Advancements in Separation Science Using “Silica Hydride” Column Technology to Break Limits in Traditional HPLC
Presented by: William Amoyal, Disruptive Technologies
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Abstract of today’s Presentation
Review Current HPLC Barriers & Limits Discuss Root Causes of these Limits Introduce You to Silica Hydride HPLC Columns What they do for you and how do you gain as a chromatographer Real Life Applications
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Common Benefits and Features of HPLC
Hydrophobic Compounds retain very well when using Reverse Phase Very reproducible Well known and easy to predict and develop methods Retain bases at high pH, Acids at low pH, or Neutral Compounds Rugged Methodologies is possible Can easily validate and automate HPLC Scale from microbore to preparative separations Works with Normal Phase or Reverse Phase Most common & successful analytical technique for quantifying analytes
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Silica Structure The backbone for over 250 brands of popular HPLC columns since the beginning of HPLC
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Silica Structure The backbone for over 250 brands of popular HPLC columns
Even today. Even your favorite columns
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Why is silica the preferred backbone of HPLC columns?
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Why is silica the preferred backbone of HPLC columns? Excellent Physical Properties!
Well suited for high pressure & constant flow of liquids
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Why is silica the preferred backbone of HPLC columns? Excellent Physical Properties! Easily modified by bonded phases
Allows for different selectivity
Mechanical strength
Allows for well packed beds-Efficiency
Rigidity
Low back pressure-stable beds
Porous, High Surface Area
Allows for small column length
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Making HPLC Grade Silica To gain some of these features, silica is “fired” or baked and is hydrated during manufacturing for HPLC to produce surface silanols and form rigidity to product features needed for HPLC.
May not be well known
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How does this surface structure impact the performance of the columns? Important Property of Silca Impacts Performance positively & negatively.
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How does this surface structure impact the performance of the columns?
Simplified Illustration for Presentation Purposes Only
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HPLC Grade Silica Attracts and holds water and will even adsorb water from your mobile phase until it has reached it’s maximum permanent hydration shell Silica is used as a dessicant for non chromatographic purposes
Hydration Shell (Adsorbed Water)
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Some separation mechanisms of silica based HPLC Columns Hydrophobic Interaction
Bonded Phase Required
Silanophilic Effects
Silanols
Partitioning
Hydration Shell/Organic Layer
Adsorption
Silica Chemistry
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Silica is not perfect. Limiting Properties! Unstable at high pH
Upper limit is typically pH 9
Surface Acidity
Problems for basic compounds
Silanols-Surface Chemistry Hydration Shell becomes virtually permanent Trapped Metals
Complex with Chelating solutes. Tailing
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Common Barriers/Limits of HPLC Summary of what we already know.
Hydrophilic Compounds do not retain in RP Loss of RT of very hydrophobic compounds using 100% Water with C18 Need to dry and control moisture in your solvents in NP Chrom Hysteresis when changing chromatographic modes on the same column Long Run Times Long Equilibration times between gradients Low pH instability Short column life when using some MP additives & some analytes Polar & Non Polar Solvent Compatibility Cannot retain bases at low pH or acids at high pH Low MS sensitivity with MP needed for retention in RP or NP On-Column Degradation of analytes due to silanols or surface water layer Bleed of end capping and bonded phases
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What are some of the root causes of the Features & Limits of silica based columns?
Why has it not advanced or improved on the limits in the last 30 years?
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HPLC Grade Silica The hydration shell is somewhat immiscible with organic solvents needed for HPLC and a bi-layer of liquids is created. The hydration shell is changeable and difficult to control between runs with changing mobile phases and or additives. Mobile Phase Short & Simple Answer Silanols are required for Bonding & Traditional HPLC
Hydration Shell (Adsorbed Water)
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A Recent Advancement in HPLC! Silica Hydride Based HPLC Columns
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What is “Silica Hydride”? Silica Hydride is a highly purified silica particle that has been manufactured to have a surface that is populated with silica hydride instead of silanols.
All physical features of HPLC silica are the same for silica hydride but the surface chemistry and resulting capabilities are different.
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Ordinary Silica O O Si O O Si O O Si
O
Silica Hydride O
OH OH OH
O Si O O Si O O Si
O O O O
O
Same basic structure with different surface chemistries
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Si O Si O Si
O
H H H
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How does this structural difference impact the performance of the columns? O O Si O O Si O O Si
O
O OH OH OH
O Si O O Si O O Si
O O O O
O
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Si O Si O Si
O
H H H
How Does this structural difference impact the performance of the columns?
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We now need to challenge our bias about HPLC column limits to benefit from this advanced and completely different column technology. Enjoy the benefits of silica hydride without limits that exist with hybride silica and polymeric columns.
What about bonding? Don’t you need silanols?
