Choosing Columns and Conditions for the Best Peak Shape
|
|
- Corey Day
- 5 years ago
- Views:
Transcription
1 Choosing Columns and Conditions for the Best Peak Shape
2 What is Good Peak Shape and Why is it Important? Good peak shape can be defined as a symmetrical or gaussian peak and poor peak shape can include both peak fronting and tailing. Good peak shape can be defined by. Tailing factor of.0 High efficiency Narrow peak width Good peak shape is important for. Improved resolution (Rs) More accurate quantitation Longer usable column lifetime (based on system suitability criteria) Page
3 Measures USP Tailing Factor at 5% of peak height* Asymmetry at 0% of peak height Skew rd moment measure column testing* Indicators Efficiency plates* Peak Width peak width at ½ height* * Available in ChemStation reports Page
4 USP Tailing Factor Efficiency peak front peak tail h N = 5.54 t R W ½ h = f W h peak maximum T = W / f Asymmetry Peak Width at ½ Height peak front peak tail h h W h / h/ W 0. = a = b 0. h W t peak maximum T f = b /a Page 4
5 Column packing factors Silica type Bonded phase and endcapping Mobile phase factors Sample factors Page 5
6 Silica type/acidity Fully hydroxylated silica and silica metal content Silica silanol ionization* Hydrogen bonding with silica silanols* Column bonding Endcapping Type of bonded phase Packing Pore Size/Structure * Controlled by mobile phase Page 6
7 H H H H H Si Si Si Si Free Silanols Geminol Silanols Associated Silanols decreasing acidity M + M + Surface Metal H Si Internal Metal (activated silanol) (most acidic) Page 7
8 Mobile Phase: 5% -Propanol in Heptane Flow Rate:.0 ml/min. Temperature: 5 C Standard Silica High Purity, Low Acidity ZRBAX Rx-SIL CH -H NH CH CH NH CH CH CH -H Improve peak shape for basic compounds with high purity, fully hydroxylated silica such as Rx-SIL Page 8
9 ZRBAX StableBond with Rx-SIL Improves Peak Shape Mobile Phase: 75% 50 mm KH P 4, ph 4.4 : 5% ACN Flow Rate:.5 ml/min Silica Type More Acidic Column: DS, 4.6 x 50 mm, 5 mm Plates: 9 USP T f (5%):.90 Silica Type High Purity, Rx-Sil Column: SB-C8, 4.6 x 50 mm, 5 mm Plates: 67 USP T f (5%):.09 Propranolol pka 9.5 H CH CHCH NHCH(CH ) The high purity Rx-SIL improves the peak shape dramatically on a C8 column Page 9
10 Potential Ion Exchange and Hydrogen Bonding Secondary Interactions Ion-exchange Si _ Na+ + R NH + Si _ N + R + Na +. Ionized silanols (Si - ) will ion-exchange with protonated bases (R NH + ) which can cause tailing and method variability. This occurs most often at mid ph where silanols are ionized. Hydrogen Bonding -SiH + RC _ -Si _... H +... _ CR. Unprotonated acids can compete for H + with protonated silanols. This can occur at low ph. Some mobile phase additives can be added to the mobile phase to reduce these interactions and this will be discussed in the mobile phase section. Page 0
11 Bonded Phase Types for Better Peak Shape Bonded phases with endcapping Eclipse XDB columns are double endcapped This minimizes the number of unreacted silanols and potential peak tailing interactions Bonded phases with embedded polar groups Bonus-RP provides unique silanol shielding reducing peak tailing for basic compounds Bonded phases for high ph Extend-C8 is stable at high ph Basic compounds can not ion-exchange with the silica thereby reducing peak tailing Bonded phases for high aqueous SB-Aq is more wettable improving analyte interactions and peak shape 00764S.PPT Page
12 Eclipse XDB is Double Endcapped for Best Peak Shape Improved peak shape for basic compounds especially at intermediate ph Double endcapped with two different reagents for more complete silanol coverage Good for all sample types acid, base, neutral Wide useable ph range ph -9 CH Si CH CH Si CH CH Si CH CH Si CH CH Si CH CH CH Page
13 Double Endcapping Improves Peak Shape ph 7 Columns: 4.6 x50 mm, 5 mm Mobile Phase: 60% ACN : 40% 0 mm phosphate buffer ph 7.0 Flow Rate:.5 ml/min. Temperature: 40 C Sample:. Nortriptyline pka 9.7. Doxepin pka 9.0. Amitriptyline pka Trimipramine Single Endcapped Waters Symmetry C8 Double Endcapped Eclipse XDB-C8 Tf Tf Each column is made from high purity silica Fewer silanol interactions on the double endcapped column reduce tailing and retention Page
14 Double Endcapping Provides Superior Peak Shape Eclipse XDB-C8 vs. Luna C8 Columns: 4.6 x 50 mm, 5 mm Mobile Phase: 50% 5 mm Na HP 4, ph 7.: 50% MeH Detection: UV 54 nm Flow Rate:.0 ml/min Temperature: 5 C Sample:. Uracil. Amlodipine. Benazepril Phenomenex Luna C8 Eclipse XDB-C8 Tf(5%) Tf(5%) Eclipse XDB shows superior peak shape for the basic compound, benazepril. Page 4
15 Bonus-RP with Embedded Polar Groups Good peak shape for basic compounds R Si PG R R Polar alkyl-amide bonded phase CH Si CH CH R Unique selectivity Si PG R Enhanced low ph stability with sterically protecting bonding R CH Si CH CH Triple endcapped R Si PG R R Page 5
16 Polar Alkyl Amide Bonded Phase Provides Better Peak Shape than Traditional Alkyl Phase Columns: 4.6 x 50 mm Mobile phase: 5 mm K HP 4, ph 7. / (MeH: ACN, 50:50), 45/5 Flow rate: ml/min. Detection: UV 54 nm Injection vol: 5 µl Sample: Anorectics ( Fen-phen ). Phentermine pka0.. Fenfluramine pka 9.. Impurity mau 5 0 Bonus-RP 5 0 mau Alkyl C min Good peak shape of highly basic compounds is readily achieved on Bonus-RP S.PPT Page 6
