SOLVED - Separations

Mystery SOLVED

HPLC

Patent Pending

THE MYSTERY IS SOLVED

For years, chromatography companies have tried to solve the great mystery

of getting the performance of silica with the chemical inertness of a polymer.

Some have tried through the compromise of a hybrid particle, others by exten-

sively bonding and endcapping—resulting in only slight improvement of stability.

Finally, a logical solution is here…Gemini® — We challenge you to find a fault.

If Gemini does not provide at least an equivalent separation as compared to a competing column of similar phase and dimensions, send in your comparative data within 45 days and keep the Gemini column for FREE.

�

Method development Achieve higher sensitivity and more retention

Improve selectivity and resolution with pHMaximize your preparative loading

2

2

1

1

3

3

2

2

1

1

3

3

Mobile Phase: A: 1.0% TFA in water; B: 1.0% TFA in Acetonitrile Gradient: 10% B to 90% B in 7 min

Mobile Phase: A: 50mM ammonium bicarbonate, pH 10/Acetonitrile (90:10); B: 50mM Ammonium Bicarbonate, pH 10.0/Acetonitrile (10:90) Gradient: 0% B to 100% B in 7 min

Increase Load 9x

Total Load = 16mg

Total Load = 150mg

Diphenhydramine 4.1mg Oxybutynin Chloride 11.2mgTerfenadine 0.7mg

Diphenhydramine �8mg Oxybutynin Chloride 105mgTerfenadine 7mg

Column: Gemini 5µm C18Dimension: 150 x �.0mm

Mobile Phase: A: 0.1% Formic Acid in Water; B: 0.1% Formic Acid in Acetonitrile

High pH: A: 10mM NH4HCO�, pH 10.0 B: Acetonitrile

Gradient: 10% B to 90% B in 10 min, hold for 2 min; re-equilibrate for 4 min

Flow Rate: 0.6mL/min

2.64

6.17

2 4 6 8 10 12 14 min

0.0

5.0e5

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1.6e6

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0.0

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4.15

2 4 6 8 10 12 14 min

0.0

4.3e6

9.07

5.05

Acebutolol pH = 10

0.1% Formic Acid

Concentration Level:

0.05-200ng/mL

Injection Volume: 5 µLDetection: API �000 LC/MS/MS

Ion Source: ESI/PIMSample: 1. Cimetidine, log P 0.4

2. Amiloride, log P 0.09�. Trimethoprim, log P 0.914. Acebutolol, log P 1.71

Retain Polar Basics

pH10.0

pH1.5

EXPAND YOUR CHROMATOGRAPHY wITH pH STAbLE (1-12) GEMINI C18 AND C6-PHENYL

Hig

h p

HLo

w p

H

bas

es

Below pKa

At pKa

Effects of Mobile Phase pH on bases

Above pKa

Effects of Mobile Phase pH on Acids

Aci

ds

Above pKa

Below pKa

At pKa

Hig

h p

HLo

w p

H

PAGE 8 PAGE 16

PAGE 13 PAGE 12

2 4 6 8 10 min

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Gemini C18 low pH

2

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2 4 6 8 10 min

2 4 6 8 10 min

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4

64

6

5

Gemini C6 Phenyl low pH

2

DifferentPhase

MethanolAcetonitrile

AP

P ID

15896

AP

P ID

15895

Improved Separation!

C6-Phenyl

C18

Phenomenex | Email: [email protected] | Web: www.phenomenex.com

4

TwO-IN-ONE TECHNOLOGY ™

(TwIN™ TECHNOLOGY)

Combining the Best of Silica and Polymers

Silica Particle

Strengths

• Mechanical Strength

• Reproducible

• Highly Efficient

Weaknesses

• Limited pH Range

• Chemically Active

Polymer Particle

Strengths

• Extended pH Range

• Chemically Inert

Weaknesses

• Lower Efficiency

• Lower Mechanical Rigidity

• Lower Reproducibility

Performance and Chemical Inertness: two key traits that every chromatog-rapher wants from an HPLC column. Silica-based materials provide excellent performance, but often have a limited pH range. Polymer materials provide wide pH stability, but fall short in delivering good efficiency and resolution.

Pore

Pore

5

1References: Waters® XTerra® Hybrid Particle Technology and Phenomenex® Luna® Advanced Silica Columns for HPLC Applications: A Critical Evaluation and Comparison.

Phenomenex. Torrance, CA 2001

During the final stage of silica manufacturing a unique silica-organic layer is grafted to create a completely new composite particle. Since the in-ternal base silica is unaltered by this manufactur-ing process, the particle retains the mechanical strength and rigidity of silica providing excellent efficiency, while the silica-organic shell protects the particle from chemical attack.

Strengths

Extended pH Stability (1-12)

Efficiency of Silica

Mechanical Strength of Silica

100% Aqueous Stable

Weaknesses We challenge you to find one!

Gemini Twin™ TechnologyHybrid ParticleA mixture of organic and silica throughout the entire particle, resulting in a compromise be-tween a silica-based and poly-mer-based material.