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Using hydrosilation on silica hydride, direct silicon carbon bonds (with 2 points of attachment) are produced when derivatizing the hydride surface. Without using Organo-Silation
Pesek Method of Bonding with Alkynes & Alkenes
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Direct Silicon-Carbon Bonds
Silica Structure & Hydride Surface remain after bonding
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What is “Silica Hydride”? For the purposes of literature search and to clear up any possible confusion using the internet or industry journals, silica hydride HPLC stationay phases are also commonly known as Cogent TYPE=C Silica which is a trademark of MicroSolv.
The term silica hydride was coined by Professor Pesek and used often in the literature interchangeably with TYPE-C Silica
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What do we gain from silica hydride HPLC columns? Think in terms of 3 modes of HPLC:
Normal Phase Reverse Phase Aqueous Normal Phase
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What do we gain from silica hydride HPLC columns?
Normal Phase
Easier to do More Reproducible
Prep Chromatography Water Free
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What do we gain from silica hydride HPLC columns?
Reverse Phase Limits are Broken
100% Water Mobile Phase pH stability More retentive for hydrophobic compounds Resistant to most additives like PIC reagents No bleed of bonded phases or end capping No on-column degradation of analytes due to acidity Fast equilibration even when using ballistic gradients Excellent for LCMS
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What do we gain from silica hydride HPLC columns?
Aqueous Normal Phase (ANP)
Normal Phase with Reverse Phase Solvents Retain polar compounds with precision No hysteresis when changing from RP to ANP Retain bases at low pH Retain acids at higher pH Excellent for LCMS Extremely fast equilibration between runs Very precise from run to run, day to day.
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Using Silica Hydride stationary phases breaks the limits and barriers of traditional HPLC which results in easier and more productive HPLC labs
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How do we use the Silica Hydride Phases? Normal Phase: Standard method development with standard MP to begin with followed by different methods to optimize method objectives by taking advantage of silica hydride column capabilities.
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How do we use the Silica Hydride Phases? Reverse Phase or ANP? Many compounds are no longer in the domain of Reverse Phase HPLC
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How do we decide between?
Rule of Thumb or Quick Start Method to determine best mode for unknown analytes. 1. Gradient 100% A to 40% B
2. Equilibrate for one column volume 3. Gradient from 95% B to 40% A with acid or base added
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More about ANP Cyano or Phenyl Columns can do it.
Precision and Column Life Times Questionable
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More about ANP What is it? Normal Phase Adsorption Ion Interaction (Not Ion Exchange) Use RP Solvents Not HILIC
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Real Life Applications of using the silica hydride columns and how you can benefit from the new technology in RP, ANP & NP
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Real Life Applications of using the silica hydride columns and how you can benefit from the new technology Nucleotide Bases
Isobaric Compounds for LCMS
Glyphosate Highly polar compound
Organic Acids
Melamine and Cyanuric Acid
Cytochrome-C
Amino Acids
Guanidine
ATP from AMP
Citric Acid from IsoCitric Acid
Succinic Acid
Leucine from Iso-Leucine
Generic Pharmaceuticals
Biogenic Amines
Metabolites from human urine
Purine Bases & Nucleosides
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Application-Reverse Phase Furazolidone: API & Related Compounds Generic Pharmaceutical
Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™ 4um, 100 Å 4.6mm i.d. x 150mm 2% Acetonitrile, 88% DI Water w/ 10ml/L acetic acid 2.0 mL/min. Isocratic
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Application-Reverse Phase Guanfacine HCl & Degradants on a C8 mAU
Guanfacine HCL
600
Column: Dimensions: Mobile phase: Flow rate:
500
Degradation Product 1
Cogent Bidentate C8™, 4um, 100 Å 4.6mm i.d. x 150mm 30% Acetonitrile, 70% DI Water w/conc/Phos Acid, SDS 1.5 mL/min. Isocratic 7 minute separation
400
300
Degradation Product 2
200
100
0
0
1
2
3
4
5
6
7
8
9
min
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Application-Reverse Phase Peptides: 1. Gly –Tyr; 2. Val – Tyr – Val; 3. Met-enkephalin 4. Leu-enkephalin; 5. Angiotensin II
Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4um, 100 Å 4.6mm i.d. x 75mm 25% acetonitrile/75% DI water + 0.1% formic acid 1.0 mL/min. Isocratic 5 minute separation
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Application-Reverse Phase Peptides: 1. Gly –Tyr; 2. Val – Tyr – Val; 3. Met-enkephalin 4. Angiotensin II 5. Leu-enkephalin; Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C8™, 5um, 300 Å 4.6mm i.d. x 75mm A: DI Water + 0.1% TFA B: Acetonitrile + 0.1% TFA 1.0 mL/min. Fast 20 minute Gradient
Gradient
Time
%B
0.0
9
5.0
21
20.0
27
21.0
9
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Application-Reverse Phase Cytochrome c: Horse & Bovine on a C8-300 Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C8™, 5um, 300 Å 4.6mm i.d. x 75mm A: DI water + 0.1% TFA B: Acn + 0.1% TFA 0.5 mL/min. Fast RP Gradient 5.0min Post Time
1.
