part i: polar compound retention...part i: polar compound retention tuesday, october 11 2016 – 3...

©2016 Waters Corporation 1

Part I: Polar Compound Retention

Tuesday, October 11 2016 – 3 pm CET / 9 am EST

Understanding What Works

and What Doesn’t Work

In Reversed-Phase Polar Compound Retention

•


Overview

Introduction

– The Problem

Background

Challenges for Reversed-Phase HPLC for Polar Molecules

Columns for Polar Compound Retention

Summary


Introduction The Problem

Conventional reversed-phase columns often do not provide

adequate retention and separation of highly polar compounds

Screening and Discovery

– Gradient - High-throughput screening (HTS) does not work for polar

compounds

o Elute un-retained in the void volume

o Co-elute at the beginning of the run – if retained at all

Method Development

– Isocratic - Many columns cannot be run under the 100% aqueous

conditions necessary for polar compound retention– columns “dewet”

(also termed - hydrophobic collapse)


Overview

Introduction

Background

– Polar molecules

– Chromatographic methods for retaining polar compounds



Summary


What is a Polar Molecule?

General chemistry definition:

– A molecule whose centers of positive and negative

charges do not coincide

– The degree of polarity is measured by

the dipole moment of the molecule

Dipole moment is the product of the

charge at either end of the dipole

times the distance between the charges

– The unequal sharing of electrons within a bond

results in a separation of positive and negative

electric charge.

Polarity is dependent on the electronegativity

difference between molecular atoms and

compound asymmetry


Examples of Polar Organic Molecules

H N

N H

O

O

Thymine (N)

O

Acetone (N)

N

N H

N H 2

O

Cytosine (B)

H N

H N

F

O

O

O

H O

5-Fluoroorotic acid (A)

O H

O H

N H

O H

Epinephrine (B) Ascorbic acid (A)

O

O H

O H

O

O H

O H

•Thiourea (N)

N N

S


Comparison of Chromatographic Methods for Retention of Polar Compounds

Technique Advantages Disadvantages

Reversed-

Phase

Familiar technique

High efficiency

Rapid equilibration

Wide selection of columns

Dewetting under aqueous conditions Poor retention of polar compounds

HILIC

High % organic mobile

phases give higher

sensitivity in MS

Eliminate evaporation of

SPE eluents

Sample solubility problems Not well understood Not widely applicable

Ion-

Pairing

Retains ionizable compounds

Ion-pairing and reversed-

phase chromatography use

same columns

Long equilibration times Difficult to run gradients Not compatible with MS Difficult method development

Ion-

Exchange

Can retain any ionizable

compound

Buffer salts or pH gradients not compatible with mass spectrometry Compounds must be ionic


Overview

Introduction

Background


– What is Dewetting

– What does not work for Polar Compound Retention


Summary


Challenge for Reversed-Phase Chromatography of Polar Compounds

To retain polar compounds on a non-polar surface we reduce

the amount of organic in the mobile phase

– make mobile phase weaker (100% aqueous mobile phase)

PROBLEM: Risk of dewetting the stationary phase particle

surface

The non-polar pore surface expels the aqueous, polar mobile

phase

(The chromatographic pores dry-out!!)

What is “dewetting” and why does it happen?


Mobile phase must be allowed into the pore in order for chromatographic retention of the analyte to take place.

Proper Wetting of Bonded Chromatographic Surface

What influence does this have on chromatography?

If the pores are dry, the analyte cannot get into the pores and it will not be retained by the chromatographic surface.

The particles are very porous, like the pores of a sponge – 99% of chromatographic

surface is inside the pores

•Analyte

•Mobile Phase


The observed reduction in V0 and sudden loss in retention after a

pressure release, indicates that material pores expel the aqueous

solvent*

Pore Dewetting: What Does It Look Like?

•*T.H. Walter, P. Iraneta and M. Capparella J. Chromatogr. A 1075 (2005) 177-183

•10% Reduction

•ACQUITY UPLC® BEH C18

•10% Reduction •in Void Volume •23% Loss

•in Retention


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•1

•2,3,4

•5

•Atlantis T3 •4.6 x 100 mm, 3 µm

•Zorbax® Eclipse Plus C18

•4.6 x 100 mm, 3.5 µm

< 7% Thymine Dewet

•Thymine Dewet: ~100%

• Compounds: • 1. Thiourea • 2. 5-Fluorocytosine • 3. Adenine • 4. Guanosine-5’-monophosphate • 5. Thymine

Chromatographically Speaking: What is Dewetting?