Strengths

• Extended pH Stability (1-12)

• Higher Efficiency than Polymer

• Higher Mechanical Strength than Polymer

Weaknesses

• Reduced Efficiency vs. Silica1

• Reduced Resolution vs. Silica1

• Reduced Peak Capacity vs. Silica1

Material Characteristics:Packing Material

Particle Shape/Size (µm)

Pore Size (Å)

Surface Area (m2/g)

Carbon Load

End Capping

Gemini C18 Spherical �,5,10 110 �75 14% TMS

Gemini C6-Phenyl

Spherical �,5 110 �75 12% TMS

Pore

Pore


min0 1 2 3 4 5 6 7 8 9

Batch #1Batch #2Batch #3 Batch #1

Batch #2Batch #3

6

REPRODUCIbLE

PHASESBatch-to-BatchReproducibility for Gemini C18

Batch-to-BatchReproducibility for Gemini C6-Phenyl

Column: Gemini 5µm C6-Phenyl

Dimension: 150 x 4.6mmMobile Phase: 20mM Phosphate buffer, pH2.5/

Methanol (97:�)Flow Rate: 1.0 mL/min

Temperature: Ambient

Detection: UV @ 220nm

Sample: 1. Tartaric Acid2. Malic Acid�. Lactic Acid4. Acetic Acid5. Citric Acid6. Propionic Acid

Gemini® C6-Phenyl incorporates the same tightly controlled silica manufacturing procedures as our world renowned Gemini® C18 phase to deliver reproducible performance you can count on: column-to-column, batch-to-batch.

Even under low buffer conditions and at high pH, Gemini provides the stability and reproducibility needed for developing and transferring critical methods. With each Gemini phase and particle size, multiple batches are available for validating methods. In addition, Gemini is available and supported in over 80 countries worldwide.

Column: Gemini 5µm C18

Dimension: 150 x 4.6mmMobile Phase: 10mM Ammonium Bicarbonate, pH

10.5/Acetonitrile (50:50)Flow Rate: 1.0 mL/min

Temperature: Ambient

Detection: UV @ 2�0nm

Sample: 1. Pendolol2. Metoprolol�. Propanolol

0

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

2 4 6 8 10 Days

7

High pH 11.5, Isocratic

Gemini® C18

Waters® XTerra® MS C18

5.5 times increased column lifetime and still going!

Gemini® Fail Line

XTerra® Fail Line

*Efficiency and lifetime comparisons based on average of two columns each run in parallel.

Accelerated High pH, Gradient

The unique engineering of Gemini® pro-vides stability and increased column life-time unrivaled by others. Whether used under isocratic or gradient conditions, Gemini® is guaranteed to out-perform and outlast “hybrid” particles. This is clearly illustrated above.

Columns: Gemini® 5µm C18Waters® XTerra® 5µm MS C18

Dimension: 150 x 4.6mmMobile Phase: Acetonitrile/50mM Methylpyrrolidine

Buffer, pH 11.5 (50:50)Flow Rate: 1 mL/min

Temperature: AmbientDetection: UV @ 254nm

Sample Analyte: Diphenhydramine

Columns fail after 50% loss in efficiency.

Dimensions: 150 x 4.6 mmMobile Phase: A: 10mM Ammonium Bicarbonate,

pH 10.0B: 90:10 Acetonitrile/Buffer

Gradient: 0% to 100% B in 10 min; hold at 100% B for 7 min; re-equilibrate at 0% B for � min

Flow Rate: 1.0 mL/minTemperature: 50˚C

Detection: UV @ 254nmSample: 1. Amitriptyline

2. Prednisolone

High Efficiency

Gemini® C18

Hybrid Particle C18

Silica Based C18

Competitor V

Competitor Z

Long Lifetime in:• High pH• Gradient Conditions• Elevated Temperature

EXTENDED COLUMN

LIFETIME


2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

4

6

2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

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6

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6

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2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

2 4 6 8 10 min

4

6

8

Gemini C18 low pH

Gemini C18 low pH

IT’S AbOUT

METHOD DEVELOPMENT OPTIONS . . .In developing methods to separate your compounds of inter-est, one set of conditions with one column may not give the desired separation of all of your analytes. Gemini, with two dif-

ferent selectivities, C18 and C6-Phenyl, AND pH 1 to 12 stabil-ity, gives a variety of options to separate your compounds of interest.

Columns: Gemini 5µm C18 Gemini 5µm C6-Phenyl

Dimension: 150 x �.0mm

Mobile Phase: Low pH Mobile Phase: A: 0.1% Formic Acid in Water B: 0.1% Formic Acid in Acetonitrile

High pH Mobile Phase: A: 10mM Ammonium Bicarbonate, pH 10.0 B: Acetonitrile

Gradient: From 10 to 90% B in 10 minutes, hold for 2 min; re-equilibrate for 4 min

Flow Rate: 0.6mL/minSample: 1. Diphenhydramine MRM 256->167

2. Lidocaine MRM 2�5->86�. Acebutolol MRM ��7->1164. Trimethoprim MRM 291->2�05. Terfenadine MRM 472->4�66. Carbamazepine MRM 2�7->194

