The Chromatographer’s Guide

To Improving UHPLC
Column Selectivity

[Link]/UHPLC
Guide to Choosing the Most Effective Selectivity

Step 1
Understanding Solid Support and Efficiency
p. 3................. Selecting the Right Solid Support
p. 4................. How Resolution is Affected by Selectivity and Efficiency
p. 5................. Efficiency Gains with Luna® Omega and Kinetex®

Step 2
How to Use Selectivity
p. 6................. Profiling the Mechanisms of Selectivity
p. 7................. Relating Selectivity to UHPLC Stationary Phases

Step 3
Our UHPLC Selectivities
p. 9................. Alkyl Phases
p. 10............... Phenyl Phases
p. 11............... Polar Retentive Phases

Step 4
Applying Column Selectivities to Your UHPLC Analysis
pp. 13-15........ Thinking Outside the Traditional C18 Phases
pp. 16-17........ Improve Polar Separations with 100 % Aqueous Stable Phases
p. 18............... Utilizing Unique Selectivities
p. 19............... Aromatic Based Selectivitiy
p. 20............... Protect your Column’s Selectivity

Step 5
Order Your New Selectivity Tools
pp. 21-23

© 2018 Phenomenex, Inc. All rights reserved.

Select the Right Solid Support Step
Phenomenex offers a full range of solid supports including core-shell and thermally modified fully porous. The
1
morphology of the solid support has a significant impact on the resulting material characteristics and column

Understanding Solid Support and Efficiency

performance.

Core-Shell and Organo-Silica Core-Shell

Unique solid silica core and porous shell that results in faster chromatogra-
phy and higher efficiencies than conventional fully porous particles.

Well suited for:

• Performance gains on ANY LC system
• Easy system-to-system and lab-to-lab method transfer
• Methods where increased sensitivity is required
• Significantly improving the productivity of older, established methods

Scalability

Capillary Minibore MidBore™ Analytical Semi-Prep Preparative Bulk Media

Particle Sizes

1.3 µm 1.6 µm 1.7 µm 2.5 µm 2.6 µm 3 µm 3.5 µm 4 µm 5 µm 10 µm 15 µm

Fully Porous – Thermally Modified Silica

Unique high efficiency and extremely robust fully porous silica that offers
astounding performance and inertness alongside versatile selectivities.
Micropores
Well suited for:
• Astounding UHPLC, HPLC, and Preparative HPLC
performance and efficiencies
• Greater separation muscle
• Better peak shape through an inert foundation
• Extreme ruggedness and dependability
Absence of Micropores

Thermal Modified Pore Structure

Most importantly, through our proprietary process, we eliminate micropores,
further improving column efficiency, inertness, and reproducibility.

Scalability

Capillary Minibore MidBore™ Analytical Semi-Prep Preparative Bulk Media

Particle Sizes

1.3 µm 1.6 µm 1.7 µm 2.5 µm 3 µm 3.5 µm 4 µm 5 µm 10 µm 15 µm

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Phenomenex l WEB: [Link]
Step How Resolution is Affected by Selectivity
1 and Efficiency
Selectivity (α) and efficiency (N) have the greatest impact on observed resolution (R), when compared to other
Understanding Solid Support and Efficiency

chromatographic parameters. Often the simplest and most effective way to improve your chromatographic
results is to change your column’s phase or solid support. Phenomenex offers a wide breadth of phase chem-
istries across multiple solid supports for simplified method development and optimization.

Selectivity is the most important

parameter for increasing resolution.
The Impact of Selectivity on Resolution
3.0

( )( )( )
2.5 Retention Selectivity Efficiency α
α -1 Ν
2.0 R=
+1 α 4
Ν
Resolution (R)

1.5

1.0

0.5

0
1.00 1.05 1.10 1.15 1.20 1.25
α
0 5,000 10,000 15,000 20,000 25,000 Ν

0 5 10 15 20 25

Retention

Efficiency
Selectivity

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Phenomenex l WEB: [Link]
Efficiency Gains with Luna Omega and Kinetex Step
The undeniably high efficiency levels found in each Luna® Omega and Kinetex® column provide you with the 1
potential of huge gains in method performance. While traditional silica and hybrid fully porous particles may

Understanding Solid Support and Efficiency

claim high performance, when compared to Luna Omega or Kinetex, they fall short and prevent HPLC/UHPLC
scientists from reaching their goals.