100% retention loss with conventional C18

Note enhanced retention

•Comparative separations may not be representative in all applications.


•Minutes

•0 •2 •4 •6 •8 •10

•Initial •(Column was wetted first with organic)

•After Flow Stoppage •(Pores “dewet” 100%)

•Mobile phase: 0.1% Acetic Acid

•Amoxicillin

•Vo: No retention of analyte

•1,500psi

•The column is not broken -- It just stopped working •Why?

•1,500psi

Conventional C18 Column Dewetting


Pore Dewetting Mechanism

Flow stoppage relieves the pressure that was forcing the aqueous

mobile phase into the pores. When pressure is reduced (e.g., pump

stopped), the hydrophobic pore surface can expel the polar mobile

phase and “dewet” the pore.

At flow, with pressure on

the mobile phase.

Stopped flow with no pressure on the

mobile phase.

Pores de-wet – restart flow – pores

still dewetted and analytes never

enter pores – resulting in no retention.

Analytes

Analytes

properly retained

Remember: Most of the

surface area (> 95%) is

inside the silica pores!


Water on C18: d = 100Å

g = 72.8 dynes/cm

θ = 110.6°*

•*B. Janczuk, T. Bialopiotrowicz and W. Wojcik, Colloids and Surfaces 36 (1989) 391-403

Methanol on C18: d = 100Å

g = 22 dynes/cm

θ = 39.9°*

Pc < 0 psi

Pc = 1,500 psi

Note: Self-wetting – No pressure required

g Equation of Young and Laplace

2 q Pc =

q

r q cos

Reducing the contact angle forces water back into the pore

Where:

Pc = Capillary pressure

γ = Surface tension

r = Capillary radius

q = Contact angle

Stationary Phase Wetting/Dewetting

Solvent dependent

H2O

MeOH

q

q

Contact Angle

•Pressure •in the pore


Re-wetting a Stationary Phase

Use a mobile phase containing > 40% methanol or other polar

organic solvent (other organic solvents may vary in % required

for wetting)

– This works by reducing the contact angle (q)

The use of pressure alone cannot force aqueous mobile

phase back into silica pores

– Not practical because column outlet is at atmospheric pressure – not

all pores see required pressure (1,500 psi)

1,500 psi 1 atm = 14.7 psi

Pressure gradient

inside column

1,500 psi

Atmospheric pressure


Dewetting Summary

Dewetting - not “hydrophobic collapse” causes the sudden loss

of retention under highly aqueous conditions

V0 reduction and sudden loss in retention after a pressure drop

indicate that the pores expel aqueous solvent (vs. ligand chains

“folding” or “matting” as described by hydrophobic collapse)

Rewetting with organic solvent can rewet the pores by reducing

the contact angle and surface tension

Ideally, it is best to avoid dewetting altogether.

How have chromatographers attempted to do this?


Embedded Polar Groups: What Doesn’t Work

Embedded polar group columns designed to:

– Improve peak shapes for basic compounds

– Provide complementary selectivity as compared to straight-

chain alkyl stationary phases

Examples of embedded polar groups include

carbamate, ether, amide, urea, etc.

Another feature of embedded polar groups is 100%

aqueous mobile phase compatibility

This feature is confused with enhanced polar compound retention.

Why?



Embedded polar groups resist pore dewetting by increasing

the water layer at the surface of the pores

Embedded polar group behaves like a “Polar Hook” – holding onto water.

Polar compound retention requires these very weak polar mobile Phases i.e., highly aqueous mobile Phases with little-or-no organic modifier

This aqueous compatibility is confused with polar compound retention.

This is false!