2

46

5 2

4 6

5

1

�

2

6

1

�

4

6

5

6

�

2

1

DifferentpH

DifferentPhase

Gemini C6-Phenyl low pH

Gemini C18 low pH

Improved Resolution

Baseline Resolution

Lidocaine

Trimethoprim

Terfenadine

Carbamazepine

2

Gemini C18 high pH

Complete Change in Selectivity

9

wITH GEMINI C6-PHENYL

THERE ARE EVEN MORE OPTIONS . . .Phenyl phases offer another group of selectivity options. p-p interactions between aromatic groups can influence retention order of aromatic compounds. Varying the organic

mobile phase can modulate such interactions: Acetonitrile re-duces aromatic interactions, while methanol increases such interactions. The use of either acetonitrile or methanol with

Gemini C6-Phenyl gives additional options to improve your separation.

AP

P ID

15896

Column: Gemini 5µm C6-Phenyl 110ÅDimension: 150 x 4.6mm

Part No.: 00F-4444-E0

Mobile Phase: 0.1% Formic Acid in Water/Organic as noted

Flow Rate: 1mL/minTemperature: Ambient

Detection: UV @ 254nm Sample: 1. Quercetin

2. Kaempferol�. Isorhamnetin

AP

P ID

15895

AP

P ID

15902

Column: Gemini 5µm C6-Phenyl 110ÅDimension: 150 x 4.6mm

Part No.: 00F-4444-E0

Mobile Phase: 20mM Potassium Phosphate, pH 2.5/ Organic as noted

Flow Rate: 1mL/minTemperature: Ambient

Detection: UV @ 220nmSample: 1. Saccharin

2. p-Hydroxybenzoic acid�. Sorbic Acid4. Dehydroacetic Acid5. Methylparaben

AP

P ID

15901

Different Selectivity!

Improved Separation!

Flavonoids Food Additives40% Acetonitrile

55% Methanol

25% Acetonitrile

35% Methanol


10

EXTENDED

pH 1-12 STAbILITY FOR ALTERNATE SELECTIVITY

pH 1.5(1.0% TFA in water)

pH 7.0(20mM phosphate buffer)

Gemini is the only silica-based column in the world to have an extended pH range of 1-12, while providing this level of perfor-mance and lifetime. Take your method development to an all-new level and conquer the demands of today’s separation needs. By

modifying the mobile phase pH, alternative selectivities can be achieved with ionizable compounds. Typically acidic compounds will have an increase in retention at a pH value below their pKa, while a basic compound will typically have longer retention when

the pH is above its pKa value. Shown below is an example of this behavior, and how by tailoring the pH one can modify selectivity.

AP

P ID

15458

pH 4.0 (20mM ammonium formate)

AP

P ID

15457

AP

P ID

15459

Poor resolution for basic compounds

11

100 TIMES INCREASE IN STAbILITY*

pH 10.0 (10mM ammonium bicarbonate buffer)

pH 12.0 (20mM triethylamine buffer)

*Compared to a traditional column with a pH 1-10 range.

Column: Gemini 5µm C18Dimension: 150 x 4.6mm

Part No.: 00F-44�5-E0Mobile Phase: Acetonitrile/Buffers at various pH’s

(see chromatograms), (50:50)Flow Rate: 1 mL/min

Temperature: 22°C


Sample: 1. Chlorpropamide (pKa 5.0)

2. Butylparaben

�. Lidocaine (pKa 7.9)

4. Triprolidine (pKa 6.5)

5. Dextromethorphan (pKa 9.2)A

PP

ID 15460

AP

P ID

15461

Good resolution for basic compounds


pH10.0 pH10.5

pH7.0pH2.5

Hig

h p

HLo

w p

H

bas

es

Below pKa

At pKa

Effects of Mobile Phase pH on bases

Above pKa

Effects of Mobile Phase pH on Acids

Aci

ds

Above pKa

Below pKa

At pKa

Hig

h p

HLo

w p

H

12

Dimension: 150 x 4.6mm



Temperature: 25°C

Sample: 1. Maleic Acid (pKa 1.8�, 6.07)2. Chlorpheniramine (pKa 4.0, 9.2)�. Triprolidine (pKa 6.5)4. Diphenhydramine (pKa 9.0)

Mobile Phase:20mM Potassium Phosphate, pH 2.5/Acetonitrile (75:25)

Cold ProductsGemini C18

pH SELECTIVITY IMPROVED PERFORMANCE FOR bASIC COMPOUNDS

AP

P ID

15010A

PP

ID 15011

Mobile Phase:20mM Ammonium Bicarbonate, pH 10.0/Acetonitrile (60:40)

Mobile Phase:20mM Phosphate Buffer, pH 7.0/Acetonitrile/Methanol (�0:�5:�5)

Tricyclic AntidepressantsGemini C6-Phenyl

AP

P ID

15860A

PP

ID 15885

Mobile Phase:10mM Ammonium Bicarbonate, pH 10.5/Acetonitrile/Methanol (�0:�5:�5)

The major tools for altering selectivity in Reversed Phase HPLC include stationary phase chemis-try, mobile phase composition (including pH) and temperature. As the majority of the pharmaceu-tically active compounds are ionizable and while uncharged species retain longer on Reversed Phase stationary phases, mobile phase is a powerful tool for manipulating analyte retention and selectivity.