Efficiency Levels (plates/m)

Kinetex 1.3 µm
Luna Omega 1.6 µm
Kinetex 1.7 µm
Conventional Fully Porous 1.7 µm
Kinetex 2.6 µm

100,000 200,000 300,000 400,000

UHPLC Performance – Cyclosporine Impurity Profile

Kinetex 2.6 μm Polar C18 Luna Omega 1.6 μm Polar C18
mAU mAU
Pressure: 70 bar Pressure: 117 bar
5 5
4
Valuable Resolution 4
Valuable Resolution
3 3

App ID 23964
App ID 23967

2 2
1 1
0 0

0 5 10 15 20 25 min 0 5 10 15 20 25 min

Conventional 1.7 μm C18

mAU Conditions for all columns same except where noted:
Pressure: 126 bar Columns: Kinetex 2.6 μm Polar C18
5
Luna Omega 1.6 μm Polar C18
4
Disappointing Conventional Fully Porous 1.7 μm C18
Resolution Dimensions: 50 x 2.1 mm
3 Mobile Phase: Acetonitrile/Tert-butyl methyl ether/Water/
App ID 23966

Phosphoric acid ([Link])

2
Flow Rate: 0.30 mL/min
1 Temperature: 80 ˚C
Detection: UV @ 210 nm
0 Sample: Cyclosporine

0 5 10 15 20 25 min

Comparative separations may not be representative of all applications.

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Phenomenex l WEB: [Link]
Step Profiling the Mechanisms of Selectivity
2 Observed selectivity is dictated by several primary molecular interactions. Below you’ll find
selectivity parameters used to help characterize reversed phase selectivity mechanisms.
How to Use Selectivity

C π-π π-π

O O

Hydrophobicity Steric Interaction

The ability of a phase to The ability of a phase to
hydrophobically interact with separate compounds based on
carbon groups structural differences

B OH
∙∙
H
∙∙
O O O O X O

Hydrogen Bond Hydrogen Bond

Donating Capacity Accepting Capacity
The ability of a phase to The ability of a phase to
hydrogen-bond with proton hydrogen-bond with proton
accepting groups donating groups

Cation Selectivity at pH 2.8

The ability of a phase to interact with
B+ cation groups at acidic pH

O O– O Cation Selectivity at pH 7.0

The ability of a phase to interact with
cation groups at neutral pH

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Phenomenex l WEB: [Link]
Relating Selectivity to UHPLC Stationary Phases Step
Follow the colors to connect functional groups to selectivity profiles! This color coordination 2
demonstrates the relationship between atomic fragments of compounds and how to relate
them to column selectivity profiles.

How to Use Selectivity

Define the Characteristics of Your Target Compounds

C
OH

O O
∙∙
O X O

Hydrophobicity
Hydrogen
Bond Accepting HO NH2
Capacity

HO
π-π π-π

π-π Interaction

Match Your Target Compounds to the 5 Primary Selectivity Parameters

Three primary mechanisms of selectivity relate to the analyte of interest above. By referencing the column
selectivity bar chart below, we can see the correlation between selectivity mechanisms and column
selectivity profiles.

Example: Luna® Omega 1.6 µm Polar C18 Selectivity Bar Chart

Hydrophobicity

Steric Interaction

Hydrogen Bond Donating Capacity

Hydrogen Bond Accepting Capacity

Cation Selectivity at pH 2.8

Cation Selectivity at pH 7.0

Low High

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Phenomenex l WEB: [Link]
Step
3 Selectivity Overview
Our UHPLC Selectivities

p. 9 Alkyl Phases
UHPLC selectivities that are best suited for the
analysis of hydrocarbons

p. 10 Phenyl Phases
UHPLC selectivities that are best suited for aromatic
compounds

p. 11 Polar Retentive Phases

UHPLC selectivities that are best suited for more
polar compounds

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Phenomenex l WEB: [Link]
Alkyl Phases Hydrophobicity
High column hydrophobicity values
Step
indicate greater retention of carbon-
containing analytes. 3
Application Spotlight

Our UHPLC Selectivities

• Cannabinoids
• Analgesics
• Pharmaceuticals (USP: L1)

Find the right amount of hydrophobic selectivity for your separa- O O

tion. Below are UHPLC columns that are recommended for the
separation of hydrocarbon-based compounds.

Hydrophobicity
Steric Interaction

Luna® Omega C18 Hydrogen Bond Donating Capacity

Hydrogen Bond Accepting Capacity
TMS TMS
Rugged and highly efficient C18 with strong focus Cation Selectivity at pH 2.8
on hydrophobic retention of non-polar and polar Cation Selectivity at pH 7.0
USP: L1 compounds. Low High

Kinetex® XB-C18 Hydrophobicity

Steric Interaction
Di-isobutyl side chains differentiate this C18 column.
Hydrogen Bond Donating Capacity
Low ligand density and an inactive surface make this Hydrogen Bond Accepting Capacity
TMS Si TMS column a great hydrogen acceptor. This phase will Cation Selectivity at pH 2.8
demonstrate improved peak shape for basic compounds Cation Selectivity at pH 7.0
USP: L1 and increased retention of acids. Low High

Hydrophobicity
Kinetex C18 Steric Interaction
Hydrogen Bond Donating Capacity
Very well balanced column providing some selectivity Hydrogen Bond Accepting Capacity
TMS TMS
through steric, hydrogen, and cationic pathways. Cation Selectivity at pH 2.8
This is a great starting point for ultra-high efficiency Cation Selectivity at pH 7.0
USP: L1 separations. Low High