Cytochrome c from Horse Heart
2.
Cytochrome c from Bovine Heart
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Application-Reverse Phase Sulfonamide Retention on a C18
Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4um, 100 Å 4.6mm i.d. x 75mm A: DI water + 0.1% formic acid B: Acetonitrile 0.4 mL/min. Fast RP gradient
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Application-Reverse Phase Dipyridamole:Tablet Formulation From Excipient on a C18
Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4um, 100 Å 4.6mm i.d. x 75mm A: DI water + 0.1% Phos acid B: 80:20 Can:0.1% PhosA 1.5 mL/min. Fast RP Gradient
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Application-Reverse Phase Diphenhydramine HCL on a Diamond Hydride
Column: Dimensions: Mobile phase: Flow rate:
Cogent Diamond Hydride™, 4um, 100 Å 4.6mm i.d. x 150mm A: DI water + 10mM pyridine B: Acn 10mM pyridine 0.4 mL/min. Fast RP Isocratic @ 25% B
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Application-Reverse Phase 100% Aqueous Mobile Phase on a C18 Column
Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4 mm, 100 Å 4.6mm i.d. x 75mm 100% 0.05% v/v H3PO4 1.0 mL/min. Isocratic
1- OXALIC ACID 2- FORMIC ACID 3- ACETIC ACID 4- SODIUM AZIDE 5- URACIL 6- FUMARIC ACID 7- PROPIONIC ACID
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Application- ANP Melamine & Cyanuric Acid on a Diamond Hydride Column
Column: Dimensions: Mobile phase:
Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 2.1mm i.d. x 150mm A: DI Water 0.1% Acetic Acid B: Acetonitrile + 0.1% Acetic Acid 0.4 mL/min. Fast 15minute Inverse Gradient
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Application- ANP Xanthine, Uric Acid & Hypoxanthine on a Diamond Hydride Column Column: Dimensions: Mobile phase: Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 2.1mm i.d. x 150mm A: DI Water 0.1% Formic Acid B: Acetonitrile + 0.1% Formic Acid 0.4 mL/min. Fast 12minute Inverse Gradient
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Application- ANP Monitoring Patient Levels of Methotrexate on a C18 Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4 mm, 100 Å 2.1mm i.d. x 150mm A: DI Water 0.5% Formic Acid B: Acetonitrile 0.4 mL/min. Fast 12minute Inverse Gradient
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Application- ANP Monitoring Patient Levels of Catecholamines on a C18 Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™, 4 mm, 100 Å 2.1mm i.d. x 75mm 90:10 Acetonitrile/DI Water w/ 0.5% FA 0.5 mL/min. Fast isocratic runs 200ng of each sample in 1ml of DI Water
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Application- ANP Choline from Acetylcholine on a UDC Cholesterol Column: Dimensions: Mobile phase: Flow rate:
Cogent UDC-Cholesterol™, 4 mm, 100 Å 4.6mm i.d. x 75mm 90:10 Acetonitrile/DI Water w/ 0.5% FA 0.5 mL/min. Fast isocratic runs
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Application- ANP Amino Acids, underivatized on a Diamond Hydride Column Column: Dimensions: Mobile phase: Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 4.6mm i.d. x 75mm A: DI Water + 0.1% Formic Acid B: Acetonitrile + 0.5% Formic Acid 0.4 mL/min. Fast Inverse Gradient
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Application- ANP Citric Acid from iso-Citric Acid on a Diamond Hydride Column Column: Dimensions: Mobile phase:
Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 4.6mm i.d. x 75mm A: DI water + 5 mM ammonium acetate; B: 90% acetonitrile/10% DI water/10 mM ammonium acetate C: 90% acetonitrile/10% DI water, D: 50% DI water/50% methanol/0.1% formic acid (wash)
0.4 mL/min. Fast 14 min Inverse Gradient
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Application- ANP Amino Acids on a Diamond Hydride Column Column: Dimensions: Mobile phase:
Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 4.6mm i.d. x 75mm A: DI water + 5 mM ammonium acetate; B: 90% acetonitrile/10% DI water/10 mM ammonium acetate C: 90% acetonitrile/10% DI water, D: 50% DI water/50% methanol/0.1% formic acid (wash)
0.4 mL/min. Fast 14 min Inverse Gradient
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Application- ANP Organic Acids on a Diamond Hydride Column Column: Dimensions: Mobile phase:
Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 4.6mm i.d. x 75mm A: DI water + 5 mM ammonium acetate; B: 90% acetonitrile/10% DI water/10 mM ammonium acetate C: 90% acetonitrile/10% DI water, D: 50% DI water/50% methanol/0.1% formic acid (wash)
0.4 mL/min. Fast 14 min Inverse Gradient
