Phase Separations Customer Education Program

Transcription

1 Phase Separations Customer Education Program Advanced HPLC Column Selection Using the Hydrophobicity Chart neutral neutral 0 base Time (min) base 50 Copyright 1998 Phase Separations

2 Course Library * Strategies and Techniques for Faster HPLC Methods Development * Methods Development Optimization Software --HIPAC * Advanced HPLC Column Selection Using The Hydrophobicity Chart * Solid Phase Extraction Technology * Selecting Optimum HPLC Column Dimensions and Stationary Phase Particle Size * Troubleshooting Common HPLC Problems * Purchasing High Performance Chromatographic Supplies * Understanding RP-HPLC Separations -- Effect of Silica Type/Activity * Reversed-Phase HPLC Methods Development Copyright 1998 Phase Separations

3 Course List - Order Number & Duration * Strategies and Techniques for Faster HPLC PSL hours Methods Development *Methods Development Optization Software -- HIPAC PSL min * Advanced HPLC Column Characterization & Selection PSL hours Using The Hydrophobicity Chart * Solid Phase Extraction Technology PSL hours * Selecting Optimum HPLC Column Dimensions and PSL hour Stationary Phase Particle Size * Troubleshooting Common HPLC Problems PSL hours * Purchasing High Performance Chromatographic PSL hours Supplies * Understanding RP-HPLC Separations PSL hours -- Effect of Silica Type/Activity *Reversed-Phase HPLC Methods Development PSL hours Copyright 1998 Phase Separations

4 ...because Chromatography is still a Science SM Strategies & Techniques for Faster HPLC Methods Development Course Abstract: This course is designed for those individuals developing new reversed-phase HPLC methods or those planning to improve existing methods in order to increase the methods' sample throughput, robustness and cost effectiveness. A logical and efficient methods development process is described which covers column selection based on analyte type and sensitivity requirements utilizing different selectivities of chromatographic packings to solve difficult application problems. We will explore solvent system optimization strategies, as well as review the role that Method Development Optimization Software can play in this process. Key examples are shown, and each attendee will receive a complete set of the slides in hard- copy form to keep for future reference. A Certificate of Achievement will be sent to all attendees, which can be used for your files as documentation of this training program. Duration: 2.5 hours Order Course # PSL Phase Separations

5 presents: Strategies & Techniques for Faster HPLC Methods Development

6 Packings for Reversed-Phase Chromatography silica-based vs. polymeric chain length/type silica quality endcapping packings with embedded polar function use a well-bonded, endcapped C8 based on a high-purity silica from a reputable manufacturer!

7 How to achieve shorter analysis times -- without sacrificing resolution and efficiency Reduce particle size Use shorter columns Maintain constant ratio of column length to particle size

8 Faster Analysis for Stability Studies Chlordiazepoxide (x) Degradation Products x a. 5 µm 0.7 ml/min Columns: Symmetry C 18 a. 5 µm 3.9 x 150 mm; b., c. 3.5 µm 4.6 x 100 mm Mobile Phase: acetonitrile/methanol/ TETA-MeCOOH ph 7.0 Detection: UV at 240 nm Sample: 100 µl of 40 µg/ml x 2 b. 3.5 µm 1.0 ml/min x c. 3.5 µm 1.4 ml/min Peak 2 Analysis USP Times Plates a. 40 min 11,240 b. 30 min 13,400 c. 20 min 12, Minutes D. J. Phillips

9 Performance of Different Diameter Columns at Equal Linear Velocities a) 0.00 b) mm x 150 mm mm x 150 mm mm x 150 mm Conditions: Column: Symmetry C 18 5 µm Mobile Phase: water/methanol/glacial acetic acid 79:20:1 Flow Rates: a) 1.4 ml/min b) 1.0 ml/min c) 0.6 ml/min d) 0.29 ml/min Sample: mixture of 6 sulfa drugs, 10 to 39 µg/ml Injection vol.: a) 14 µl, b) 10 µl, c) 6 µl and d) 3 µl c) mm x 150 mm d) 0.00 Minutes USP Plates USP Tailing Peak 4 a) 4.6 mm ID b) 3.9 mm ID c) 3.0 mm ID d) 2.1 mm ID D. J. Phillips