Hydrophobicity
Kinetex C8 Steric Interaction
Hydrogen Bond Donating Capacity
Brings the benefits of core-shell technology to Hydrogen Bond Accepting Capacity
TMS TMS
USP L7 methods. The phase will provide moderate Cation Selectivity at pH 2.8
hydrophobicity and good steric and hydrogen donating Cation Selectivity at pH 7.0
USP: L7 selectivity. Low High

Hydrophobicity
Steric Interaction
Hydrogen Bond Donating Capacity
TMS TMS
Kinetex EVO C18 Hydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8
Novel pH 1-12 stable C18 that delivers robust methods Cation Selectivity at pH 7.0
USP: L1 and improved peak shape for bases. Low High

Denotes stationary phases that are 100 % aqueous stable

Material Characteristics
Particle Pore Size Effective Surface Effective pH Pressure
Phase Sizes (µm) (Å) Area (m²/g) Carbon Load % Stability Stability
Luna Omega C18 1.6 100 260 11 1.5 - 8.5*
Kinetex XB-C18 1.7, 2.6, 3.5, 5 100 200 10 1.5-8.5*
1,000/600†
Kinetex C18 1.3, 1.7, 2.6, 5 100 200 12 1.5-8.5*
bar
Kinetex C8 1.7, 2.6, 5 100 200 8 1.5-8.5*
Kinetex EVO C18 1.7, 2.6, 5 100 200 11 1.0-12.0

* pH stability under gradient conditions. pH stability is 1.5 - 10 under isocratic conditions.

†
2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.
When using Kinetex 1.3 µm or 1.7 µm, increased performance can be achieved, however high pressure-capable instrumentation is required.

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Phenomenex l WEB: [Link]
Step Phenyl Phases Aromaticity
3
Column chemistries that contain ring
structures interact with aromatic or
phenyl containing compounds via pi-pi
Application Spotlight interactions (π stacking).
Our UHPLC Selectivities

• Taxanes
• Mycotoxins
• Opiates

Aromatic interactions greatly promote steric interactions. Below

are UHPLC columns that have the highest potential for pi-pi bond π-π π-π
interaction.

Hydrophobicity
Steric Interaction
Hydrogen Bond Donating Capacity
TMS TMS
Kinetex® Biphenyl Hydrogen Bond Accepting Capacity
Cation Selectivity at pH 2.8
100 % aqueous stable reversed phase chemistry with Cation Selectivity at pH 7.0
USP: L11 hydrophobic‚ aromatic‚ and enhanced polar selectivity. Low High

Hydrophobicity
Steric Interaction

Kinetex Phenyl-Hexyl Hydrogen Bond Donating Capacity

Hydrogen Bond Accepting Capacity
TMS TMS
Aromatic and moderate hydrophobic selectivity result Cation Selectivity at pH 2.8
in the great retention and separation of aromatic Cation Selectivity at pH 7.0
USP: L11 hydrocarbons. Low High

F Hydrophobicity
F F
Kinetex F5 Steric Interaction
Hydrogen Bond Donating Capacity
F F This pentafluorophenyl propyl column provides a very Hydrogen Bond Accepting Capacity
TMS TMS
high degree of steric selectivity to separate structural Cation Selectivity at pH 2.8
isomers. The electronegative fluorine groups offer high Cation Selectivity at pH 7.0
USP: L43 selectivity for cationic compounds. Low High

Denotes stationary phases that are 100 % aqueous stable

Material Characteristics
Particle Pore Size Effective Surface Effective pH Pressure
Phase Sizes (µm) (Å) Area (m²/g) Carbon Load % Stability Stability
Kinetex Biphenyl 1.7, 2.6, 5 100 200 11 1.5-8.5*
1,000/600†
Kinetex Phenyl-Hexyl 1.7, 2.6, 5 100 200 11 1.5-8.5*
bar
Kinetex F5 1.7, 2.6, 5 100 200 9 1.5-8.5
* pH stability under gradient conditions. pH stability is 1.5 - 10 under isocratic conditions.
†
2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.
When using Kinetex 1.3 µm or 1.7 µm, increased performance can be achieved, however high pressure-capable instrumentation is required.

Method Develoment Tip!

Try using methanol for the organic portion of the mobile phase.
It can help promote pi-pi bond interaction!

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Phenomenex l WEB: [Link]
Polar Retentive Phases Hydrogen Bond
Accepting Capacity
Step
Hydrogen bond accepting groups on
the silica surface interact with hydrogen 3
Application Spotlight bond donating functionalities on
analytes.