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Application- ANP Carbohydrates on a Diamond Hydride Column Column: Dimensions: Mobile phase:
Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 4.6mm i.d. x 75mm A: DI water + 5 mM ammonium acetate; B: 90% acetonitrile/10% DI water/10 mM ammonium acetate C: 90% acetonitrile/10% DI water, D: 50% DI water/50% methanol/0.1% formic acid (wash)
0.4 mL/min. Fast 14 min Inverse Gradient
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Application- ANP Glyphosate, un-derivatized on a Diamond Hydride Column Column: Dimensions: Mobile phase Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 2.1mm i.d. x 150mm A: DI water + 5 mM ammonium acetate; B: 90% ACN/10% DI water/10 mM amm acetate 0.5 mL/min. Fast Inverse Gradient
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Application- ANP ATP from AMP on a Diamond Hydride Column Column: Dimensions: Mobile phase Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 2.1mm i.d. x 100mm A: DI water + 0.1% ammonium acetate; B: 90% ACN/10% DI water/0.1% amm acetate 0.3 mL/min. Fast Inverse Gradient
1 = adenosine-3’,5’-cyclic monophosphate; 2 = adenosine 5’-monophosphate
3 = adenosine 5’-triphosphate.
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Application- ANP Mepiquat from Chlormequat on a Diamond Hydride Column Column: Dimensions: Mobile phase Flow rate:
Cogent Diamond Hydride™, 4 mm, 100 Å 2.1mm i.d. x 100mm A: DI water + 20mM ammonium acetate; pH 3 B: Acetonitrile 0.5 mL/min. Fast isocratic run
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Application-ANP Organic Acids on a Diamond Hydride Column Column: Dimensions: Mobile phase: Flow rate:
Cogent Diamond Hydride™ HPLC Column, 4 mm, 100 Å 2.1mm i.d. x 150mm A: DI Water 0.1% Am Formate B:90% ACN/10% DI Water/0.1%Am Formate 0.4 mL/min. Fast 8minute Inverse Gradient
1- MALEIC ACID 2- trans ACONITIC ACID 3- cis ACONITIC ACID 4- IMPURITY 5- FUMRAIC ACID 6- CITRIC ACID 7- OXALOACETIC ACID
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Application-Normal Phase Pro Drugs in NP on a C18 Column Column: Dimensions: Mobile phase: Flow rate:
Cogent Bidentate C18™ HPLC Column, 4 mm, 100 Å 4.6mm i.d. x 75mm 95:5 Hexane/Ethyl Acetate 1.0 mL/min. Isocratic
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Review of Previous Barriers/Limits of HPLC Summary of what we already knew.
Hydrophilic Compounds do retain in RP No Loss of RT of very hydrophobic compounds using 100% Water with C18 No Need to dry and control moisture in your solvents in NP Chrom No Hysteresis when changing chromatographic modes on the same column Shorter Run Times then before Very Short Equilibration times between gradients Low pH Stability Long column life even when using some MP additives & some analytes Polar & Non Polar Solvent Compatibility Retain bases at low pH & acids at high pH Increased MS sensitivity Minimal On-Column Degradation of analytes due to silanols or surface water layer No Bleed of end capping and bonded phases
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Change your bias about HPLC Limits
The possibilities for new methods are limitless Questions?
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About MicroSolv
MicroSolv started in 1992 Formed to work with technology transfer departments at universities Role MicroSolv plays in product development
Tech Transfer Product Development and Marketing R&D Production Design QC Marketing and Sales Distribution of Wide Array of Products Education and Technical Support Product Line Extension
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About Disruptive Technologies
Disruptive Technologies started in 2005 Distribute innovative products including instruments and consumable
LDTD ionsource for MS to increase throughput 10x to 100x without the need for separation CaptiveSpray ion source for MS to increase sensitivity by 100x vs ESI Automated sample preparation systems (PLE, Power-Prep, SPE, SPME …) High throughput separation systems such as 24 and 96 capillary electrophoresis instruments Dissolution testing systems (bath, media preparation …) Rapid Microbiology instrument and methods Automated extraction system for DNA, RNA and proteins Control of PCR fragments and quantitation at high throughput High quality HPLC columns, vials, filters …
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Thank for your attention Contact details Disruptive Technologies 3 allée des Camélias 94440 Villecresnes, France William Amoyal Sales and Marketing Director Phone : +33 6 98 64 98 81 Email
[email protected] Web www.disruptechno.com
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