17 ZRBAX Extend-C8 for High ph Superior high ph stability up to ph.5 with silica particles Si C8 Excellent reproducibility Patented bidentate, C8 bonding Double endcapping Si C8 Si C8 Si C8 Page 7
18 Extend-C8 Improves Retention and Peak Shape of Basic Compounds at High ph Column: ZRBAX Extend-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 0% buffer: 70% MeH ph 7 buffer=0 mm Na HP 4 ph buffer = 0 mm TEA Flow Rate:.0 ml/min Temperature: RT Detection: UV 54 nm Sample:. Maleate. Scopolamine pka 7.6. Pseudoephedrine pka Doxylamine pka Chlorpheniramine pka Triprolidine pka Diphenhydramine pka 9.0 Extend-C8 ph 7 4, 7 Extend-C8 ph t R = 8.5 t R = The retention of this sample of basic compounds increases at high ph on Extend-C8. Page 8
19 Large Molecules Need Large Pore Sizes for Good Peak Shape Wide-pore 00Å totally porous columns can be selected for efficient peaks when separating proteins and peptides. Select wide-pore columns for lower molecular weight molecules with large hydrodynamic volumes. Select Poroshell columns for more rapid mass transfer and improved efficiency of large peptides and proteins at higher flow rates. Page 9
20 Choose 00Å Columns for Good Peak Shape with Peptides and Proteins Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations SB-C8 (00Å) SB-C8 (80Å) 0.4 PW / Leu Enkephalin M.W. = 556 Angiotensin II M.W. = 046 Insulin B M.W. =,496 Cyt C M.W. =,7 RNase M.W. =,684 Lysozyme M.W. =,900 Proper pore size selection results in sharper peaks for large molecules Page 0
21 Improved Peak Shape for Large Molecules in Solution with 00Å Columns Columns: 4.6 x 50 mm, 5 mm Mobile Phase: 60% MeH: 40% 0.% TFA Flow Rate: 0.75 ml/min Temperature: RT Detection: UV 8 nm Sample: Tylosin (MW 96) SB-C (80Å) H CH CH H C CH CH CH CH H C CH CH CH H H C H N CH CH 00SB-C (00Å) H CH H CH Pw / = 0.44 Pw / = The size of a molecule in solution determines which pore size column is best. The narrower peak width indicates unrestricted access to the pores. Page
22 Poroshell Particles Provide More Efficient Peaks for Peptides and Proteins Porous Shell SLID CRE 5 µ m Poroshell 00SB-C8 is for the separation of large peptides and proteins Poroshell particles have a solid silica core with a superficially porous surface with a 00Å pore size This results in more efficient mass transfer and sharper peaks with large proteins and peptides Page
23 Fast, High Resolution Separation of Peptides and Proteins With Poroshell 00SB-C8... In Seconds Columns: Poroshell 00SB-C8. x 75 mm, 5 mm Mobile Phase: A: 0.% TFA B: 0.07% TFA in ACN Gradient: 5 00% B in.0 min. Flow Rate:.0 ml/min. Temperature: 70 C Pressure: 50 bar Detection: UV 5 nm Sample:. Angiotensin II. Neurotensin. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.valbumin nly Poroshell can provide high efficiency at higher flow rates for extremely rapid separations of proteins and peptides. This is due to more rapid mass transfer of the superficially porous particle Page
24 Effect of Increasing Flow Rate in Protein Analysis with use of Totally Porous Silica Agilent 00 WPS with AutoBypass; Piston Stroke: 0 µl, Temperature: 70 C Detection: UV 5 nm Mobile Phase: A= 95% H, 5% ACN with 0.%TFA; B= 5% H, 95% ACN, with 0.07%TFA 0.5 ml/min 0-00%B in 4 min ml/min 0-00%B in min ml/min 0-00%B in min Totally Porous.x75mm, 5µm. Neurotensin. Rnase A. Lysozyme 4. Myoglobin ml/min 0-00%B in 0.67 min Efficiency and resolution are lost at high flow rates on a totally porous 00Å RP column Page 4
25 High Efficiency at High Flow Rates for Ultra-Fast Protein Analyses with Poroshell 0.5 ml/min 5 00%B in 4 min 0 ml/min 5 00%B in min ml/min 5 00%B in min ml/min 5 00%B in 0.67 min 4 ml/min 5 00%B in 0.5 min Agilent 00 DAD Agilent 00 WPS with ADVR Column: Poroshell 00SB-C8. x 75 mm, 5 mm Mobile Phase: A: 95% H, 5% ACN with 0.% TFA B: 5% H, 5% ACN with 0.% TFA Temperature:70 C Detector: UV 5 nm Sample:. Neurotensin. Lysozyme. RNaseA 4. Myoglobin min Resolution is maintained at high flow rates with Poroshell, allowing for efficient peaks and rapid analysis times. Page 5
26 Mobile Phase Factors for Improved Peak Shape ph Buffers rganic modifiers Additional mobile phase modifiers (TEA, TFA) Page 6
27 Good Peak Shape at Low ph ph 7.0 ph.0 USP TF( 5%) 4..5 USP TF (5%) 4.. Column: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 mm Mobile Phase:85% 5 mm Na HP 4 5% ACN Flow Rate:.0 ml/min. Temperature: 5 C Sample: Amphetamines. Phenylpropanolamine pka 9.4. Ephedrine pka 9.6. Amphetamine pka Methamphetamine pka Phentermine pka These basic compounds have good peak shape when the ph is lowered and the silica silanols are protonated. Page 7
28 Effect of ph on Peak Shape at or Near the Sample pka Column: ZRBAX SB-C8 4.6 x 50 mm, 5 mm Mobile Phase: 40% 5 mm KH P 4 : 60% ACN Flow Rate:.0 ml/min. Temperature: RT ph 4.4 ph.0 CH CHCH CH CH(CH ) Ibuprofen pk a = Inconsistent and tailing peaks may occur when operating close to an analyte pka and should be avoided. Page 8