Our UHPLC Selectivities

• Peptide Mapping
• Pesticides
• Nucleosides

Column phases with hydrogen bond accepting/donating capacity have

OH
increased polar selectivity and retention of polar compounds. Below are
UHPLC columns that offer increased polar selectivity/retention.
∙∙
O X O

Luna® Omega Polar C18 Hydrophobicity

Steric Interaction
100 % aqueous stability and enhanced selectivity/
Hydrogen Bond Donating Capacity
retention for polar analytes without diminishing useful Hydrogen Bond Accepting Capacity
TMS TMS
non-polar retention. The C18 ligand provides general Cation Selectivity at pH 2.8
Polar Polar
hydrophobic interactions while a polar modified particle Cation Selectivity at pH 7.0
USP: L1 surface provides enhanced polar compound retention. Low High

Luna Omega PS C18

Unique, 100 % aqueous stable mixed-mode phase
that provides both polar and non-polar retention. The
surface contains a positive charged ligand which aids Hydrophobicity
Steric Interaction
in the retention of acidic compounds through ionic
Hydrogen Bond Donating Capacity
TMS TMS
interactions, while the C18 ligand promotes general Hydrogen Bond Accepting Capacity
+ +
reversed phase hydrophobic retention. The positively Cation Selectivity at pH 2.8
USP: L1
charged surface also improves basic compound peak Cation Selectivity at pH 7.0
shape through ionic repulsion. Low High

Hydrophobicity
Steric Interaction

Kinetex® Polar C18 Hydrogen Bond Donating Capacity

Hydrogen Bond Accepting Capacity
TMS TMS
Combined C18 and polar modified surface that provide Cation Selectivity at pH 2.8
Polar Polar
polar and non-polar retention alongside 100 % aqueous Cation Selectivity at pH 7.0
USP: L1 stability. Low High

Denotes stationary phases that are 100 % aqueous stable

Material Characteristics
Particle Sizes Pore Size Surface Area Carbon Load pH Pressure
Phase (μm) (Å) (m²/g) (%) Stability Stability
Kinetex Polar C18 2.6 100 200 9 1.5 - 8.5*
1,000/600†
Luna Omega Polar C18 1.6, 3, 5 100 260 9 1.5 - 8.5*
bar
Luna Omega PS C18 1.6, 3, 5 100 260 9 1.5 - 8.5*
* pH stability under gradient conditions. pH stability is 1.5 - 10.0 under isocratic conditions.
†
2.1 mm ID Kinetex columns are pressure stable up to 1000 bar.

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Step
4
Applying Column Selectivities to
Your UHPLC Analysis
Applying Selectivity

pp. 13-15 Thinking Outside the Traditional C18 Phases

Advances over traditional C18 selectivity
pp. 16-17 Improve Polar Separations with 100 % Aqueous Stable Phases
The selectivity power of using aqueous stable UHPLC columns
p. 18 Utilizing Unique Selectivities
Comparison of unique stationary phases
p. 19 Aromatic Based Selectivity
Aromatic (pi-pi interaction)
p. 20 Protect the Column’s Selectivity
Extend your column’s lifetime

Browse thousands of applications at:

[Link]/applications
™

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Phenomenex l WEB: [Link]
Thinking Outside the Traditional C18 Phase Step
The extent to which resolution depends on selectivity becomes evident when a chromatogra-
pher takes advantage of technologic advances in polar stationary phases in order to improve
4
their analysis. Below is an example of resolution improvements obtained by switching from a

Applying Selectivity
traditional C18 to a column with additional selectivity mechanisms.

Polar Selectivity of Catecholamines

24613
6
1.5e5
5
Polar Modified
1.4e5
1.3e5 Luna® Omega 1.6 μm Polar C18
1.2e5
1.1e5
1.0e5
9.0e4 HO NH2
Intensity, cps

8.0e4
7.0e4
Dopamine
6.0e4 2 HO

5.0e4 3
4
4.0e4

App ID 24611
TMS TMS
3.0e4
Polar Polar
2.0e4
1 USP: L1
1.0e4

0
0 0.5 1 1.5 2 2.5 3 3.5 min

24609

1.3e5
Traditional C18
6

1.2e5

1.1e5 Kinetex® 1.7 µm C18

1.0e5 Improved Selectivity for Polar Analytes
9.0e4

8.0e4
Intensity, cps

7.0e4
5
6.0e4

5.0e4 2, 4
3
4.0e4
App ID 24609

3.0e4 TMS TMS

2.0e4

1.0e4 1 HILIC
0
0 0.5 1 1.5 2 2.5 3 3.5 min

Conditions for both columns: Improved Separation with Polar C18

Columns: Luna Omega 1.6 µm Polar C18 The increased hydrogen bond accepting capacity of the Luna Omega Polar
Kinetex 1.7 µm C18 C18 column improves polar selectivity for analytes such as dopamine.
Dimensions: 50 x 2.1 mm
Mobile Phase: A: 10 mM Ammonium Formate with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic Acid Hydrophobicity
Gradient: Time (min) % B Steric Interaction
0 0 Hydrogen Bond Donating Capacity
3 90 Hydrogen Bond Accepting Capacity
Flow Rate: 0.4 mL/min Cation Selectivity at pH 2.8
Injection Volume: 1 µL Cation Selectivity at pH 7.0
Temperature: 22 °C Low High
Detection: MS/MS (SCIEX API 4000™)
Sample: 1. Norepinephrine
2. Epinephrine
3. Normetanephrine
4. Dopamine
5. Metanephrine
6. Serotonin

Comparative separations may not be representative of all applications.