10 Method Development Strategy Gradient at low ph Starting Solvent Gradient at high ph Starting Solvent Gradient with Second Slope Calculate Isocratic Mobile Phase Isocratic Run in Starting Solvent Calculate Isoeluotropic Mobile Phases Isocratic Run in Second Solvent Isocratic Run in Third Solvent Mixture Optimization

11 Method Validation Criteria Precision Accuracy Method Validation Limit of Detection Limit of Quantitation Specificity Linearity and Range Ruggedness Robustness

12 Method Ruggedness Analyst to Analyst Instrument to Instrument Lab to Lab Column to Column Batch to Batch

13 Method Robustness Content of Organic Solvent Ionic Strength ph Temperature Other Additives

14 ...because Chromatography is still a Science SM Methods Development Optimization Software HIPAC Course Abstract: One of the most significant trends today is the need for rapid HPLC Methods Development efforts. Recently, a tool to aid the analytical chemist in reducing the time necessary developing new applications has become available. This tool utilizes the chromatographic experience of analytical chemists combined with desktop computers and software, based on chromatographic behavior models, to rapidly optimize application conditions. In addition, this tool can be used to assess the robustness of potential applications, without the need for many multiple trial runs. A review of the HIPAC brand software designed for the optimization of binary, isocratic reversed- phase applications, and system optimization is given. Detailed use of the software is shown, as well as examples of the thorough, tutorial information included in the software. This tutorial information, and the use of the built- in chromatographic optimization examples, can also be effectively used in the training of lab personnel new to HPLC technology. Key examples are shown, and each attendee will receive a complete set of the slides in hard- copy form to keep for future reference. A Certificate of Achievement will be sent to all attendees, which can be used for your files as documentation of this training program. Duration: 45 minutes Order Course # PSL Phase Separations

15 HPLC Methods Development Optimization Software HIPAC TM B and S Binary-Isocratic/ inary-isocratic/system ystem Optimization

16 HIPACTM Chromatography Optimization Software Software Package Designed To Combine A Chromatographers' Experience, Experimental Data And HPLC Theory To Speed Methods Development Process Chromatographers' Experience Experimental Fast HPLC Data Theory Processing (Software)

17 HIPACTM Chromatography Optimization Software Complete Chromatography Methods Development Software Package Available In Individual Modules Targeted For The Type Of Applications In Your Lab * HIPAC B Isocratic Reversed-Phase And Binary Normal-Phase * HIPAC S Optimize System And Column Parameters * HIPAC G Reversed-Phase Binary Gradients * HIPAC TQ Ternary And Quarternary Isocratic

18 HIPACTM Chromatography Optimization Software Quickly Determines The Optimum Conditions For Your Application Based On Your Inputs

19 HIPACTM Chromatography Optimization Software

20 HIPACTM Chromatography Optimization Software Isoeluotropic Mobile Phase Selection

21 HIPACTM Chromatography Optimization Software

22 HIPACTM Chromatography Optimization Software

23 ...because Chromatography is still a Science SM Advanced HPLC Column Characterization & Selection Using the Hydrophobicity Chart Course Abstract: Today, analytical chemists are frequently faced with the very challenging assignment of developing new HPLC methods, which are simple, rugged and robust, all in a short period of time. Traditional methods development techniques, coupled with the improper selection of HPLC column, could result in the spending of much unnecessary time to achieve the goals of the development effort, thus delaying the introduction of new products, which can be very costly. Phase Separations has developed a new, reversed- phase HPLC Column Selection chart, which scientifically differentiates many brands of HPLC columns. The resulting HYDROPHOBICITY Chart provides the analytical chemist with a performance ranking for each column. This allows the chemist to quickly select the best column for his/her application. With proper use, this selection can result in much faster methods development success, due to use This course provides the chemist with a complete understanding of chromatographic particle technology and performance. A wide range of HPLC columns from different vendors were evaluated. The development of the performance test, and how the chart is constructed is explained in detail. Examples of using the chart to solve application problems are given. A complete booklet of the course presentation materials will be furnished for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. Duration: 1.5 hours Order Course # PSL Phase Separations