Operating 2 pH units above (for bases) or below (for acids) pKawill:

• Improve peak shape

• Increase retention

For recommended buffer choices, please contact your Phenomenex Technical Consultant and request your FREE pH method development poster.

Dimension: 150 x 4.6mm



Temperature: 25°C

Sample: 1. Nortriptyline (pKa 9.7)2. Imipramine (pKa 9.5)�. Amitriptyline (pKa 9.4)4. Clomipramine (pKa 9.5)

pH1.5

pH10.0

pH2.0

pH10.0min0 0.5 1 1.5 2 2.5 3 3.5

1

2

min0 1 2 3 4 5

1

2

2

2

1

1

3

3

2

2

1

1

3

3

min0 0.5 1 1.5 2 2.5 3 3.5

1

2

min0 1 2 3 4 5

1

2

1�

Mobile Phase: A: 1.0% TFA in Water; B: 1.0% TFA in Acetonitrile Gradient: 10% B to 90% B in 7 min

Mobile Phase: A: 50mM Ammonium Bicarbonate, pH 10/Acetonitrile (90:10); B: 50mM Ammonium Bicarbonate, pH 10/Acetonitrile (10:90) Gradient: 0% B to 100% B in 7 min


Injection Volume: 420µL

Sample: 1. Propanolol2. Diphenydramine

Mobile Phase: A: 0.5% TFA in Water; B: 0.5% TFA in Acetonitrile Gradient: 5% B to 95% B in 4 min

Mobile Phase: A: 50mM Ammonium Bicarbonate, pH 10; B: Acetonitrile Gradient: 5% B to 95% B in 5 min

Injection Volume: 1.2mLSample: 1. Diphenhydramine

2. Oxybutynin Chloride�. Terfenadine

Improve Resolution Increase Load 9x

Total Load = 16mg

Total Load = 150mg

Diphenhydramine 4.1mg Oxybutynin Chloride 11.2mgTerfenadine 0.7mg

Diphenhydramine �8mg Oxybutynin Chloride 105mgTerfenadine 7mg

HIGHER LOADAbILITY

bASIC COMPOUNDS AT HIGH pHBefore increasing the size of your preparative column to in-crease sample loading, consider switching to a high pH pu-rification using Gemini for increased retention and loading of basic compounds. Most pH stable hybrid medias have signifi-

cantly less surface area1- limiting sample loading and reducing the benefit of performing separations at high pH. Gemini is an ideal media for high pH purifications with: • High surface area2 for extreme loadability

• Extended pH stability (1-12) for long lifetime • High Efficiency for sharper peaks which provides o Smaller fraction volumes reducing dry down times o Improved resolution

Column: Gemini 5µm C18Dimension: 50 x 21.2mmFlow Rate: �4 mL/minDetection: UV @ 254nm

Column: Luna 5µm C18 Axia Packed (pH 2)Gemini 5µm C18 Axia Packed (pH 10)

Dimension: 50 x 21.2mmFlow Rate: �0 mL/min

Improved Resolution will allow for higher load!

1XBridge™, 185 m2/g; XTerra® 175m2/g. Waters 2006-2007 Catalog, page 66. 2Gemini® �75m2/g, Phenomenex 2006 Catalog, page �77.


0

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2000

5 5.5 6 min

mAu

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Area Enlarged

14

HIGH-THROUGHPUT PREPARATIVE SEPARATIONS

INTRODUCING Axia™

Axia™ is an advanced column packing and hardware design that eliminates bed collapse as a source of failure in short prep columns. Using patent-pending Hydraulic Piston Compression technology, several fundamental problems faced daily by preparative chromatographers have been solved including the collapse and voiding of the column, variability in bed density and efficiency and peak asymmetry. The result is:

• Longer lifetime, guaranteed • Improved column-to-column reproducibility • Analytical-like efficiency

For more information and to view the technology in action, please visit www.AxiaPrep.com or request your free Axia brochure.

Gemini® 10µm C18 Axia™ Packed

Waters® XTerra® 5µm Prep MS C18 OBD™

Column: Gemini® 10µm C18 Axia™ PackedWaters® XTerra® 5µm Prep MS C18 OBD™

Dimension: 50 x 21.2mm (Gemini®)50 x 19mm (XTerra®)

Mobile Phase: A: (95:5) 50mM Ammonium Bicarbonate, pH 10.0 / AcetonitrileB: (5:95) 50mM Ammonium Bicarbonate, pH 10.0 / Acetonitrile

Gradient: Linear from 100% A to 100% B over 7 min, re-equilibrate for � min

Injection Volume: 500 μLFlow Rate: �0 mL/min (Gemini®)

24 mL/min (XTerra®)*Temperature: Ambient

Detection: UV @ 254nmSample: 1. Methacycline 16mg

2. Triprolidine 1.6mg�. Amitriptyline 5.25mg*Same linear velocity

Conditions same for both columns except where noted.