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Atlantis T3

•1 •2

•3 •4

•5

•Vo = 1.2min

Zorbax Bonus RP (amide)

•1 •2 •3

•4 •5

GL Sciences Inertsil ODS EP

•Vo = 1.2min

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•Vo = 1.3min



Embedded Polar Groups – What Doesn’t Work

•Atlantis® T3

•1 •2

•3 •4

•5

•Supelco ABZ+ •(amide)

•Vo = 1.3min

•1 •2 •3

•4

•5

•Metachem •Polaris C18-A

•Vo = 1.3min

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•Vo = 1.3min

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•Dionex Acclaim® •Polar Advantage




Why less retention of polar compounds with

embedded polar group columns? Reason 1 Water layer “shields” silanols and reduces cationic interactions and H-bonding

This “water shield” results in less retention of polar compounds - These are also the reasons for the excellent peak shape observed for bases

+

N

N

N

N

H

N H 3

Reason 2 Shorter alkyl chain length in order to obtain higher ligand density


Embedded Polar Groups: Not the Answer

Embedded polar groups – summary

– Embedded polar groups were designed for improved peak shape

– Embedded polar groups provide aqueous compatibility

– For some compounds, embedded polar groups can provide increased

retention (e.g., polyphenolics)

– For highly polar compounds, however, embedded polar groups

produce less retention

Now that we’ve defined what is not an ideal column for polar retention, let’s define what an ideal column is for polar compound retention.


Overview

Introduction

Background



– Designing the ideal column

– Particle and Bonding Considerations

– Introduction to the T3 Column Family

Summary


Retaining Polar Analytes Using Reversed-Phase HPLC

•O-

•O- •Si

•Si

•OH

•O- •Si

•O- •Si

•OH

•O- •Si •OH

•O- •Si

•O- •Si

•O- •Si

•OH

•O- •Si

•O- •Si

•Non-polar •Stationary phase

•Polar •Mobile phase

•Analyte X

•Polar

Analyte Y Non-Polar

•How can a column simultaneously retain •and separate analytes of different polarities?

•Flow

•Poorly •Retained •(like attracts like)

•Well retained •(like attracts like)


Peak Shape Dewetting

The “Ideal” Column

Retention Stability

Optimization of pore size, ligand type, ligand density and

endcapping results in a good balance among peak shape,

stability, dewetting and polar retention.


Designing the Ideal Column

Q: How can reversed-phase columns designed for polar

compound retention be improved?

– Increase retention

– Improve column lifetime at low pH

– Provide better peak shape at neutral pH

– Little-to-no MS bleed

– Seamless scalability from UPLC-UHPLC-HPLC-Preparative separations


Bonded Phase Hydrolysis: Limitations of Monofunctional Ligands

+ HCl

+

+

Hydrolysis at low pH (monofunctional bond broken)

SiC

CC

CC

CC

CH3

CH3

H3C

Cl

OHSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

OSi

O

O

O

SiC

CC

CC

CC

CH3

CH3

H3C

HO

OHSi

O

O

O

Monofunctional = Attached to silica at one point Highly susceptible to low pH hydrolysis (column bleed)

Synthesis (monofunctional bond created)

Single siloxane bond

C8 Monochlorosilane Ligand


Making a Bonded Phase Material: dC18 Difunctional Synthesis

•+

•+ •HCl

•C8 Dichlorosilane Ligand

OHOH

OHSi

SiSi

O

O

O

O

O O

O

•Cl

•Si

•Cl

•CH3

•CH3

O

S i

O

OH

Si

Si Si

O

O

O

O

O O

O

•CH3

•CH3

•Difunctional = Attached to silica at two points •Less susceptible to low pH hydrolysis (column bleed)

•Synthesis

•Two siloxane bonds


Making a Bonded Phase Material: T3 Trifunctional Synthesis

•+

•+ •HCl

•C8 Trichlorosilane Ligand

OHOH

OHSi

SiSi

O

O

O

O

O O

O

•Cl

•Si

•Cl

•Cl

•CH3

O

Si

O

O

Si

Si Si

O

O

O

O

O O

O

•CH3

Trifunctional = Attached to silica at three points Much less susceptible to low pH hydrolysis (column bleed) Not quite as simple as this….

•Synthesis

•Three siloxane bonds


Effect of Ligand Phase Functionality on Synthesis

Trifunctional C18-Bondings

– 1-2 surface silanol group reacts with one silane ligand

– More resistant to hydrolysis at pH <2

– Reproducible, but more difficult reaction (polymeric side-

reaction)

– Higher silanol activity (on ligands)

– Capable of higher ligand density

(CH

2) 1

7C

H3

SiHO O

O

(CH

2) 1

7C

H3

Si

OO

(CH

2) 1

7C

H3

SiOH

OO

(CH

2) 1

7C

H3

SiHO O

O

(CH

2) 1

7C

H3

Si

OO


Low pH (0.1%TFA) Lifetime Study T3 Bonding - Atlantis T3 columns

Excellent stability during entire study with little-to-no RT loss

1000 injections over 2 weeks (35000 mL/14590 c.v. of mobile phase)