24 ...because Chromatography is still a Science SM Reversed-Phase HPLC Methods Development Course Abstract: This is a full day course is designed to provide critical information important to those developing new methods, as well as those redeveloping existing methods to improve performance in the areas of sensitivity, robustness and sample throughput. Examples are given, and a complete set of handouts is provided for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. Key topics include: Impact of Particle Technology on Chromatographic Performance Resolution Suggested Methods Development Strategy Benefits of Optimizing Column Dimensions and Particle Size Optimization Software Troubleshooting Tips Duration: 7 hours Order Course # PSL Phase Separations

25 Advanced HPLC Column Characterization & Selection Using the Hydrophobicity Chart neutral base neutral base 0 Time (min) 50

26 Silica Pore Structure * Porous Silica > 99% of Surface Area in the Pores 10nm 100 Å * Pore Size Surface Area SA ~ 3 SV PD SA - Surface Area, m 2 /g SV - Specific Pore Volume, ml/g PD - Pore Diameter, Å or nm Typical Surface Area m 2 /g

27 Silanol Types H H Vicinal O O Si Si O O O Geminal OH OH Si O O H Si O O Lone Si Si Si O O O O O

28 Bonded Phase: Trifunctional Synthesis Cl Si OH + Cl Si Cl c c c c c c c c (Hydrolysis) OH Si Si o o Si o Si o c c c c c c c c c c c c c c c c + HCl Si Si o o Si c c c c c c c c

29 Bonded Pore C18 CH3 Si - O - Si -CH3 CH3 Å 50 CH3 Si - O - Si -CH3 CH3 Si-CCCCCCCCCCCCCCCCCC Si - OH Endcapped Silanol Si-CCCCCCCCCCCCCCCCCC Si-CCCCCCCCCCCCCCCCCC Si - OH CH3 Si - O - Si -CH3 CH3 CH3 Si - O - Si -CH3 CH3 CH3 Si - O - Si -CH3 CH3 Si - OH Si - OH Si-CCCCCCCCCCCCCCCCCC Si-CCCCCCCCCCCCCCCCCC Si-CCCCCCCCCCCCCCCCCC Si-CCCCCCCCCCCCCCCCCC Si-CCCCCCCCCCCCCCCCCC Si - OH Si-CCCCCCCCCCCCCCCCCC = C18 25 Å Si - OH = Silanol Group

30 Ligand Density (Surface Coverage) * Better measure of material's characteristics χ = %C [ %C 100 SA 1 - MW - 1 ] 100 nc 12 = µmoles/m2 SA - Specific Surface Area %C - % Carbon Load MW - Molecular Weight of Ligand nc - # of Carbon Atoms in Ligand Ligand Density Primary Ligand Density Silanols Ligand Density Surface Area Hydrolytic Stability Ligand Density

31 ION EXCHANGE (-) ph 14 Cation (+)pka 10.6 ph % Ionized 12.6 Neutral (+) ph 7 Anion 8.6 ~100% 6.7 ~100% (-) pka 4.7 ph Neutral

32 Terminology Surface Silanol Groups * Changes surface charge as ph 2 7 OH O + H + Si Si Behaves as a Cation Exchanger (ph 2) (ph 7)

33 Tailing vs. Buffer ph Tailing Factor Conventional C 18 Modern C 18 Ideal Buffer ph B. A. Alden, T. H. Walter

34 Effect of Mobile Phase ph on Selectivity 12 base 8 Ret. Time 4 0 acid neutral Buffer ph Base ph 3 ph 8 Acid Neutral Acid Neutral Base

35 Reversed-Phase Selectivity Chart 20 Waters Spherisorb ODS 1 Resolve C 18 (Log Scale) 10 Platinum C 18 Zorbax Rx C 8 µbondapak C 18 Zorbax SB C 8 Alltima C 8 YMC J'Sphere L 80 Lichrosorb Select B Waters Spherisorb C 8 Lichrospher Select B Waters Spherisorb ODS 2 Purospher RP 18 Nucleosil C 18 YMC J'Sphere M 80 Alltima C 18 Silanol Activity YMC-Basic Prodigy C 8 Nova-Pak C 8 Hypersil BDS C 8 Kromasil C 8 Zorbax XDB C 8 Inertsil C 8 Symmetry C 8 Hypersil ODS Zorbax SB C 18 Nova-Pak C 18 Hypersil BDS C 18 Hypersil Hypurity Elite Zorbax Rx C 18 Zorbax XDB C 18 Inertsil ODS-3 Inertsil ODS-2 Kromasil C 18 Prodigy C 18 Symmetry C 18 YMC J'Sphere H SymmetryShield RP Hydrophobicity (Log Scale)