29 Use Buffered Mobile Phases for Best Peak Shape and Retention Column: ZRBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm,.5 µm Mobile Phase: 44% A : 56% methanol Flow Rate:.0 ml/min Temperature: 5 C Detection: UV 50 nm Sample:. ketoprofen. ethyl paraben. hydrocortisone pka Fenoprofen pka propyl paraben 6. Propranolol pka Ibuprofen pka 4.4 A = ph 7.0 water A = ph 7.0, 5 mm phosphate buffer, 4, 6, Buffered mobile phases enhance retention, resolution, and peak shape. Page 9
30 Increasing Buffer Concentration Decreases Tf 0 mm Phosphate ph mm Phosphate ph 7.0 USP Tf USP Tf Columns: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 µm Mobile Phase: 40% phoshpate buffer 60% ACN Flow Rate:.5 ml/min Temperature: 40 C Sample: Tricyclic Antidepressants. Desipramine. Nortriptyline 6. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine Higher ionic strength at mid ph is very effective at masking silanol interactions and reducing peak tailing. Page 0
31 Mobile Phase rganic Modifier Acetonitrile vs. Methanol 40% 5 mm Na HP 4 ph % ACN 40% 5 mm Na HP 4 ph % MeH 4 5 USP TF 5% USP TF 5% Columns: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 µm Flow Rate:.5 ml/min. Temperature: 40 C Sample: Tricyclic Antidepressants. Desipramine. Nortriptyline. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine Changing the organic modifier may improve peak shape due to secondary interactions. Page
32 Mobile Phase Modifiers Effect of TEA on Peak Shape of Basic Compounds Column: ZRBAX Eclipse XDB-C8, 4.6 x 50 mm, 5 µm Mobile Phase: 85% 5 mm Na HP 4 : 5% ACN Flow Rate:.0 ml/min. Temperature: 5 C Sample: Amphetamines. Phenylpropanolamine. Ephedrine. Amphetamine 4. Methamphetamine 5. Phenteramine ph 7 No TEA 0 mm TEA 4 5 USP TF 5% USP TF 5% Page
33 Mobile Phase Modifiers Effects of Competing Acids on the Peak Shape of Acidic Compounds A: ph 5 mm NaH P 4 ph 5 mm NaH P 4 % acetic acid ph.5 0.% TFA Column: ZRBAX StableBond SB-C8 4.6 x 50 mm, 5 µm Mobile Phase: 40% A: 60% ACN Flow Rate:.0 ml/min. Temperature: Room Temperature Sample: Ibuprofen pka 4.4 T f =.8 T f =.0 T f =.09 Both acetic acid and TFA (trifluoroacetic acid) act as competing acids. TFA can be used at a lower concentration and is the preferred choice. Page
34 Sample and Additional Considerations for Good Peak Shape Sample overload (mass/volume) Sample solvent/injection solvent Metal complexation Page 4
35 Sample verload May Cause Peak Fronting and Tailing Peak fronting from sample mass overload more sample than can effectively partition results in some sample preceding the rest of the peak Peak tailing from overload of silanols with basic compounds may be seen as peak tailing Reduce sample load to eliminate 00 mau for compounds with average absorptivity Page 5
36 Strong Injection Solvent May Cause Poor Peak Shape Column: ZRBAX StableBond SB-C8, 4.6 x 50 mm, 5 µm Mobile Phase: 8% H : 8% ACN Injection Volume: 0 µl Sample:. Caffeine. Salicylamide A. Injection Solvent 00% Acetonitrile B. Injection Solvent Mobile Phase Injecting in a solvent stronger than the mobile phase can cause peak shape problems, such as peak splitting or broadening. Page 6
37 Metal Complexation May Cause Poor Peak Shape Analytes that may complex with metals may show poor peak shape Both tailing and fronting may result from metal complexation Metals are present in LC systems from solvents, tubing, and stainless steel frits High purity silica eliminates silica as a source of metals Page 7
38 Metal Sensitive Compounds Can Chelate Hint: Look for Lone Pair of Electrons on :: or N Which Can Form 5 or 6 Membered Ring with Metal : C :H : H C M + H + M + : : H Salicylaldehyde 6-membered ring complex N: M + H : : C C : : N : H M + 8-hydroxyquinoline 5-membered ring complex α-benzoinoxomine 5-membered ring complex Page 8
39 Acid Wash Can Improve Peak Shape Before Acid Wash After Acid Wash mls % H P 4 H H H H H.... H H H Columns: ZRBAX SB-Phenyl 4.6 x 50 mm Mobile Phase: 75% 5 mm ammonium phosphate buffer 5% ACN Flow Rate:.0 ml/min. Temperature: RT Sample Size: 5 µl Tf:.7 Tf:. A % H P 4 solution is used on SB columns, 0.5 % can be used on endcapped columns. Page 9
40 Purpurin Column: Zorbax SB-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 0% 0.0% TFA in water: 80% MeH Flow Rate: ml/min Detection: UV 54 nm Temperature: 4 C A. Before Acid Wash B. After Acid Wash H % phosphoric acid, 0 min. Purpurin H H Tf=.6 Tf= Both fronting and tailing are evident on purpurin before the acid wash Page 40
41 Guidelines for Improved Peak Shape Select columns based on high purity fully hydroxylated silica Zorbax Rx-Sil based columns, such as StableBond, Eclipse XDB, Bonus-RP and Extend Select double endcapped columns for mid ph or difficult basic compounds, such as Eclipse XDB Select special bonded phases (Bonus-RP, Extend-C8) for better peak shape at mid and high ph Select wide-pore columns for high molecular weight analytes Use buffered low ph mobile phases to reduce secondary interactions Use 5 50 mm buffered mobile phases at every ph Use additional additives only when needed Check sample solvents Page 4