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Phenomenex l WEB: [Link]
Step Thinking Outside the Traditional C18 Phase
4 Traditional C18 phases may not always be the best option. A UHPLC column
that has both polar and hydrophobic versatility allows for great method devel-
opment flexibility.
Applying Selectivity

Acids/Bases/Neutrals
23707

0.55

0.50
4
Positive
0.45 Nortriptyline Surface Charge
0.40 Luna Omega
NH 1.6 μm PS C18
0.35
6
0.30
AU

0.25
2 5
0.20
7
0.15

App ID 23707
3 TMS TMS

0.10 + +
0.05 1
USP: L1

0 1 2 3 4 5 min

Better Resolution and Peak Shape

23714

0.55

0.50
Traditional C18
0.45 Waters® ACQUITY® BEH
0.40
1.7 μm C18

0.35

0.30
6
0.25
AU

5
0.20 4

0.15
App ID 23714

7
2 TMS TMS

0.10
1 3
0.05
HILIC
0

0 1 2 3 4 5 min

Conditions for both columns: Improved Separation with PS C18

Columns: Luna Omega 1.6 μm PS C18
The increase in steric interaction of the Luna Omega PS C18 column
ACQUITY BEH 1.7 μm C18
improves resolution for aromatic analytes such as nortriptyline.
Dimensions: 50 x 2.1 mm
Mobile Phase: A: Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic Acid Hydrophobicity
Gradient: Time (min) % B Steric Interaction
0 5 Hydrogen Bond Donating Capacity
5 95 Hydrogen Bond Accepting Capacity
5.01 5 Cation Selectivity at pH 2.8
8 5 Cation Selectivity at pH 7.0
Flow Rate: 0.4 mL/min Low High
Temperature: 22 °C
Detection: UV @ 254 nm
Sample: 1. Uracil
2. Pindolol
3. Chlorpheniramine
4. Nortriptyline
5. 3-Methyl-4-nitrobenzoic acid
6. 5-Methyl salicylaldehyde
7. Hexanophenone

Comparative separations may not be representative of all applications.

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Phenomenex l WEB: [Link]
 Thinking Outside the Traditional C18 Phase Step
Traditional UHPLC C18 phases can be prone to peak tailing for highly basic
compounds and this tailing can become exacerbated when higher loadability is
4
required. The Luna® Omega PS C18’s combination of a high surface area and

Applying Selectivity
novel surface chemistry allows for better peak shape as loading increases.

Why is Loadability Important?

• Analytical conformation parallel to purification
• Dealing with high API concentrations when conducting stability tests
• High sample loading to visualize low-level analytes of interest

Amitriptyline
0.80
Loading Study
0.70
Positive Surface
0.60
Charge
0.50
Luna® Omega
0.40 1.6 µm PS C18
AU

0.30

App ID 23737
0.20
TMS TMS

0.10 + +
USP: L1
0
1.5 2 2.5 min

Better Peak Shape

Over All Loads

0.80

0.70 Traditional C18 Conditions for both columns:

Columns: Luna Omega 1.6 μm PS C18
ACQUITY BEH 1.7 μm C18
0.60 Waters® ACQUITY® BEH
Dimensions: 50 x 2.1 mm
1.7 μm C18
Mobile Phase: A: Water with 0.1 % Formic Acid
0.50
B: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min) % B
AU

0.40 0 5
5 80
0.30
Flow Rate: 0.4 mL/min
App ID 23738

Temperature: 22 °C
0.20
TMS TMS
Detection: UV @ 254 nm
Sample: Amitriptyline
0.10

0.00 HILIC
1.5 2 2.5 min

Luna Omega PS C18 50 x 2.1 mm vs. Other Columns

0.09
Luna 1.6 µm PS C18
App ID 23739

0.08
Advanced Materials Technology
0.07 HALO® 2 µm C18

Waters ACQUITY® BEH

Peak Width at 50 %

0.06
1.7 µm C18
0.05
Thermo Hypersil GOLD®
0.04 1.9 µm C18

0.03

0.02 Better Peak Shape Over All

0.01 Sample Loadings
0
0 0.1 0.2 0.3 0.4 0.5 0.6

Comparative separations may not be representative of all applications.

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Phenomenex l WEB: [Link]
Step Improve Polar Separations with
4 100 % Aqueous Stable Phases
Applying Selectivity

A powerful tool in the chromatographer’s toolbox is the ability to use 100 %

aqueous conditions to promote polar selectivity and increased retention. Traditional
C18 phases are known to collapse under 100 % aqueous conditions, causing a loss
of retention and method development headaches.