Expect even sharper peaks with Gemini 5µm Axia Packed!

15

Humanplasma blank

SPE ConditionsSample: 0.5mL Human Plasma + 0.5mL Water

+ 0.1mL std (2.0µg/mL in Water)

Sorbent: Strata-X �0mg/1mL tubesPart No.: 8B-S100-TAK

Condition: 1mL Methanol followed by 1mL WaterWash: 1mL Water, 1mL of 40% Methanol

Elution: 1mL Methanol, evaporated to dryness under gentle N2 flow

Reconstitute: with 100µL water

Benzodiazepines spikedin human plasma

2µg/mL benzodiazepinestandard

Sample:

1. Chlordiazepoxide 4. Clonazepam2. Flurazepam 5. Temazepam�. Oxazepam 6. Diazepam

2

1

�4 5 6

AP

P ID

15454, 15456

12

3

4 56

7

89

10

0 2 4 6 8 10 12 14 16 18 min

Clinical SolutionBenzodiazepines in Plasma

Toxicology Solution Corticosteriods in Plasma

HPLC ConditionsColumn: Gemini 5µm C18

Dimension: 150 x �.0mmPart No.: 00F-44�5-Y0

Mobile Phase: A: 0.1% Formic Acid in WaterB: 0.1% Formic Acid in Acetonitrile

Gradient: 80:20 (A/B) linear to 20:80 over 8 min, Hold for 1 min

Flow Rate: 0.8 mL/minTemperature: 22°C


HPLC ConditionsColumn: Gemini 5µm C18

Dimension: 150 x �.0mmPart No.: 00F-44�5-Y0

Mobile Phase: A: 0.1% Formic Acid in WaterB: 0.1% Formic Acid in 95%

Acetonitrile/WaterGradient: Hold at 40% B for 4 min then from

40% B to 75% B over 11 minutesFlow Rate: 0.6 mL/minDetection: UV @ 254nm

SPE ConditionsSample: 1mL spiked human serum

(diluted 1:1 with water)

Sorbent: Strata-X �0mg/1mL tubesPart No.: 8B-S100-TAK

Condition: 1mL Methanol followed by 1mL WaterWash: 1mL Water, 1mL of 40% Methanol

in waterElution: 1mL Methanol

Reconstitute: under Nitrogen stream; 100µL Acetonitrile/Water 40:60

COMPLETE

SOLUTIONSTN

-1026

Human plasma blank

500 ng/mL standards spiked in human plasma

500 ng/mL standards

Sample: 1. Triamcinolone 6. Triamcinolone Acetonide 2. Prednisolone 7. 11-a-Hydroxyprogesterone �. Cortisone 8. Cortisone Acetate 4. Betamethasone (IS) 9. 11-Ketoprogesterone 5. Corticosterone 10. Betamethasone 17-valerate


2.64

6.17

2 4 6 8 10 12 14 min

0.0

5.0e5

2 4 6 8 10 12 14 min

0.0

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1.6e6

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0.0

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4.15

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0.0

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9.07

5.05

2.64

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2 4 6 8 10 12 14 min

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1.6e6

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2 4 6 8 10 12 14 min

0.0

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0.0

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9.07

5.05

16

EXPLORE HIGH pH IN

POSITIVE ION MODE (PIM) ELECTROSPRAY

Column: Gemini 5µm C18Dimension: 150 x �.0mm

Mobile Phase: Low pH Mobile Phase: A: 0.1% Formic Acid in Water B: 0.1% Formic Acid in Acetonitrile

High pH Mobile Phase: A: 10mM Ammonium Bicarbonate, pH 10.0 B: Acetonitrile

Gradient: 10% B to 90% B in 10 min, hold for 2 min; re-equilibrate for 4 min


Amiloride

Acebutolol

10mM Ammonium Bicarbonate, pH 10.0

0.1% Formic Acid, pH 2.7

Cimetidine

Trimethoprim

Concentration Level:

0.05-200ng/mL

Injection Volume: 5µLDetection: API �000 LC/MS/MS

Ion Source: ESI/PIMSample: 1. Cimetidine, logP 0.4

2. Amiloride, logP 0.09�. Trimethoprim, logP 0.914. Acebutolol, logP 1.71

Retain Polar Basics

Contrary to common expectations, using high pH mobile phases does NOT suppress the ionization of basic compounds in ESI+; positive ions are formed abundantly, and analyte responses are comparable, or often better at high pH compared

to acidic mobile phases. The results show that the use of high pH mobile phases for the analysis of basic compounds offers a good alternative to the current practice of using low pH mobile phases in ESI+ LC/MS for:

• Better Retention of Polar Basic Compounds • Increased Sensitivity (S/N) • Sharper Peaks for Improved Quantitation

17

EXPLORE HIGH pH IN

POSITIVE ION MODE (PIM) ELECTROSPRAYIncreased SensitivityLC/MS/MS Response for Basic Compounds in Acidic and Basic Mobile Phases

Sharper Peaks to Improve Quantitation Peak Asymmetry of Basic Compounds in Acidic and Basic Mobile Phases

