Mystery SOLVED HPLC Patent Pending
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
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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
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1.6e6
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4.15
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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
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Below pKa
At pKa
Hig
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H
PAGE 8 PAGE 16
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Gemini C18 low pH
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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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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
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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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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
Detection: UV @ 254nm
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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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
Flow Rate: 1.5mL/min
Detection: UV @ 210nm
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
Flow Rate: 1.5mL/min
Detection: UV @ 254nm
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
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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
Detection: UV @ 254nm
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.
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
0
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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
Detection: UV @ 254nm
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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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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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
Flow Rate: 0.6mL/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% Formic Acid, pH 2.7
10mM Ammonium Bicarbonate, pH 10.0
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.
0.1% Formic Acid, pH 2.7
10mM Ammonium Bicarbonate, pH 10.0
Over 50% increase in LC/MS/MS Response at pH10! Major improvement in Peak
Asymmetry at pH 10!
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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
Temperature: AmbientDetection: UV @ 254nm
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
Temperature: AmbientDetection: UV @ 254nm
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
Temperature: AmbientDetection: UV @ 254nm
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)
Flow Rate: 1.0 mL/minTemperature: Ambient
Detection: UV @ 254nmSample: 1. Sulfathiazole
2. Sulfamerazine�. Sulfamethoxazole
Rs = 4.0
Rs = 1.0
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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)
Flow Rate: 1 mL/minTemperature: Ambient
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
Column: Gemini 5µm C18
Dimension: 100 x �.0mmPart No.: 00D-44�5-Y0
Mobile Phase: 0.1% Formic Acid in Water / Acetonitrile (50:50)
Flow Rate: 0.5 mL/minTemperature: Ambient
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
Column: Gemini 5µm C18
Dimension: 150 x 4.6mmPart No.: 00F-44�5-E0
Mobile Phase: 10mM Ammonium Bicarbonate, pH 10.5 / Acetonitrile (50:50)
Flow Rate: 1 mL/minTemperature: 25°C
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
Flow Rate: 1 mL/minTemperature: 25°C
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)
Flow Rate: 1 mL/minTemperature: Ambient
Detection: UV @ 260nmSample: 1. Uracil
2. Cytosine�. Guanine4. Thymine5. Adenine
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
SecurityGuard™KJ0-4282
22
ORDERING INFORMATION
GEMINI®
SecurityGuard™ requires holder, Part No.: KJ0-4282
SecurityGuard™ requires holder, Part No.: KJ0-4282
SecurityGuard™ requires holder, Part No.: KJ0-4282
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
Phenomenex | Email: [email protected] | Web: www.phenomenex.com
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
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mail:
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email:
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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].