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37 ...because Chromatography is still a Science SM Solid Phase Extraction Technology Course Abstract: Solid Phase Extraction (SPE) is a powerful tool for sample preparation and has been growing in popularity because of the significant benefits in reduced sample preparation cost and increased sample throughput it can bring to your laboratory. In addition, utilizing a chromatographic bed in a sample preparation protocol can solve the difficult problems of increasing method sensitivity, accuracy and precision for your analytical applications. This seminar has been specifically designed for the chromatographer and will include: 1. Introduction and theory 2. Strategies for use cleanup, fractionation and trace concentration 3. Chemistries and device configurations 4. Experimental techniques and demonstrations 5. Method development and application assistance resources 6. Method troubleshooting tips 7. Applications using the latest in SPE technology A complete booklet of the course presentation materials will be furnished for future reference. A Certificate of Achievement will be issued to all attendees. Duration: 4 hours Order Course # PSL Phase Separations

38 Solid Phase Extraction Technology A Powerful Tool in Sample Preparation

39 Solid Phase Extraction (SPE) Technology Goals of Sample Preparation * Remove Interferences * Make Matrix Compatible with Analysis * Concentrate Sample to Increase Sensitivity * Derivatization * Protect Analytical Column + Simpler + Improve Accuracy + More Convenient + More Reproducible + More Cost Effective + Improve Safety

40 Solid Phase Extraction (SPE) Technology Precipitation Chemical Technique Utilizing the Addition of a Solvent to the Sample which causes One, or Some of the Compounds to Fall Out of Solution as Particulates

41 Solid Phase Extraction (SPE) Technology Liquid-Liquid Extraction (LLE) Chemical Technique Where an Immiscible Solvent is Added to the Sample which then Separates into 2 Distinct Liquid Phases. Some Sample Analytes will go into the Bottom Phase (Aqueous), Some will Separate into the Top Phase, (Organic)

42 Solid Phase Extraction (SPE) Technology How Does Chromatography Work? Stationary Phase Mobile Phase Analyte A Analyte B

43 Solid Phase Extraction (SPE) Technology Housing/Body Filters/Frits Chromatographic Bed (Sorbent) Luer Tip

44 Solid Phase Extraction (SPE) Technology Vacuum Manifolds

45 Solid Phase Extraction (SPE) Technology SPE Strategies Elute the product of interest, retain interferences want k 0 for analyte want k large for interferences Elute interferences, retain product want k 0 for interferences want k large for analyte * Concentrate product of interest ^ want k large for analyte / load large sample volume ^ elute concentrated analyte ^ enhanced sensitivity

46 Solid Phase Extraction (SPE) Technology Solid Phase Extraction (SPE) The Chromatographic Bed In The Cartridge Can Perform Three Critical Functions: ) Chemical Clean-up (Pure Red) 2) Pre-Concentration (Dark Purple) 3) Fractionation (Pure Blue & Pure Red)

47 Solid Phase Extraction (SPE) Technology Breakthrough Study * Series of Experiments -- Passing Increasing Volumes of Sample Matrix Thru SPE Device * Follow Elution Protocol * Determine Analytical Results (% Recovery) * Plot Results vs Sample Volume * Determine Maximum Volume for Each of the Analytes -- Find Maximum Volume for Method

48 Solid Phase Extraction (SPE) Technology % Recovery as a Function of k % Recovery k = 10 k = Volume Thru SPE Device (ml)

49 Solid Phase Extraction (SPE) Technology Mass Balance -- Measuring Analyte Concentration in all Fractions -- Will Show Breakthrough During Loading Proper Load Sample Analyte Fully Retained During Loading OverLoad Sample Analyte Breaks Through During Loading Poor Conditioning Drying Out Sample Analyte Breaks Through During Loading Poor Recovery