42 HPLC Column Technical Support (phone: US & Canada) * (phone) * * Select option 4, then option (fax) Page 4
William E. Barber, Ph.D. Applications Chemist October
William E. Barber, Ph.D. Applications Chemist ctober 0 00 Secrets of Good Peak Shape in HPLC Time: :00 p.m. CET Telephone Number: + 44 0 76 088 Chairperson: John Vis The Secrets of Good Peak Shape in HPLC
More informationThe Secrets of Rapid HPLC Method Development. Choosing Columns for Rapid Method Development and Short Analysis Times
The Secrets of Rapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times Rapid Analysis Is More Than Run Time It is developing a method to meet a goal and developing
More informationLC and LC/MS Column Selection Flow Chart
LC and LC/MS Column Selection Flow Chart To use the column selection diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the
More informationBarry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 10, 2000
Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 0, 000 HPLC Column Troubleshooting What Every HPLC User Should Know :00 a.m. EST Telephone Number: 86-650-06 Chair Person: Tim Spaeder
More informationTips and Tricks of HPLC System Troubleshooting
Tips and Tricks of HPLC System Troubleshooting Agilent Technologies, Inc. LC Tips And Tricks Seminar Series Page 1 Trouble Shooting Steps You Have Recognized There is a Problem! How Do You Fix It? 1 st
More informationAdvanced Topic: Tips and Tricks of HPLC Separations and Troubleshooting. Rita Steed LC Advanced Topics Seminar Series August 25, 2010.
Advanced Topic: Tips and Tricks of HPLC Separations and Troubleshooting Rita Steed LC Advanced Topics Seminar Series August 25, 200 Page Troubleshooting Steps You Have Recognized There is a Problem! How
More informationA Look at Column Choices. Ed Kim Application Engineer Agilent Technologies, Inc February 12, 2009
A Look at Column Choices Does one-size-fit-all? Ed Kim Application Engineer Agilent Technologies, Inc February 12, 2009 Seminar utline lica Surface Chemistry & Physical Characteristics ti lica Bonding
More informationTips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think!
Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think! Agilent Technologies Page Tips and Tricks of HPLC System Troubleshooting Agilent Technologies Page Trouble
More informationHPLC Method Development with Eclipse Plus: Standard Practices and New Columns. Agilent Technologies
HPLC Method Development with Eclipse Plus: Standard Practices and New Columns Agilent Technologies What are Some Standard Method Development Practices?. Follow preferred method development scheme and do
More informationHICHROM. Chromatography Columns and Supplies. LC COLUMNS Zorbax. Catalogue 9. Hichrom Limited
HICHROM Chromatography Columns and Supplies LC COLUMNS Zorbax Catalogue 9 1 The Markham Centre, Station Road Theale, Reading, Berks, RG7 4PE, UK Tel: +44 (0)118 930 3660 Fax: +44 (0)118 932 3484 Email:
More informationHPLC Column Troubleshooting
HPLC Column Troubleshooting Gulf Coast Conference 04 John Palmer Page HPLC System Components Pump Injector/Autosampler Column Detector Data System/Integrator Problems Can Be Related to All Components in
More informationHPLC Column Troubleshooting: Is It Really The Column? LC Columns and Consumables. Robyn C. Woolley Online Application Engineer July 24, 2007.
HPLC Column Troubleshooting: Is It Really The Column? LC Columns and Consumables Robyn C. Woolley Online Application Engineer July 24, 2007 Page 1 Troubleshooting in HPLC ma U 20 00 15 00 10 005 0 0 0
More informationToo Polar for Reversed Phase What Do You Do?
Too Polar for Reversed Phase What Do You Do? June 20, 2013 Mark Powell Columns and Consumables Technical Support Page 1 C8 or C18 Doesn t Always Do the Job Typical reversed phase conditions involve water/buffer
More informationEliminating Unwanted Variability in LC Methods: More Uptime, More Sample Throughput
Eliminating Unwanted Variability in LC Methods: More Uptime, More Sample Throughput HPLC Separation Robustness and Ruggedness Group/Presentation Title June 20, Agilent 2017Month Restricted ##, 200X Page
More informationHPLC Method Development: Standard Practices and New Columns
HPLC Method Development: Standard Practices and New Columns Edward Kim Application Engineer Agilent Technologies, Inc. March 8, 29 What are Some Standard Method Development Practices?. Follow preferred
More informationReversed Phase Solvents
Part 1. General Chromatographic Theory Part 2. verview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use Reversed Phase Solvents 2 Solvents for RP Chromatography
More informationHypersil BDS Columns TG 01-05
TG 0-0 Hypersil BDS Columns Introduction Hypersil BDS columns have gained a reputation over the years as one of the most robust, reproducible and reliable HPLC column brands available. This Technical Guide
More informationRegis Technologies Ion Pair Reagents and Buffers
egis Technologies Ion Pair eagents and Buffers Ion Pair HPLC greatly extends the applicability of HPLC by making the efficient octadecylsilyl columns amendable to use with ionized or ionizable samples.