Catecholamines
4.0e5

3 % Initial Organic Gradient

3.0e5 Luna® Omega 1.6 µm Polar C18
Intensity, cps

2.0e5

App ID 24410
TMS TMS
1.0e5
Polar Polar

USP: L1

0.0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min

Greater Retention and Resolution

Under 100 % Aqueous Conditions

2.5e5
100 % Aqueous Organic Gradient
2.0e5 Luna® Omega 1.6 µm Polar C18
Intensity, cps

2.5e5

1.0e5
App ID 24408

TMS TMS

Polar Polar
1.5e5
USP: L1

0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min

Conditions for both separations:

Column: Luna Omega 1.6 µm Polar C18 Temperature: 40 °C
Dimension: 50 x 2.1 mm Detection: MS/MS (SCIEX API 4000™) (ambient)
Part No.: 00B-4748-AN Sample: 1. Norepinephaine
Mobile Phase: A: Water with 0.1 % Formic Acid 2. Epinephrine
B: Acetonitrile with 0.1 % Formic Acid 3. Normetanephrine
Gradient: Time (min) % B 4. Dopamine
0 3 (except where noted) 5. Metanephrine
3 100 6. 3-Hydroxytyramine
Flow Rate: 0.4 mL/min

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Phenomenex l WEB: [Link]
 Improve Polar Separations with Step
100 % Aqueous Stable Phases 4
Dipeptides in 100 % Aqueous Conditions

Applying Selectivity
mAU 3

Column: Luna Omega 1.6 µm Polar C18

1750
Luna® Omega Dimensions: 50 x 2.1 mm
1.6 µm Polar C18 Part No.: 00B-4748-AN
1500
Mobile Phase: A: Water with 0.1 % TFA
B: Acetonitrile with 0.1 % TFA
1250
Gradient: Time (min) % B
4 0 0
1000
3 75
2 Flow Rate: 0.6 mL/min

App ID 24612
5
750 Injection Volume: 1 µL
Temperature: 40 °C
500 Detection: UV @ 210 nm (ambient)
Sample: 1. Arg-Glu
250 2. Gly-Tyr
1 3. Trp-Gly
0 4. Gly-Trp
5. Pro-Trp
0 1 2 3 4 min

100 % Aqueous Conditions Increase

Retention and Selectivity of Polar Analytes

23586
1700 Column: Competitor 1.7 µm C18
3 Traditional Dimensions: 50 x 2.1 mm
1600
Competitor 1.7 µm C18 Mobile Phase: A: Water with 0.1 % TFA
1400 B: Acetonitrile with 0.1 % TFA
Gradient: Time (min) % B
1200 0 3
1000 4 3 75
Flow Rate: 0.6 mL/min
800 Injection Volume: 1 µL
App ID 24613

5
Temperature: 40 °C
600
Detection: UV @ 210 nm (ambient)
400 Sample: 1. Arg-Glu
2 2. Gly-Tyr
200 1 3. Trp-Gly
0 4. Gly-Trp
5. Pro-Trp
0 1 2 3 4 min

Water Soluble Vitamins under 100 % Aqueous Conditions

Conditions same for both separations:
2 4
2.0e6 Standards Column: Luna Omega 1.6 µm Polar C18
1.8e6 Dimensions: 50 x 2.1 mm
1.6e6
Intensity, cps

1.4e6 Part No.: 00B-4748-AN

1.2e6 1 Mobile Phase: A: 10 mM Ammonium Formate with 0.1 % Formic Acid
App ID 23584

1.0e6 B: Acetonitrile with 0.1 % Formic Acid

8.0e5 Gradient: Time (min) % B
6.0e5 0 0
4.0e5 5 7 4 90
2.0e5 3 6 4.1 0
0 7 0
1 2 3 4 min Flow Rate: 0.4 mL/min
Temperature: 40 °C
Detection: MS/MS (SCIEX API 4000™) @ 450 °C
4.5e6 4 Sample: 1. Pyridoxamine 5. Pantothenic acid
Vitamin Tablet 2. Thiamine 6. Folic acid
4.0e6
3. Nicotinic acid 7. Riboflavin
Intensity, cps

5 4. Pyridoxine
3.0e6 2
App ID 23579

2.0e6

1.0e6
7
1 6
0
1 2 3 4 min

Comparative separations may not be representative of all applications.

17
Phenomenex l WEB: [Link]
Step Utilizing Unique Selectivities
4 The elution order of analytes can change depending upon the predominate selectivity characteris-
tics of a UHPLC column and the utilized mobile phase. Therefore, by altering the stationary phase
Applying Selectivity

or mobile phase conditions, we can observe a unique elution order and extend retention of more
polar analytes.

Luna® Omega 1.6 µm C18

23443
pH 2.5

Strong hydrophobic retention

and steric interaction

App ID 23443
TMS TMS

USP: L1

0 1 2 3 4 min

Kinetex® 1.7 µm Biphenyl

23445

pH 2.5

Increased aromatic selectivity

and polar retention

App ID 23445
TMS TMS

USP: L11

0 1 2 3 4 min

Conditions for both columns:

Columns: Luna Omega 1.6 µm C18
Kinetex 1.7 µm Biphenyl
Dimensions: 50 x 2.1 mm
Part No.: 00B-4742-AN
00B-4628-AN
Mobile Phase: A: Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic Acid
Gradient: Time (min) % B
0 5
4 95
5 95
5.1 5
Flow Rate: 0.4 mL/min
Temperature: 40 °C
Detection: MS/MS (SCIEX API 4000™) (ambient)
Sample: Drugs of Abuse

18
Phenomenex l WEB: [Link]
 Aromatic Based Selectivity Step
Compounds with aromatic ring structures offer a specific type of selectivity associated with pi-pi bond 4
interaction. The compound’s aromaticity provides pi electrons that have the potential to interact with

Applying Selectivity
pi bonds, which can be found on phenyl-based stationary phases. This provides a unique, orthogonal
selectivity compared to a traditional C18 phase.