Injection 5

Injection 100

Injection 200 Injection 300 Injection 400

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Injection 500 Injection 600 Injection 700 Injection 800 Injection 900 Injection 1000

Compounds: 1. Adenine 2. Acetaminophen 3. Caffeine 4. 2-acetamidophenol 5. Acetanilide



0 5 10 15 20 25 30 35 40 45 50 55 60

Inertsil ODS-SP

Zorbax Eclipse Plus C18

Phenomenex Aqua C18

Kinetex EVO C18

Atlantis dC18

HALO RP Amide

Synergi Polar-RP

Synergi Hydro-RP

XBridge BEH Shield RP18

CORTECS Shield RP18

CORTECS T3

XSelect HSS T3

Atlantis T3

% Loss in Retention Factor (Methyl Paraben)

% Loss in Retention Factor for Methyl Paraben after Exposure to 0.5% TFA at 60°C

T3 Bonding Technology: Longer Column Lifetimes at Low pH


How Do T3 Columns Work?

T3 columns are designed to maximize the dominant reversed-

phase (van der Waals forces – hydrophobic attraction) retention

mechanism

– Retention maximized using 100% aqueous mobile phases

– Retention maximized by using reduced C18 coverage

o Polar analytes can “fit” between C18 ligands and interact with

surface silanols and alkyl chains

Secondary interactions due to residual silanols that are more

accessible due to reduced C18 coverage

– Cation-exchange interactions

– Hydrogen bonding interactions


How Does T3 Bonding Work for both Nonpolar and Polar molecules?

NonPolar

– Retention mechanism is classic reversed-phase, hydrophobic interaction with the C18 stationary phase

Polar

– Dominant retention mechanism is still reversed-phase

o Retention maximized using 100% aqueous mobile phases (enabled by having the wider pore diameter)

– Retention maximized by using reduced C18 coverage

o Polar analytes can “fit” between C18 ligands and interact with surface silanols and alkyl chains

– Secondary interactions due to residual silanols that are more accessible due to reduced C18 coverage

– Cation-exchange interactions

– Hydrogen bonding interactions


T3 Particle Technology

•Full aqueous compatibility and excellent peak shape • across the entire usable silica pH 2 – 8 range

Highest efficiency

•Greater Speed and Throughput

•Reduced

backpressure

High retention

Seamless Scalability

Compatible with UPLC operating

pressures

Exceptional loading capacity

•Superior basic

peak shape

•Low pH Stability

T3 bonding is a combination of a tri-functionally bonded,

intermediate ligand density ligand and a proprietary endcap

– C18 column chemistry designed to provide superior retention of small, water-soluble polar organic compounds


Atlantis T3 Columns: Superior Polar Compound Retention

Atlantis T3

Conditions Columns: 4.6 x 150 mm, 5 µm Mobile Phase: 10 mM NH

4 COOH, pH 3.0

Flow rate: 1.2 mL/min Injection volume: 7 µL Detection: UV@254 nm

Compounds 1. Thiourea 2. 5- Fluorocytosine

3. Adenine 4. Guanosine -5’- monophosphate 5. Thymine

Aqueous Separation of Polar Compounds for Atlantis T3 vs. Conventional High Coverage C18 Column

0 Minutes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Luna C18(2)

1 2

3 4

5

1 2

3 4 5



Atlantis T3 Columns:

Superior Retention and Peak Shape • Compounds: 1. Thiourea 2. 5-Fluorocytosine 3. Adenine 4. Guanosine-5'-monophosphate 5. Thymine

•0.00 •1.00 •2.00 •3.00 •4.00 •5.00 •6.00 •7.00 •8.00 •9.00

•1 •2 •3 •4

•5

•Atlantis T3

•1 •2 •3

•1 •2 •4 •3 •5

•GL Sciences Inertsil® ODS-SP

•1 •2 •3 •4

•5

•Shiseido Capcell PAK C18 AQ

•Thermo Hypurity Aquastar

•1 •2 •4

•3

•5

•Phenomenex Aqua C18

•4 •5



Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

1) uracil, 2) propranolol, 3) butylparaben, 4) naphthalene, 5) dipropylphthalate, 6) acenaphthene, 7) amitriptyline

