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

1967 57µm (pellicular) 1,000

1972 10µm 6,000

1985 5µm 12,000

1992 3.5µm 22,000

2003 ☻ <2µm >30,000

2007/2008 2.7 µm 32,000

Particle Technologies

2.7 and 4 µm superficially porous

Superficially Porous Column Technologies
Poroshell 120 columns:
0.5um
• Efficiency ≈ 90% of sub-2 µm
• Pressure ≈ 40-50% of sub-2 µm
1.7um
• N ≈ 2X 3.5 µm (totally porous)

• dp = 2.7µm
• 2 µm frit to reduce clogging 0.5um

• Plimit = 600 bar for HPLC or UHPLC

• Particles
• 1.7 μm solid core
• 0.5 μm diffusion path
• 2.7 μm total diameter
Comparing Efficiency and Pressure with Different
Types of Columns
Particle Size/Type Pressure Efficiency LC Compatibility

5 µm All 400 bar

80 bar 5,000
Totally Porous instruments
3.5 µm All 400 bar
123 bar 7,800
Totally Porous instruments
2.7 µm All LCs/UHPLCs
180 bar 12,000
Poroshell 120 (up to 600 bar)
1.8 µm All LCs/UHPLCs
285 bar 12,500
Totally Porous (up to 1200 bar)

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!

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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

Month ##, 200X

Group/Presentation Title
Agilent Restricted
The Surface of Silica Supports

OH HO OH OH OH

Si Si Si Si

Free Geminol Associated

Silanols Silanols Silanols

decreasing acidity

OH
+ +
M M Si
Surface Metal Internal Metal
(activated silanol)
(most acidic)

Month ##, 200X

Group/Presentation Title
Agilent Restricted
Potential Secondary Interactions

Ion-exchange
SiO Na+ + R3NH+
_ _
SiO N+R3 + Na+

1. Ionized silanols (SiO-) will ion-exchange with protonated bases (R3NH+)

which can cause tailing and method variability. This occurs most often at
mid pH where silanols are ionized.

Hydrogen bonding
-SiO . . . H + . . . OOCR
_ _ _
-SiOH + RCOO

2. Unprotonated acids can compete for H+ with protonated silanols.

This can occur at low pH.

Month ##, 200X

Group/Presentation Title
Agilent Restricted
Highly Purified Zorbax Rx-Sil

Original ZORBAX, 1973 and other type A silicas

• basic compounds can tail

Conditions: Flow Rate: 2.0 mL / min.

Mobile Phase: 5% 2-Propanol in Heptane

ZORBAX Rx-Sil, 1987 and other Type B

silicas
• basic compounds have less tailing
• lower effective silanol pKa

Month ##, 200X

Group/Presentation Title
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Agilent Restricted
Poroshell 120 Column Chemistries
Multiple bonded phases for flexibility in method development
Poroshell 120 EC-C18 and C8 Poroshell 120 SB-Aq
• Robust end-capped C18/C8 for • Proprietary bonded phase is an
best peak shape at pH 2-9 excellent choice for polar analytes
Poroshell 120 Stablebond C18 Poroshell 120 Bonus-RP
and C8 • Embedded polar group provides unique
• Robust chemistries for pH<2 selectivity for polar compounds
• Non-endcapped for alternate Poroshell 120 EC-CN
selectivity • Flexible end-capped CN chemistry for
Poroshell HPH-C18 and HPH-C8 normal and reversed-phase separations
• Long lifetime at high pH Poroshell 120 HILIC
Poroshell 120 Phenyl-Hexyl • Bare silica HILIC for use in hydrophilic
• Excellent choice for pi-pi interactions interaction chromatography of polar
• Selectivity similar to phenyl, diphenyl, or
molecules
other phenyl-hexyl columns Poroshell 120 PFP
• Pentafluorophenyl chemistry for
orthogonal selectivity relative to C18
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Porshell 120 Column Chemistries

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

5.00 Increasing Alpha

Increasing k'
Resolution

4.00 Rs = N½/4  (-1)/  k’/(k’+1)

3.00

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
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Method Development Scheme

Changing selectivity to improve resolution:

Mobile phase (1st choice to change because it’s easy)

• Mobile phase – organic modifier (ACN, MeOH etc.)
• Mobile phase – pH – over a wide pH range – pH 1-12 if needed

Bonded phase (optimization for robust methods)

• Phases other than C18/C8
• Phenyl-Hexyl, Polar-embedded, CN, PFP
Why Is Changing the Bonded Phase Effective?