H3C
O
N
H
O N N
Cl N
O

H3CO

O NH3 CH3
3.0e6 O H
CH3 H
2.8e6 O N
CH3
2.6e6
O
2.4e6
2.2e6
2.0e6
1.8e6 H3C N
Intensity, cps

1.6e6 N
N
1.4e 6
O CH3
1.2e6 NH3
O Cl N
1.0e6
8.0e5

App ID 22584
6.0e5
4.0e5
2.0e5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

Column: Kinetex® 1.7 µm Biphenyl Flow Rate: 0.5 mL/min

Dimensions: 50 x 2.1 mm Injection Volume: 10 µL
Part No.:00B-4628-AN Temperature: 40 °C
Mobile Phase: A: Water with 0.1 % Formic Acid Detection: MS/MS (SCIEX API 4000™) (ambient)
B: Acetonitrile with 0.1 % Formic Acid Sample: Drugs of Abuse
Gradient: Time (min) % B
0 5
4 100
5 100
7 5

Increased Separation of Aromatic Compounds Using a Biphenyl Phase

Kinetex 1.7 µm C18 Kinetex 1.7 µm Biphenyl
4.8e4 4.4e4
1 1
4.0e4
4.0e4
2

3.0e4
3.0e4 Greater Separation
Intensity, cps

Intensity, cps

2
Power
2.0e4
2.0e4
App ID 24638
App ID 24637

1.0e4 1.0e4

0 0
0 0.5 1.0 1.5 min 0 0.5 1.0 1.5 min
Conditions for both columns:
Columns: Kinetex® 1.7 µm C18 Flow Rate: 400 µL/min
Kinetex® 1.7 µm Biphenyl Injection Volume: 10 µL
Dimensions: 50 x 2.1 mm Temperature: 50 °C
Part No.: 00B-4628-AN Backpressure: 450 Bar
00B-4475-AN Detection: MS/MS (SCIEX API 4000™) (ambient)
Mobile Phase: A: Water with 0.1 % Formic Acid Sample: 1. Morphine
B: Methanol with 0.1 % Formic Acid 2. Hydromorphone
Gradient: Time (min) % B
0 10
4 100
4.1 10

19
Phenomenex l WEB: [Link]
Step Protect Your Column’s Selectivity
4
ULTRA
Applying Selectivity

UHPLC Column Protection

Save Time and Money
It’s a fact! Chemical contaminants and particulates are a natural part
of any chromatographic analysis. The easiest way to extend column SecurityGuard
performance is to remove these contaminants and particulates with ULTRA
SecurityGuard ULTRA before they reach your UHPLC column and
degrade your chromatography. For all core-shell and/or
< 3 μm particle columns
With SecurityGuard ULTRA, you will experience: (< 20,000 psi / 1,378 bar)

• Increased UHPLC column lifetime

• Better column performance
• More reproducible chromatography
• Fewer wasted columns

With SecurityGuard ULTRA

Inlet Frit Column Media

No contaminant Intact media

or particle buildup

(24000 times magnification)

Without SecurityGuard ULTRA

We used to have
Inlet Frit Column Media
to change out our
columns every 2 to
3 months and ever
since we started using
Contaminant Crushed media the SecurityGuard
and particle buildup and fines
cartridges we can
(24000 times magnification)
do at least 6 months
Cartridge Holder before changing a
column out.
T. Serviss

The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.

20
Phenomenex l WEB: [Link]
 Step
4

Applying Selectivity
You have things to do.
How can we help?

Chat Now!
[Link]/chat
A Phenomenex Technical Specialist is here
to help nearly 24 hours a day!

21
Phenomenex l WEB: [Link]
Step
5 As Voted by You
Kinetex® Core-Shell LC Columns Earned the
SelectScience Gold Seal of Quality Award
Ordering Information

Amgen® GlycosBIO® Food Sciences

The Kinetex column has worked great for our validated I really love the Kinetex columns. I now have shorter HPLC

assays. We easily converted our HPLC methods to UPLC method runs and nice peak resolution. Methods that used

methods using the Kinetex column and have enjoyed being to take 30 to 40 minutes on other columns now take 15−20

able to run fast UPLC chromatography... minutes. I’m enjoying the fact that my samples are analyzed

more quickly without compromising the quality of the

peaks in the chromatograms.

University of Texas MD
Anderson Cancer Center
We have drastically improved sensitivity, reproducibility,

and lifetime on our column after switching to

the Kinetex technology. Core-Shell Technology

The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.