1 2 3 4 5

6 7 Atlantis T3 Ami Ace

Phenomenex Aqua C18

Ami

•Zorbax StableBond-AQ Ami

Synergi™ Hydro-RP Ace

Ami - missing

Ace

Ace

pH 7 Peak Shape & Selectivity Test MeOH/20mM K2HPO4/KH2PO4, pH 7 (65/35)



Superior Peak Shape and Retention: Superior Polar Compound Loading

Mobile phase A: 0.1% formic acid in water

Mobile phase B: 0.1% formic acid in acetonitrile

Compounds

1. Atenolol 40 mg/mL in DMSO

2. Metoprolol 200 mg/mL in DMSO

3. Propranolol 200 mg/mL in DMSO

Atlantis® T3

5 µm, 4.6 x 100 mm

1

2

3 Flow rate: 1.0 mL/min

Gradient: Time Profile

(min) %A %B

0.0 100 0

1.0 100 0

2.0 82 18

6.0 28 72

8.0 28 72

8.1 0 100

10.0 0 100

Inj. Vol.: 15 µL (6.6 mg total mass load)

Atlantis® Prep T3 OBD™

5 µm, 19 x 100 mm

V0 = 1.5 min

Flow rate: 17.06 mL/min


(min) %A %B

0.0 100 0

2.0 100 0

3.0 82 18

7.0 28 72

9.0 28 72

9.1 0 100

11.0 0 100


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1.0

2.0

3.0

0.0

1.0

2.0

3.0

0.0

1.0

2.0

3.0

Atlantis® Prep T3 OBD™

5 µm, 30 x 100 mm

Flow rate: 42.53 mL/min


(min) %A %B

0.00 100 0

2.04 100 0

3.04 82 18

7.04 28 72

9.04 28 72

9.14 0 100

11.04 0 100


0 2 4 6 8 10 12



T3 Bonding Technology: High Strength Silica (HSS) Particles

What is an HSS Particle?

– ACQUITY UPLC and XSelect High Strength Silica (HSS)

particles are the only 100% fully porous silica particles that are

certified for use in applications up to 18000 psi/1240 bar

CORTECS T3 ACQUITY HSS T3

XSelect HSS T3

Atlantis T3

Intended Use High Efficiency Wide Retention Maximum Loading

Particle Type Solid-Core Silica Fully Porous High Strength

Silica

Fully Porous Silica

Maximum Rated

Pressure

18,000PSI (1240

bar)

18,000PSI (1240 bar) 6,000 PSI (400 bar)

Available Particle

Sizes

1.6 µm, 2.7 µm 1.8 µm, 2.5 µm, 3.5 µm,

5 µm

3 µm, 5 µm, 10 µm

Pore

Diameter/Volume

120 Å 100 Å 100 Å

Pore Volume 0.3 mL/g 0.7 mL/g 1.0 mL/g

Surface Area 100 m2/g 230 m2/g 330 m2/g


HSS T3 Technology Speed & Resolution – Without Compromise

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•2.1 x 150 mm, 5 µm •Rs (2,3) = 4.29

•1 •2

•2.50

•2.1 x 50 mm, 1.7 µm •Rs (2,3) = 4.28

•1

•2

•3

•8X Speed •3.4X Sensitivity •Same Resolution

•3

•2.1 x 100 mm, 1.7 µm •Rs (2,3) = 6.38

•1

•2

•3

HPLC

•4.5X Speed •2X Sensitivity •1.5X Resolution

•4.50

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•Absorb

ance a

t 270 n

m

•0.00

•0.26

•Absorb

ance a

t 270 n

m

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•Absorb

ance a

t 270 n

m

•0.00

UPLC UPLC

•Fastest & Most Sensitive Methods •Same Resolution

•Faster & More Sensitive Methods •Highest Resolution

Increase Speed

Increase Resolution


HSS T3 Bonding Technology: Comparative Chromatograms

•AU

•0.00

•0.05

•0.10

•0.15

•AU

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•A

U

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1. 1. Propyl Gallate 4. Butylated Hydroxyanisole 2. 2. 2,4,5-Trihydroxybutyrophenone 5. 2,6 di-tert-butyl-4-hydroxymethylphenol 3. 3. tert-Butylhydroquinone 6. Octyl Gallate

•1

•2

•3

•4

•5

•6 •Atlantis T3 •Average Peak [email protected]% = 0.075 •Peak Capacity = 100

•XSelect HSS T3 XP •Average Peak [email protected]% = 0.066 •Peak Capacity = 114

•CORTECS T3 •Average Peak [email protected]% = 0.058 •Peak Capacity = 129

•+14%

•+13%

•+29%

Increased peak capacity!