More informationzorbax Packed by Phenomenex Material Characteristics High efficiency Guaranteed quality Phenomenex columns packed with Zorbax
Packed by Phenomenex High efficiency Guaranteed quality Phenomenex columns packed with 7 μm materials offer the advantages of lower cost as well as lower operating backpressures, which result in long column
More informationKromasil. for your analytical HPLC. Kromasil. The way to peak performance in liquid chromatography
Kromasil for your analytical HPLC Kromasil The way to peak performance in liquid chromatography Kromasil is known, worldwide, for its high performance and excellent total economy in preparative and industrial
More informationProntoSIL C18-EPS Reversed-Phase HPLC Columns
ProntoSIL C8-EPS Reversed-Phase HPLC Columns Provides excellent separation of polar compounds Better peak shape for acids and bases Stabilized bonded phase for rugged, robust HPLC methods More retentive
More informationImprove Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application
Improve Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application Pharmaceuticals Authors John W. Henderson Jr., Nona Martone, and Cliff Woodward
More informationEfficiently analyze your most challenging compounds. Deliver high-quality results faster. Agilent ZORBAX Column Selection Guide for HPLC
Efficiently analyze your most challenging compounds. Deliver high-quality results faster. Agilent ZORBAX Column Selection Guide for HPLC Agilent ZORBAX Column Selection Guide for HPLC Here it is your single
More informationWelcome to our E-Seminar: Choosing HPLC Columns for Faster Analysis Smaller and Faster
Welcome to our E-Sear: Choosing HPLC Columns for Faster Analysis Smaller and Faster High Throughput/Fast LC Requires. Short columns 0 mm or shorter Small particle sizes. µm Rapid Resolution or new.8 µm
More informationNew Product Update. HPLC Columns
New Product Update HPLC Columns ZORBAX MicroBore HPLC Columns ZORBAX Extend-C18 Columns ZORBAX Bonus-RP Columns Inertsil ODS-2 Cartridge Columns ZORBAX Carbohydrate Analysis Columns ZORBAX Eclipse dsdna
More informationDoes one-size-fit-all?
A Look at Column Choices: Does one-size-fit-all? Bill Champion Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 william_champion@agilent.com Page 1 Separation Techniques Hydro - philic Volatile
More informationHow proteins separate on reverse-phase HPLC
1 Reverse Phase How proteins separate on reverse-phase HPLC RP chromatography separates proteins through the interaction of the hydrophobic foot of the protein with a nonpolar surface of the particle RP
More informationpenta-hilic UHPLC COLUMNS
penta-hilic UHPLC COLUMNS penta-hilic Highly retentive, proprietary penta-hydroxy-ligand Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and
More informationHigh Resolution Fast LC
High Resolution Fast LC Easier Than You Think Rita Steed LC Columns Application Engineer May 9, 2013 What is High Resolution Fast LC? Maintain Resolution with Faster Run Time Increased Resolution with
More informationState-of-the-art C18 HPLC Columns
An HPLC GL Sciences Newest and Most Advanced ODS Phase-New For 00 State-of-the-art C HPLC s Improved Peak Shapes and Heights Enhancing Sensitivity High Resolution Fast Equilibration Compatible with 00%
More informationACE SuperC18. ACE UHPLC and HPLC Columns. Ultra-Inert UHPLC and HPLC Columns with Extended ph Stability
ACE SuperC8 TM Ultra-Inert UHPLC and HPLC Columns with Extended ph Stability Exploit selectivity at low, intermediate and high ph Use with LC/MS compatible buffers between ph.. High efficiency µm, µm,
More informationLC Column Troubleshooting. Do you have an equivalent column for my LC column?
LC Column Troubleshooting Do you have an equivalent column for my LC column? LC columns from different suppliers may have very different retention properties even if the bonding is the same. Although there
More informationHPLC Separation Robustness and Ruggedness Assessing the Effects of Experimental Variables During Method Development
HPLC Separation Robustness and Ruggedness Assessing the Effects of Experimental Variables During Method Development Rita Steed Application Engineer Agilent Technologies, Inc. November 19, 2015 Method Development
More informationProduct Bulletin. ACE LC/MS and Rapid Analysis HPLC Columns. HPLC Columns
Product Bulletin HPLC Columns ACE LC/MS and Rapid Analysis HPLC Columns 0 mm, 30 mm, 35 mm and 50 mm column lengths.0,., 3.0, 4.0 and 4.6 mm column diameters Configured for High Sample Throughput Specially
More informationAlltech Alltima HP Introduction
Alltech Introduction High-Stability, High-Purity, High-Performance, Low-Bleed Columns for Demanding Applications Better Peak Symmetry high-purity silica eliminates peak tailing problems Long Column Life
More informationA Look at Agilent HPLC Columns Choices
A Look at Agilent HPLC Columns Choices Brno & Prague, May 29-30 2018 Giorgio Ferlat IDO Product Specialist Chemistries and Consumable 1 Considerating Column Properties for Column Selection Silica Particle