Differences in interactions between polar and non-polar compounds.

Other types of interactions with a bonded phase can be exploited (pi -pi
interactions etc.)

These all change with bonded phase!

Changing the bonded phase can improve selectivity/resolution, reduce

analysis time

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

Acetylsalicylic acid (pka 3.5)

10 Pyridine (pKa 5.2)
Codeine (pKa 8)
8
Retention Time

Procainamide (pKa 9.2)

Amphetamine (pKa 9.9)
6 Caffeine (pKa 14)

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)

• Silanols on silica ionize at mid-pH, increasing retention of basic

analytes (i.e possible ion-exchange interactions)

• Choose mobile phase pH to optimize retention and selectivity during

method development

• Poroshell 120 EC-C18 and C8 can be used over a wide pH range

• Other choices exist for high pH

First Steps in a Method Development Scheme
 Select a high quality (Poroshell 120 EC
• Poroshell 120 EC-C18 or or Eclipse Plus) C18 bonded phase first
Eclipse Plus C18 for good retention and resolution with
typical acidic, basic and neutral samples.
• Low pH
• Adjust %ACN/MeOH for 0.5 <k < 20
 Optimize the organic component of the
More resolution needed mobile phase to change selectivity

Change % organic  Choose alternate bonded phases to

completely optimize method if needed
More resolution needed

• Change organic modifier  Evaluate other bonded phases and

• Adjust % organic for 0.5 < k <20 conditions for most robust method

More resolution needed  With superficially porous or sub-

2um column choices, steps can be
• Change bonded phase done quickly
•Phenyl-Hexyl, Bonus-RP, CN, SB-C18,
SB-C8, PFP, HILIC • Makes it possible to find a robust
..method
Method Development Scheme – Evaluating Mid pH
From low pH

Mid pH can provide better

• Poroshell 120 EC-C18 or Eclipse selectivity
Plus C18
• pH 7 (6-9) acetate or other buffer, It may be more compatible with
•Adjust % ACN for 0.5 <k < 20
your sample
More resolution needed
The process for investigating mid
Change % organic pH is the same as for low pH
More resolution needed
Poroshell 120 EC- and Eclipse
• Change organic modifier (MeOH) Plus deliver outstanding peak
• Adjust % organic for 0.5 < k <20 shape and lifetime performance at
mid pH
More resolution needed
Alternate bonded phases should
• Try Phenyl-Hexyl, Bonus-RP, etc also be considered if improved
selectivity is desired
Separation of 8 Steroids with Methanol Gradient
Best Resolution of all analytes with Poroshell 120 Phenyl-Hexyl
3 4 7
mAU
1 8
150

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

1.Hydrocortisone, 2. β-Estradiol, 3. Androstadiene 3,17 dione, 4. Testosterone,

5. Ethinylestradione, 6. Estrone, 7. Norethindone acetate, 8. Progesterone

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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 4 Poroshell 120 EC-C18

0

-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

1.Atenolol, 2. Pindolol, 3. Naldolol, 4. Metoprolol, 5. Acebutolol, 6. Propranolol, 7. Alprenolol

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Beta Blockers with Methanol Gradient

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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

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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
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NSAID Separation Poroshell 120 with a Methanol
Gradient

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Method Development at High pH
From Mid pH

Reasons to Consider High pH

Increase retention of basic
• Poroshell 120 HPH-C8 or C18 compounds by analyzing them in non-
• pH 10.5 (9-12) 5 mM ammonia, or TEA, charged form
or 10 – 50 mM organic or borate buffers
Improve selectivity
• T = 25°C (ambient – 40°C)
• Adjust MeOH for 0.5 <k < 20

More resolution needed

• Change organic modifier

• Adjust for 0.5 < k<20

Try different HPLC mode - HILIC

Poroshell HPH-C18 Low, Mid, & High pH
Why use multiple pH’s?