Catecholamines & Metabolites Transfer: Atlantis T3 to ACQUITY UPLC HSS T3

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•5

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Compounds: 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-methoxyphenylacetic acid (HVA)

•6

•ACQUITY UPLC HSS T3

•Atlantis T3



In Vitro Incubated Amitriptyline Hepatocyte Samples

ACQUITY UPLC BEH C18 1.7 µm, 2.1x100mm

ACQUITY UPLC HSS T3 1.8 µm, 2.1x100 mm

2x increased retention compared to

conventional C18!!

Amitriptyline + 16 Metabolites

Amitriptyline + 16 Metabolites



•Minutes •0.00 •0.50 •1.00 •1.50 •2.00 •2.50 •3.00 •3.50 •4.00 •4.50 •5.00

ACQUITY UPLC HSS T3 Columns: Highest Retentivity – Polar Compounds

•Waters ACQUITY UPLC HSS T3, 1.8 µm

•Thermo Hypersil Gold AQ, 1.9 µm

•Agilent ZORBAX Eclipse Plus C18, 1.8 µm

•Agilent ZORBAX SB-C18, 1.8 µm

•Thermo Hypersil Gold,1.9 µm

•kThy = 6.3

•kThy = 3.5

•kThy = 3.1

•kThy = 5.2

•kThy = 5.2

•Elution order: thiourea, 5-fluorocytosine, adenine, thymidine-5-monophosphate, thymine •10 mM NH4COOH pH 3.0, 0.20 mL/min •Comparative separations may not be representative in all applications.


T3 Bonding Technology: CORTECS T3

Solid core particle with

– Larger pore diameter 120Å

– Lower ligand density when compared to typical C18 columns

– Chemically stable at low pH and reproducible

– LC/MS compatible

– Balanced retention of both polar and non-polar compounds

under RPLC conditions

– Increased retention for polar compounds over typically bonded

solid-core C18 phases


Increased CORTECS T3 Efficiency

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Test Flow Rate (mLs / min)

•CORTECS UPLC T3 1.6 µm •ACQUITY UPLC HSS T3 1.8 µm

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Test Flow Rate (mLs / min)

•CORTECS T3 2.7 µm •XSelect HSS T3 2.5 µm

•57% Higher Efficiency •CORTECS UPLC T3 1.6 µm

compared to •ACQUITY UPLC HSS T3 1.8 µm

32% Higher Efficiency CORTECS T3 2.7 µm

compared to XSelect HSS T3 2.5 µm


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Flow Rate (mL/min)

CORTECS XSelect

More Efficiency per Unit Pressure Measured N/ΔP*

Faster flow rates for higher throughput

Lower backpressures for 100% aqueous mobile phases

Longer columns for added resolution

•*Comparison of particles of equivalent particle size


T3 Particle Efficiency: Atlantis T3 Versus CORTECS T3

1. 1. Propyl Gallate 2. 2. 2,4,5-Trihydroxybutyrophenone 3. 3. tert-Butylhydroquinone 4. 4. Butylated Hydroxyanisole 5. 5. 2,6 di-tert-butyl-4-hydroxymethylphenol 6. 6. Octyl Gallate

•CORTECS T3 •3.0 x 75 mm, 2.7 µm

•Atlantis T3 •4.6 x 150 mm, 5 µm



Summary: Reversed-Phase HPLC

Reversed-Phase HPLC for Polar Compounds

– Embedded polar groups were designed for peak shape, not polar

compound retention

– The sudden loss in retention observed under highly aqueous

conditions is due to pore dewetting not hydrophobic collapse

– A properly designed straight alkyl chain C18 column can provide the

perfect balance of polar and non-polar compound retention

T3 particle technology gives you:

– Superior retentivity for polar compounds

– Resistance to pore dewetting

– Ultra-low MS bleed

– Improved pH stability

– Improved pH 7 peak shape


Thank You!

Live Q&A Session

part i: polar compound retention...part i: polar compound retention tuesday, october 11 2016 – 3...

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