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

Res

po

nse

- LC

/MS

/MS

Lidocaine Carbam-azepine

Tetracaine Haloperidol Diltiazem Terfenadine Procainamide Amiloride DPHA Atropine Acebutolol Verapamil Cimetidine Trimethoprim

Compound NameLOQ (S/N = 10, fg on column)

Acidic pH Basic pH

Lidocaine Carbamazepine Tetracaine Haloperidol Diltiazem Terfenadine Procainamide Amiloride Diphenhydramine Atropine Acebutolol Verapamil Cimetidine Trimethoprim

>500 >1750>500 >125 >250 >500 >500 >1250 >250 >500 >250 >1250 >500 >1750

>50 >1250>65 >85 >250 >250 >250 >750 >100 >1250 >100 >85 >65 >500

At a signal to noise ratio of 10, the limit of quantitation (LOQ) is listed to the right in femtograms (fg). In most cases, the LOQ is lower, allowing for increased sensitivity.



0

1

2

3

4

5

6

7

Pea

k A

sym

met

ry -

LC

/MS

/MS

Lidocaine Carbam-azepine

Tetracaine Haloperidol Diltiazem Terfenadine Procainamide Amiloride DPHA Atropine Acebutolol Verapamil Cimetidine Trimethoprim

Performing basic compound analysis in high pH mobile phases by ESI+ LC/MS/MS can allow for higher sensitivity, good linearity, precision and accuracy when using the high performance Gemini 5µm C18 HPLC column.

For details on the improved limits of detection (LOD), quantitation (LOQ), precision and accuracy, please request TN-10�1, “LC/MS/MS Analysis of Basic Drugs with Positive Ion Electrospray (ESI+) using a High pH Stable RP C18 in High pH Mobile Phase” by contacting your Phenomenex Technical Consultant.



Over 50% increase in LC/MS/MS Response at pH10! Major improvement in Peak

Asymmetry at pH 10!


18

EXCEPTIONAL PERFORMANCEGemini® vs. XBridge™Tricyclic Antidepressants

Gemini® 5µm C18

AP

P ID

15851

XBridge™ 5µm MS C18

AP

P ID

15852

Gemini® vs. Zorbax® Extend-C18Analgesics

Gemini® 5µm C18

AP

P ID

15606

Extend-C18 5µm

AP

P ID

15607

Gemini® vs. ACE®

Tricyclic Antidepressants

Gemini® 5µm C18

AP

P ID

15595

ACE® 5µm C18

AP

P ID

15602

Dimension: 150 x 4.6mmMobile Phase: 20mM Phosphate Buffer, pH 7.0 /

Acetonitrile / Methanol (�0:�5:�5)Flow Rate: 1.5 mL/min


Sample: 1. Nortriptyline2. Imipramine�. Amitriptyline4. Clomipramine

Rs = 2.09

Rs = 1.40

N = 13715

N = 2540

Dimension: 150 x 4.6mmMobile Phase: 20mM Phosphate Buffer, pH 2.5 /

Acetonitrile (50:50)Flow Rate: 1.0 mL/min

Temperature: AmbientDetection: UV @ 2�0nm

Sample: 1. Ethylparaben2. Naproxen�. Diflunisal4. Indomethacin5. Ibuprofen

Dimension: 150 x 4.6mmMobile Phase: Acetonitrile / Methanol / 10mM

Ammonium Bicarbonate, pH 10.5 (25:45:�0)Flow Rate: 1.5 mL/min


Sample: 1. Nordoxepin2. Doxepin�. Nortriptyline4. Imipramine5. Amitriptyline6. Clomipramine7. Trimipramine

As = 1.19

As = 2.12

19

Gemini® vs. XTerra®

Tricyclic Antidepressants

Gemini® 5µm C18

AP

P ID

150��

XTerra® 5µm MS C18

AP

P ID

150�4

Gemini® vs. SunFire™Aromatic Metabolites

Gemini® 3µm C18

AP

P ID

15824

SunFire™ 3.5µm C18

AP

P ID

15826

Gemini® vs. Varian Pursuit®

Sulfa Drugs

Gemini® 5µm C6-Phenyl

AP

P ID

1590�

Pursuit® 5µm DiPhenyl

AP

P ID

15908

15908

0 5 10

12

3

15 min

mAU

0

20

40

60

N = 11549

N = 5925

Baseline Resolution!

Dimension: 150 x 4.6mmMobile Phase: 10mM Ammonium Bicarbonate,

pH 10.5 / Acetonitrile (25:75)Flow Rate: 1.5 mL/min


Sample: 1. Imipramine2. Nortriptyline�. Amitriptyline4. Clomipramine

Dimension: 150 x 4.6mmMobile Phase: A: 20mM Sodium Phosphate, pH �.2

B: MethanolGradient: A/B (75:25) to A/B (25:75) over 5 min

Flow Rate: 1.0 mL/minTemperature: Ambient

Detection: UV @ 225nmSample: 1. Mandelic Acid

2. Hippuric Acid�. 2-Methyl hippuric Acid4. �-Methyl hippuric Acid

Dimension: 150 x 4.6mmMobile Phase: 0.1% Formic Acid / Methanol (70:�0)