More informationpenta-hilic UHPLC COLUMNS
penta-hilic UHPLC COLUMNS Highly retentive, proprietary penta-hydroxy-ligand penta-hilic Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and
More informationHPLC Method Development: Standard Practices and New Columns. Agilent Technologies February 2007
HPLC Method Development: Standard Practices and New Columns Agilent Technologies February 7 What are Some Standard Method Development Practices?. Follow preferred method development scheme and do hands-on
More informationLogical Method Development for Peptides and Proteins Using RP-HPLC and SEC
Logical Method Development for Peptides and Proteins Using RP-HPLC and SEC Slide 4 001284P1.PPT Seminar Outline 1. Gel-filtration separations by SEC 2. Developing methods for protein and peptide separations
More informationInertsil ODS-EP Technical Information
Technical Information Advantages of The selectivity is completely different from those of conventional columns such as ODS column due to its specific polar group in the stationary phase. Durability can
More informationProntoSIL HPLC Columns
ProntoSIL HPLC Columns For Technical Assistance Call -800--708 analytical inc. To Order and For More Information: Call FAX -800--708-0-8-99 -0-8-99 We ship nearly 00% of your orders the same day they are
More informationCOMPARISON GUIDE. to C18 Reversed Phase HPLC Columns. Comparison Data on 60 Commonly Used C18 Phases. Stationary Phase Specifications
COMPARISON GUIDE to C18 Reversed Phase HPLC Columns Comparison Data on 60 Commonly Used C18 Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Phases Compared According
More informationACE Ultra Inert Base Deactivated HPLC Columns
ACE Ultra Inert Base Deactivated HPLC Columns Contents Page ACE HPLC Columns...- Independent Comparison of HPLC Columns #... Independent Comparison of HPLC Columns #... ACE 00Å HPLC Columns Specifications...-
More informationThermo Scientific Accucore XL HPLC Columns. Technical Manual
Thermo Scientific Accucore XL HPLC Columns Technical Manual Thermo Scientific Accucore XL HPLC Columns Based on Core Enhanced Technology using µm solid core particles, Accucore XL HPLC columns allow users
More informationACE Excel SuperC18. ACE UHPLC and HPLC Columns. Ultra-Inert UHPLC and HPLC Columns with Extended ph Stability
ACE Excel SuperC8 TM Ultra-Inert UHPLC and HPLC s with Extended ph Stability Exploit selectivity at low, intermediate and high ph Use with LC/MS-compatible buffers between ph.. High efficiency µm, µm,
More informationmau 200 ph min
mau ph - 6 8 TAKE A LOOK INSIDE TWIN Technology TWIN (Two-In-One ) Technology is what gives Gei its superior performance edge. During the final stage of silica manufacturing, a unique silica-organic layer
More informationComparison Guide to C18 Reversed Phase HPLC Columns
Comparison Guide to C18 Reversed Phase HPLC Columns Comparison Data on Commonly Used C18 Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Phases Compared According
More informationACE SuperC18. ACE UHPLC and HPLC Columns. Ultra-Inert UHPLC and HPLC Columns with Extended ph Stability
ACE SuperC8 TM Ultra-Inert UHPLC and HPLC s with Extended ph Stability Exploit selectivity at low, intermediate and high ph Use with LC/MS compatible buffers between ph.. High efficiency µm, µm, µm and
More informationSunniest C18 Sunniest RP-AQUA Sunniest C8 Patent pending
Patent pending Website : www.chromtech.net.au E-mail : info@chromtech.net.au telno : 0 97 0... in AUSTRALIA A Novel Bonding Technique (patent pending) An unique trifunctional silyl-reagent was developed
More informationValidated HPLC Methods
Validated HPLC Methods To Tweak or Not to Tweak Slide 4 Adjustments of operating conditions to meet system suitability requirements may be necessary. USP 23 p1776 Slide 5 001816S1.PPT What is the line
More informationUptisphere CS Evolution Core Shell columns for fast & highly efficient identification & quantification of small molecules
Uptisphere CS Evolution Core Shell columns for fast & highly efficient identification & quantification of small molecules www.interchim.com www.blog.interchim.com www.forum.interchim.com www.facebook.com/interchim
More informationBringing uhplc Performance to the Separation of Peptides
P e p t i d e E S - C 1 8 U H P L C C o l u m n s Bringing uhplc Performance to the Separation of Peptides Compatible with UHPLC and conventional HPLC equipment. Sumpfstr. 3, CH-6300 Zug; Fax: 041 748
More informationHICHROM. Chromatography Columns and Supplies. LC COLUMNS HALO and HALO-5. Catalogue 9. Hichrom Limited
HICHROM Chromatography Columns and Supplies LC COLUMNS HALO and HALO- Catalogue 9 The Markham Centre, Station Road Theale, Reading, Berks, RG PE, UK Tel: + (0)8 90 0 Fax: + (0)8 9 8 Email: sales@hichrom.co.uk
More informationPackings for HPLC. Packings for HPLC
Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied.