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Column Lifetime with High pH Methods

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Column Lifetime with High pH Methods

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Column Lifetime with High pH Methods

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HILIC
Hydrophilic Interaction Chromatography
• HILIC offers more retention than reversed-phase for very polar bases

•Polar stationary phase:

• Silica
• Amine
• Amide

• Polar mobile phase:

• Water is the strong solvent
•Typically ACN/water
•Buffer controls ionization of analyte and stationary phase
•Typically ammonium acetate or ammonium formate

•Retention/elution is from least to most polar

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

Time(min) %B Flow Rate= 0.6 mL/min

150 Initial 97 2.1 x 100 mm
4 85
4.67 85

3.970
125
5 97

3.635
6.67 97
100

75 1. Dopamine
2. Epinephrine
3. Norepinephrine
50
1.659

Approx. 1 mg/mL each in 66:33 DMSO:MeCN

25
0.824

0.5 µl injection

2 4 6 8 min
Poroshell 120 Options

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Method Development Kits

Method Development Kits Description (One of each) Dimension Part No.

Poroshell 120 Selectivity EC-C18, Phenyl-Hexyl, Bonus-RP 2.1 x 50 mm 5190-6155
Poroshell 120 Selectivity EC-C18, Phenyl-Hexyl, Bonus-RP 4.6 x 50 mm 5190-6156
Poroshell 120 Aqueous SB-Aq, Phenyl-Hexyl, Bonus-RP 2.1 x 50 mm 5190-6157
Poroshell 120 Aqueous SB-Aq, Phenyl-Hexyl, Bonus-RP 4.6 x 50 mm 5190-6158
Poroshell 120 USP L1, L7, and L10 EC-C18, EC-C8, EC-CN 4.6 x 100 mm 5190-6159
Poroshell 120 USP L1, L7, and L10 EC-C18, EC-C8, EC-CN 3.0 x 100 mm 5190-6160
ZORBAX RRHD pH SB-C18, Eclipse Plus C18, and Extend-C18 2.1 x 50 mm 5190-6152
ZORBAX Eclipse Plus C18, C8, Phenyl-Hexyl 2.1 x 50 mm 5190-6153

ZORBAX RRHD Aqueous SB-Aq, Bonus-RP, Eclipse Plus Phenyl-Hexyl 2.1 x 50 mm 5190-6154

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Poroshell 120 Options

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Benefits of 4 µm Poroshell 120

1. Naproxen 50:49:1 MeCN:water:acetic acid

2. Butyrophenone Flow rate: 1.2 mL/min 5991-5408EN
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Consistent Selectivity Across Particle Sizes

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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

Avoiding Beginner Pitfalls Agilent

Restricted
4/8/2015
Summary
What to consider when choosing a column?
• Make sure you choose the best pore size

• Consider newer particle technologies

• Poroshell 120 2.7 µm
• Poroshell 120 4.0 µm

• Take advantage of selectivity changes

• Mobile phase
• pH
• Bonded phase

Thank you!

[email protected]
AdvanceBio RP-mAb

Particle The optimum large molecule

resolution for use with both HPLC
• 3.5 μm SP particle
and UHPLC systems
• 0.25 μm porous layer depth
0.25 um
• 450Å pore diameter
3.0 um 3.5 um
Phases
• C4
• SB-C8
• Diphenyl
The most popular phases for proteins,
plus a choice for unique selectivity
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AdvanceBio RP-mAb

Column dimensions: 2.1 x 100 mm, 3.5 μm

Mobile phase A: 0.1% TFA in water:IPA (98:2) Mobile phase B: IPA:acetonitrile:MPA (70:20:10)
Flow rate: 1.0 mL/min Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B
Temperature: 80 °C Detection: UV @ 254 nm
Sample: 5 μL injection of humanized recombinant Herceptin IgG1 intact from Creative Biolabs (1 mg/mL)

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