Detection: UV @ 254nmSample: 1. Sulfathiazole

2. Sulfamerazine�. Sulfamethoxazole

Rs = 4.0

Rs = 1.0


15903

0

15903

2

2

3

1

4 6 8 10 12 14 16 18 min

AP

P ID

1590�A

PP

ID 158�6

AP

P ID

150�1A

PP

ID 155��

20

APPLICATIONSVersatility of Gemini

Sulfa Drugs

Column: Gemini 5µm C6-PhenylDimensions: 150 x 4.6mm

Part No.: 00F-4444-E0

Mobile Phase: 0.1% Formic Acid in Water/Methanol (70:�0)

Flow Rate: 1 mL/minTemperature: Ambient

Detection: UV @ 254nmSample: 1. Sulfathiazole

2. Sulfamerazine�. Sulfamethoxazole

Green Tea

Column: Gemini �µm C18

Dimension: 100 x 4.6mmPart No.: 00D-44�9-E0

Mobile Phase: 10mM Ammonium Bicarbonate, pH 10.5 / Acetonitrile (50:50)


Detection: UV @ 210nmSample: 1. Epigallocatechin

2. Catechin�. Epicatechin4. Gallocatechin gallate5. Epicatechin gallate6. Catechin gallate

Beta Blockers

Column: Gemini 5µm C18Dimensions: 150 x 4.6mm

Part No.: 00F-44�5-E0

Mobile Phase: A: 20mM Ammonium Acetate, pH 7.5B: Acetonitrile

Gradient: 98:2 (A/B) linear to 80:20 over 15 min, Hold for � min

Flow Rate: 1 mL/minTemperature: 25°C

Detection: UV @ 27�nmSample: 1. Pindolol

2. Metoprolol�. Propranolol

Zearalone


Dimension: 100 x �.0mmPart No.: 00D-44�5-Y0

Mobile Phase: 0.1% Formic Acid in Water / Acetonitrile (50:50)


Ion Source: ESI/PIMDetection: API �000 LC/MS/MS

Concentration: 200 ng/mLInjection Volume: 0.5µL in methanol

Sample: 1. beta-Zearalenol2. alpha-Zearalenol�. Zearalenone

AP

P ID

15001A

PP

ID 14999

AP

P ID

15014

21

AP

P ID

15828

Estradiol - USP Method

Column: Gemini 5µm C18Dimension: 150 x 4.6mm

Part No.: 00F-44�5-E0

Mobile Phase: Water/Acetonitrile (45:55)Flow Rate: 1 mL/min

Temperature: 25°CDetection: UV @ 205nm

Sample: 1. Estrone2. Ethylparaben�. Estradiol

Local Anesthetics


Dimension: 150 x 4.6mmPart No.: 00F-44�5-E0

Mobile Phase: 10mM Ammonium Bicarbonate, pH 10.5 / Acetonitrile (50:50)


Detection: UV @ 210nmSample: 1. Procaine

2. Lidocaine�. Tetracaine

Caffeine & Metabolites

Column: Gemini 5µm C18Dimensions: 150 x 4.6mm

Part No.: 00F-44�5-E0

Mobile Phase: A: 20mM Ammonium Acetate, pH 7.5 B: Acetonitrile

Gradient: 98:2 (A/B) linear to 80:20 over 15 min, Hold for � min


Detection: UV @ 27�nmSample: 1. Xanthine

2. Theobromine�. Theophylline4. Caffeine

Nucleic Acid Bases

Column: Gemini �µm C18

Dimension: 100 x 4.6mmPart No.: 00D-44�9-E0

Mobile Phase: 20mM Ammonium Acetate, pH 4.5 / Acetonitrile (99:1)


Detection: UV @ 260nmSample: 1. Uracil

2. Cytosine�. Guanine4. Thymine5. Adenine


SecurityGuard™KJ0-4282

22

ORDERING INFORMATION

GEMINI®

SecurityGuard™ requires holder, Part No.: KJ0-4282




3µm Minibore and Narrow Bore Columns (mm) SecurityGuard™ Cartridges

20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 75 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0

C18 00M-44�9-B0 00A-44�9-B0 00B-44�9-B0 00D-44�9-B0 00F-44�9-B0 00B-44�9-Y0 00C-44�9-Y0 00D-44�9-Y0 00F-44�9-Y0 AJ0-7596C6-Phenyl 00M-444�-B0 00A-444�-B0 00B-444�-B0 00D-444�-B0 00F-444�-B0 00B-444�-Y0 Inquire 00D-444�-Y0 00F-444�-Y0 AJ0-7914

for ID: 2.0-�.0mm

3µm Analytical Columns (mm) SecurityGuard™ Cartridges

20 x 4.0 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0

C18 00M-44�9-D0 00A-44�9-E0 00B-44�9-E0 00D-44�9-E0 00F-44�9-E0 00G-44�9-E0 AJ0-7597C6-Phenyl 00M-444�-D0 00A-444�-E0 00B-444�-E0 00D-444�-E0 00F-444�-E0 00G-444�-E0 AJ0-7915

for ID: �.2-8.0mm

5µm Minibore and Narrow Bore Columns (mm) SecurityGuard™ Cartridges

30 x 2.0 50 x 2.0 150 x 2.0 250 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0