More informationGeneral Principles of HPLC Method Development
General Principles of PLC Method Development Philip J. Koerner, Ph.D. Thailand September 213 Part 1. General Chromatographic Theory Part 2. The Stationary Phase: An verview of PLC Media Part 3. Role of
More information(HILIC) Bill Champion Agilent Technologies, Inc opt 3/opt3/opt 2 HILIC - Agilent Restricted
Hydrophilic Interaction Chromatography (HILIC) Bill Champion Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 william_champion@agilent.com Page 1 HILIC A method of recent attention Bill Champion
More informationHamilton. Reversed-Phase Anion Exchange Cation Exchange Ion Exclusion
HPLC Columns Agilent for Small Molecule InfinityLab Poroshell 0 ZORBAX Eclipse Plus ZORBAX Eclipse XDB ZORBAX 80Å StableBond ZORBAX 80Å Extend-C8 ZORBAX Bonus-RP ZORBAX Eclipse PAH ZORBAX HILIC Plus ZORBAX
More informationShodex TM ODP2 HP series columns
HPLC Columns Shodex TM ODP2 HP series columns Better retention of highly polar substances Technical notebook No. 6 Contents 1. Introduction 1-1. Specifications 1-2. Eluent Compatibility of ODP2 HP Series
More informationSuperficially Porous Silica Particles with Wide Pores for Biomolecular Separations
Superficially Porous Silica Particles with Wide Pores for Biomolecular Separations B. M. Wagner, S. A. Schuster, B. E. Boyes, J. J. Kirkland Advanced Materials Technology, Inc. Wilmington, DE, USA Study
More informationPeptide and protein analysis by capillary HPLC Optimization of chromatographic and instrument parameters. Application. Angelika Gratzfeld-Huesgen
Peptide and protein analysis by capillary HPLC Optimization of chromatographic and instrument parameters Application Angelika Gratzfeld-Huesgen Abstract This application has been verified using an Agilent
More informationcolumns Acclaim Mixed-Mode WCX-1 for Separating Basic Molecules
columns Acclaim Mixed-Mode WCX- for Separating Basic Molecules The Acclaim Mixed-Mode WCX- is a novel, high-efficiency, silica-based column specially designed for separating various basic analytes. This
More informationP R O D U C T B U L L E T I N. Fused-Core particle technology for hyper-fast and super-rugged HPLC columns
P R O D U C T B U L L E T I N Fused-Core particle technology for hyper-fast and super-rugged HPLC columns HALO column packings are not made the typical way. Instead, the particles packed into HALO columns
More informationHPLC Column Classification
HPLC Column Classification Introduction 1978 USP XIX Fourth supplement : L1 designation for C18 column 1980 USP XX : 7 columns were classified -Classified HPLC column to 56 descriptions -More than 220
More informationA C18 Silica Column With Exceptional Temperature and ph Stability
A C18 lica Column With Exceptional Temperature and ph Stability Brian A. Jones 1, Stephanie J. Marin 1, Jody Clark 1, Nathan L. Porter 1, J. Andreas Lippert 2, and Todd M. Johnson 2 1. Selerity Technologies,
More informationOrosil HPLC Columns. OroSil HPLC columns are designed for the separation of polar, semi-polar, and nonpolar compounds at low to medium ph.
Orosil OroSil HPLC columns are designed for the separation of polar, semi-polar, and nonpolar compounds at low to medium ph. Excellent organic base selectivity with very low asymmetry values Compatible
More informationAgilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX
Agilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX Technical Note Agilent s SampliQ WAX provides Applications for strongly acidic, acidic and neutral compounds Excellent
More informationOptimisil HPLC & UPLC Column catalogue
Optimisil HPLC & UPLC Column catalogue Analytical Hub Plot No: 37, P & T Colony, Vikrampuri, Secunderabad 500009 Tel: 040-64621313, Mobile: +91 7799931313 Website: www.analyticalhub.com email: contact@analyticalhub.com
More informationAgilent 1290 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases
Agilent 129 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract The Agilent
More informationReversed Phase Chromatography: Mobile phase Considerations
Reversed Phase: Mobile Phase Considerations Dr. www.forumsci.co.il/hplc Example of a Reversed Phase Method with UV Detection 1 Chromatographic Process B+A Mobile phase Elution through the Column-movie
More informationPerformance characteristics of the Agilent 1290 Infinity Quaternary Pump
Performance characteristics of the Agilent 129 Infinity Quaternary Pump Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract This Technical Overview presents Proof
More informationRonald E. Majors, Ph.D. Product Marketing Specialist Consumables and Accessories Business Unit May 31, 2000
Ronald E. Majors, Ph.D. Product Marketing Specialist Consumables and Accessories Business Unit May 31, 2000 Logical Method Development for Small Molecular Weight Ionizable Compounds using RP-HPLC 11:00
More informationHPLC UHPLC Columns. Dikma Technologies Inc.
Technologies Inc. About HPLC UHPLC Columns has over years of R&D experience in HPLC / UHPLC columns. HPLC / UHPLC columns have been widely used by pharmaceutical manufacturers. We offer columns of ultimate
More informationAcclaim Mixed-Mode WCX-1
Acclaim Mixed-Mode WCX-1 Product Manual for the Acclaim Mixed-Mode WCX-1 Column Page 1 of 28 PRODUCT MANUAL for the Acclaim Mixed-Mode WCX-1 Columns 4.6 x 150 mm, P/N (068353) 4.6 x 250 mm, P/N (068352)
More informationPart 2. Overview of HPLC Media
Part 1. General Chromatographic Theory Part 2. Overview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use 1 HPLC Particle Technology Core-Shell Particle Fully
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 1 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationfor Acclaim Mixed-Mode WCX-1
for Acclaim Mixed-Mode WCX- Product Manual for the Acclaim Mixed-Mode WCX- Column Page of 8 Product Manual for Acclaim Mixed-Mode WCX- Columns Acclaim Mixed-Mode WCX-, 3µm, Analytical column, 3.0 x 50mm
More informationInertSustainSwift C8
Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :ES (Evolved Surface) Silica Gel :.9 μm, μm, μm :00 m /g :00 Å (0 nm)
More informationOverview Applications of NUCLEODUR HPLC Phases
verview Applications of HPLC Phases Phase Specification Characteristics* Stability Structure A B C octadecyl phase, high density coating multi-endcapping 8% C USP L ph stability -, suitable for LC/MS (Si-