Kinetex Core-Shell UHPLC Column Ordering Information

SecurityGuard™
1.7 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
EVO C18 –– 00B-4726-AN 00D-4726-AN 00F-4726-AN AJ0-9298
F5 –– 00B-4722-AN 00D-4722-AN 00F-4722-AN AJ0-9322
Biphenyl –– 00B-4628-AN 00D-4628-AN 00F-4628-AN AJ0-9209
XB-C18 00A-4498-AN 00B-4498-AN 00D-4498-AN 00F-4498-AN AJ0-8782
C18 00A-4475-AN 00B-4475-AN 00D-4475-AN 00F-4475-AN AJ0-8782
C8 00A-4499-AN 00B-4499-AN 00D-4499-AN 00F-4499-AN AJ0-8784
HILIC 00A-4474-AN 00B-4474-AN 00D-4474-AN –– AJ0-8786
Phenyl-Hexyl –– 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788
for 2.1 mm ID
SecurityGuard
1.7 μm MidBore™ Columns (mm) ULTRA Cartridges‡
Phases 30 x 3.0 50 x 3.0 100 x 3.0 3/pk
XB-C18 00A-4498-Y0 00B-4498-Y0 00D-4498-Y0 AJ0-8775
C18 –– 00B-4475-Y0 00D-4475-Y0 AJ0-8775
C8 00A-4499-Y0 00B-4499-Y0 00D-4499-Y0 AJ0-8777
HILIC –– 00B-4474-Y0 –– AJ0-8779
for 3.0 mm ID

1.7 μm Microbore Columns (mm) 1.3 μm Minibore Columns (mm)

Phases 50 x 1.0 100 x 1.0 150 x 1.0 Phases 30 x 2.1 50 x 2.1
EVO C18 00B-4726-A0 00D-4726-A0 00F-4726-A0 C18 00A-4515-AN 00B-4515-AN
Biphenyl 00B-4628-A0 00D-4628-A0 00F-4628-A0

SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000

‡

22
Phenomenex l WEB: [Link]
 Step
Luna Omega Offers Better UHPLC Column Lifetimes
®

5
Phenomenex columns are engineered for durability and are able to withstand high system pressure. For example,
lifetime studies are conducted to ensure superior performance and long column lifetimes.

Ordering Information
Less Increase
Accelerated Lifetime Study in Pressure Over Time

10%
OMEGA
9%

8%
% Increase over Initial Backpressure

7%
Conditions for both columns:
6% Columns: Luna Omega 1.6 µm C18
ACQUITY BEH 1.7 µm C18
Dimensions: 50 x 2.1 mm
5% Mobile Phase: A: Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic Acid
4% Gradient: Time (min) % B
0 5
4 95
3% 4.1 5
Luna® Omega 1.6 µm C18 (AVG) Flow Rate: 0.4 mL/min
2%
App ID 23610
Waters® ACQUITY® BEH 1.7 µm C18 (AVG) Temperature: 25 °C
Detection: UV @ 210 nm
1% Sample: Protein Matrix

0%
0 10 20 30 40 50 60
Injection Cycle

Luna Omega UHPLC Column Ordering Information

1.6 μm Microbore Columns (mm)
Phases 50 x 1.0 100 x 1.0 150 x 1.0
Polar C18 00B-4748-A0 00D-4748-A0 00F-4748-A0
C18 00B-4742-A0 00D-4742-A0 00F-4742-A0
PS C18 00B-4752-A0 00D-4752-A0 -

SecurityGuard™
1.6 μm Minibore Columns (mm) ULTRA Cartridges‡
Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
Polar C18 00A-4748-AN 00B-4748-AN 00D-4748-AN 00F-4748-AN AJ0-9505
PS C18 00A-4752-AN 00B-4752-AN 00D-4752-AN 00F-4752-AN AJ0-9508
C18 00A-4742-AN 00B-4742-AN 00D-4742-AN 00F-4742-AN AJ0-9502
for 2.1 mm ID

‡
SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000

If Phenomenex analytical columns do not provide at least

an equivalent separation as compared to a competing
column of the same particle size, similar phase and
dimensions, return the Phenomenex column with
comparative data within 45 days for a FULL REFUND.

23
Phenomenex l WEB: [Link]
 The Chromatographer’s Guide
To Improving UHPLC
Column Selectivity

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Terms and Conditions

Subject to Phenomenex Standard Terms & Conditions, which may be viewed at
www. [Link]/TermsAndConditions.
Trademarks
Luna and Kinetex are registered trademarks and MidBore and SecurityGuard are trademarks of
Phenomenex. Waters and ACQUITY are registered trademarks of Waters Technologies Corporation.
HALO is a registered trademark of Advanced Materials Technology, Inc. Hypersil GOLD is a registered
trademark of Thermo Hypersil-Keystone LLC. API 4000 is a trademark of AB SCIEX Ltd. AB SCIEX ™
is being used under license. SelectScience is a registered trademark of SelectScience LLC.
Disclaimer
Phenomenex is not affiliated with Advanced Materials Technology, Thermo, or Waters. Comparative
BR55580917_W

separations may not be representative of all applications.

Kinetex EVO is patented by Phenomenex. U.S. Patent Nos. 7,563,367 and 8,658,038 and foreign
counterparts.
The opinions stated herein are solely those of the speaker and not necessarily those of any company
or organization.
© 2018 Phenomenex, Inc. All rights reserved.