C18 00A-44�5-B0 00B-44�5-B0 00F-44�5-B0 00G-44�5-B0 00B-44�5-Y0 00D-44�5-Y0 00F-44�5-Y0 00G-44�5-Y0 AJ0-7596C6-Phenyl 00A-4444-B0 00B-4444-B0 00F-4444-B0 00G-4444-B0 00B-4444-Y0 Inquire 00F-4444-Y0 00G-4444-Y0 AJ0-7914

for ID: 2.0-�.0mm

5µm Analytical Columns (mm) SecurityGuard™ Cartridges

30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0

C18 00A-44�5-E0 00B-44�5-E0 00D-44�5-E0 00F-44�5-E0 00G-44�5-E0 AJ0-7597C6-Phenyl 00A-4444-E0 00B-4444-E0 00D-4444-E0 00F-4444-E0 00G-4444-E0 AJ0-7915

for ID: �.2-8.0mm

/10pk

/10pk

/10pk

/10pk

2�

XTerra® is a registered trademark of Waters® Corp. SunFire, XBridge and OBD are trademarks of Waters Corp. Pursuit® is a registered trademark of Varian. Zorbax® is a registered trademark of Agilent Technologies. ACE® is a registered trademark of Advanced Chromatography Technologies (ACT). strata™X, Twin™, Two-in-One Technology™, Axia™ and SecurityGuard™ are trademarks of Phenomenex®. Luna® and Gemini® are registered trademarks of Phenomenex, Inc. in the USA and other countries. Phenomenex is not associated in any way with Waters, Varian, Agilent Technologies, or ACT. Data on previous pages may not be representative of all applications. © 2006 Phenomenex Inc. All rights reserved.

SecurityGuard™ requires holder, Part No.: KJ0-4282; Semi-prep SecurityGuard™ requires holder, Part No.: AJ0-7220; PREP SecurityGuard™ requires holder, Part No.: AJ0-78�8

Semi-prep SecurityGuard™ requires holder, Part No.: AJ0-7220; PREP SecurityGuard™ requires holder, Part No.: AJ0-78�8

PREP SecurityGuard™ requires holder, Part No.: AJ0-78�8

10µm Semi-Prep and Preparative Columns (mm) SecurityGuard™ Cartridges PREP In-line Filters

C18250 x 4.6 250 x 10 250 x 21.2 250 x 30 250 x 50 4 x 3.0 10 x 10 15 x 21.2 2.0µm x 21.2mm Dia

00G-44�6-E0 00G-44�6-N0 00G-44�6-P0 00G-44�6-U0 00G-44�6-V0 AJ0-7597 AJ0-7598 AJ0-7846 AF0-7866for ID: �.2-8.0mm for ID: 9-16mm for ID: 17-�0mm

5µm Semi-Prep and Preparative Columns (mm) SecurityGuard™ Cartridges PREP In-line Filters

50 x 10 150 x 10 250 x 10 150 x 21.2 250 x 21.2 250 x 30 10 x 10 15 x 21.2 2.0µm x 21.2mm Dia

C18 00B-44�5-N0 00F-44�5-N0 00G-44�5-N0 00F-44�5-P0 00G-44�5-P0 00G-44�5-U0 AJ0-7598 AJ0-7846 AF0-7866C6-Phenyl Inquire Inquire 00G-4444-N0 00F-4444-P0 00G-4444-P0 00G-4444-U0 AJ0-7�14 AJ0-7541 AF0-7866

for ID: 9-16mm for ID: 17-�0mm

5µm Axia™ Packed High-Throughput Columns (mm) SecurityGuard™ Cartridges PREP In-line Filters

50 x 21.2 100 x 21.2 50 x 30 75 x 30 100 x 30 15 x 21.2 2.0µm x 21.2mm Dia

C18 00B-44�5-P0-AX 00D-44�5-P0-AX 00B-44�5-U0-AX 00C-44�5-U0-AX 00D-44�5-U0-AX AJ0-7846 AF0-7866C6-Phenyl 00B-4444-P0-AX 00D-4444-P0-AX 00B-4444-U0-AX 00C-4444-U0-AX 00D-4444-U0-AX AJ0-7841 AF0-7866

for ID: 17-�0mm

ORDERING INFORMATION

PREP SecurityGuard™

/ea /ea

/10pk /�pk /ea /ea

/�pk /ea /ea


If Gemini does not provide at least and equivalent separation as compared to a competing column of similar phase and dimensions, send in your com-parative data within 45 days and keep the Gemini column for FREE.

SOLVED

4�96

_IUSA411 Madrid Ave.Torrance, CA90501-1430USA

(310) 212-0555(310) [email protected]

mail:

tel.:fax:

email:

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(800) 541-HPLC(310) [email protected]

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01 30 09 21 1001 30 09 21 [email protected]

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[email protected]

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01 247 5405+44 [email protected]

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[email protected]

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[email protected]

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or e-mail: [email protected].

SOLVED - Separations

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