More informationApplication Note. Author. Abstract. Pharmaceutical QA/QC. Siji Joseph Agilent Technologies, Inc. Bangalore, India
Effective use of pharmacopeia guidelines to reduce cost of chromatographic analysis Optimized, cost-effective HPLC analysis of atorvastatin by varying column dimensions within the USP allowed limts
More informationSTUDY OF LOADABILITY AND SELECTIVITY OF PHARMACEUTICAL COMPOUNDS ON RPLC COLUMNS
STUDY OF LOADABILITY AND SELECTIVITY OF PHARMACEUTICAL COMPOUNDS ON RPLC COLUMNS Fang Xia, Jie Y. Cavanaugh, Uwe Neue, Jeffrey R. Mazzeo, Diane M. Diehl Waters Corporation, Milford, MA USA OVERVIEW- INTRODUCTION-
More informationBreaking the speed limit: Fast, high-resolution peptide and tryptic digest separations using fused-core particles
Breaking the speed limit: Fast, high-resolution peptide and tryptic digest separations using fused-core particles Stephanie Schuster 1, Barry Boyes 1,2, and Darryl Johnson 2 1 Advanced Materials Technology,
More informationZirconia: the Ideal Substrate for Ion-Exchange LC and LC-MS
Zirconia: the Ideal Substrate for Ion-Exchange LC and LC-MS EAS 2005 Bingwen Yan 1, Clayton V. McNeff 1, Richard A. Henry 2, and David S. Bell 2 1 ZirChrom Separations, Inc., 617 Pierce Street, Anoka,
More informationNew To HPLC Avoiding Beginner Pitfalls. Rita Steed LC Columns Application Engineer April 24, 2013
New To HPLC Rita Steed LC Columns Application Engineer April 24, 2013 Outline Instrument Connections Performance Detector Column Characteristics Lifetime Method Conditions Mobile phase ph Temperature Sample
More informationState - of - the -art C18 HPLC Columns
An HPLC GL Sciences Newest and Most Advanced ODS Phase -New For State - of - the -art C HPLC s Improved Peak Shapes and Heights Enhancing Sensitivity High Resolution Fast Equilibration Compatible with
More informationNovel LC/MS-Compatible Stationary Phase with Polar Selectivity
Novel LC/MS-Compatible Stationary Phase with Polar Selectivity Carmen T. Santasania and David S. Bell Supelco 595 N. Harrison Rd., Bellefonte, PA 16823 T403168 GJU Introduction Stationary phases based
More informationExceptional Selectivity of Agilent ZORBAX Eclipse Plus Phenyl-Hexyl Columns to Separate Estrogens
Exceptional Selectivity of Agilent ZORBAX Eclipse Plus Phenyl-exyl Columns to Separate Estrogens Application Note Pharmaceutical, Environmental Authors John W. enderson Jr. and William J. Long Agilent
More information( )( ) Selectivity Choices in Reversed-Phase Fast LC. Introduction. R s = 1 a 1 k 4 a 1 + k
Selectivity Choices in Reversed-Phase Fast LC Luisa Pereira, Monica Dolci, Thermo Fisher Scientific, Runcorn, Cheshire, UK Technical Note 20543 Key Words Accucore, Hypersil GOLD, Syncronis, Column Chemistry,
More information8. Methods in Developing Mobile Phase Condition for C18 Column
I. HPLC Columns Technical Information 8. Methods in Developing Mobile Phase Condition for C18 Column Introduction In reversed phase HPLC, octadecyl group bonded silica columns (C18, ODS) are the most widely
More informationPacked Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS. Table of Contents
No. 089 SEPARATION REPORT Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS Table of Contents 1. Introduction 1 2. Column Specification 1 3. Features of Packing Materials
More informationZirconia-Based Phases as a Powerful Complement to Silica-Based Phases for LC and LC-MS under Non-extreme Mobile Phase Conditions
Zirconia-Based Phases as a Powerful Complement to Silica-Based Phases for LC and LC-MS under on-extreme Mobile Phase Conditions Richard A. Henry, Shawn R. Wyatt, Carmen T. Santasania and David S. Bell
More informationACE. For increased polar retention and alternative selectivity. Alternative selectivity for method development
ACE C8-Amide For increased polar retention and alternative selectivity Alternative selectivity for method development Improved separations with polar, acidic, basic and phenolic compounds High efficiency
More informationThermo Scientific Syncronis HPLC Columns. Remarkable separations guaranteed time after time
Thermo Scientific Syncronis HPLC Columns Remarkable separations guaranteed time after time Syncronis HPLC and UHPLC Columns Remarkable separations guaranteed time after time When developing a new method,
More informationDo you need trouble-free speed? The NEW Chromolith HR
Do you need trouble-free speed? The NEW Chromolith HR EMD Millipore is a division of Merck KGaA, Darmstadt, Germany ... then speed-up to Chromolith HighResolution... Revolutionary monolithic silica columns
More informationfor Acclaim Mixed-Mode HILIC-1 Column
for Acclaim Mixed-Mode HILIC-1 Column Product Manual for ACCLAIM Mixed-Mode HILIC-1 Page 1 of 17 Product Manual for ACCLAIM Mixed-Mode HILIC-1 Column 5µm, 4.6 x 250mm, P/N 066844 5µm, 4.6 x 150mm, P/N
More informationRP C18 column with feature of a silanol group
RP C8 column with feature of a silanol group lanol Activity Controlled C8 Column Sunrise ctacosyl C8) Sunrise ctadecyl C8) Sunrise ctadecyl-sac C8-SAC has an interaction of silanol groups ChromaNik Sunrise
More informationMethod Development Kits
Method Development Kits sales representative for ordering information or contact...77 Method Development Kits First column choice for Pharma Development USP L C Method Development Kits Maximize retention
More informationTechnical Guide Thermo Scientific Syncronis HPLC Columns
Technical Guide Thermo Scientific Syncronis HPLC Columns Consistent, predictable separations, Column after column, time after time Thermo Scientific Syncronis Columns When developing a new method, one
More information"Theory and Practice of High Speed Chromatography for Bioanalysis" Stuart Coleman March 20, 2007
"Theory and Practice of High Speed Chromatography for Bioanalysis" Stuart Coleman March 20, 2007 General Observations about HPLC Separations A good separation is necessary for good bioanalytical quantitation.
More informationUPLC Method Development and Validation
UPLC Method Development and Validation Rev. 2 2008 Waters Corporation Challenges of Method Development Methods are developed throughout the drug development process Samples vary in complexity Redundancy
More information