50 Solid Phase Extraction (SPE) Technology Fraction 1 (Clear) Water Polar Analytes (Clear) Fraction 2 8% IPA Pure Red Fractionation Fraction 3 35% IPA Pure Blue Fraction 4 70% IPA Non-Polar Analytes (Clear)

51 Solid Phase Extraction (SPE) Technology Low Concentration Analyte (Light Purple) Initially Retained and Concentrated as Large Sample Volume is Processed Interferences Discarded Then Concentrated Analyte is Eluted by a Different Elution Solvent Trace Concentration

52 Methods Development Approach Determine Nature of Analytes, and Sample Matrix Similar to Existing Method in Lab? Yes No Try Conditions - Evaluate for Capacity/ Breakthru, Recovery Reproducibility, Robustness and Ruggedness Meets Goals? Yes Validate Method No Yes Review SPE Bibliography, and Literature References for Exact or Similar Applications Any? No Determine Method Goals, and Strategy Call SPE Vendor Chromatography Mode Develop Method Conditions

53 Solid Phase Extraction (SPE) Technology Some Causes of Poor Recovery * Poor/No Condtioning * Drying Out Before Loading * Poor Chromatographic Conditions - k too Low (Breahthrough) - k too Large (Still Adsorbed) * Basic Compounds Strongly Retained By Deprotonated Silica Silanols of Sorbent * Metals in Silica Based Sorbents Can Interact With Metal Chelator Analytes

54 Room Temperature Stability of Sample in Solutions Betamethasone Valerate (20 µg/ml) a t=0 stock 3 Day 5 Day b b % Nominal Concentration Saline Acetonitrile Methanol Spiked Solutions b b Minutes a: betamethasone valerate b: breakdown products t=5 days saline t=5 days MeOH t=5 days MeCN

55 Results: Tetracyclines Compound Concentration % Recovery % RSD Minocycline 2.5 µg/ml Tetracycline 2.5 µg/ml AU Column: SymmetryShield RP8, 5 µm, 3.0 x 150 mm Mobile Phase: 0.1% TFA in Water:Acetonitrile: Methanol (91:7:2) Detection: UV at 270 nm Flow Rate: 0.9 ml/min. Injection Volume: 20 µl Sample Identification: Peak 1: Minocycline Peak 2: Tetracycline Peak 3: Demeclocycline (I.S.) Minutes Cheng

56 ...because Chromatography is still a Science SM Selecting Optimum HPLC Column Dimensions and Stationary Phase Particle Size Course Abstract: In order to speed the introduction of new products to market, analytical chemists are now often faced with two additional challenges; analyzing compounds at lower concentration levels, as well as analyzing more samples per unit time. Developing new HPLC methods with high sensitivity and high sample throughput is becoming a significant priority. Phase Separations has created a new course that covers the key elements of optimizing HPLC column dimensions, and stationary phase particle size to meet these methods development goals. A discussion of the impact of the ratio of column length to particle size is given. This critical ratio determines the optimum performance of the column in the method, as it relates to efficiency, analysis time and back pressure. Examples are given, and a complete set of handouts is provided for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. Duration: 1 hour Order Course # PSL Phase Separations

57 Selecting Optimum HPLC Column Dimensions and Stationary Phase Particle Size

58 Benefits of Optimizing Column Dimensions Reduction in analysis time with decreasing column length Increase in mass sensitivity with decreasing column diameter Increase in resolution with increasing ratio of column length to particle size Increase in loadability with increasing column diameter

59 How to Achieve Shorter Analysis Times -- Without Sacrificing Resolution and Efficiency Reduce particle size Use shorter columns Maintain constant ratio of column length to particle size

60 Ratio of Column Length to Particle Size Determines 3 Key Performance Measures: * Maximum Column Efficiency * Shortest Analysis Time for Given Pressure * Pressure Drop at Given Analysis Time

61 Selection of Proper L/dp Ratio Type of Analysis L/dp Difficult > 50,000 Normal ~ 30,000 Fast < 15,000 Column 300mm -- 5µm 150mm -- 5µm 50mm -- 3µm

62 Constant Ratio of Column Length to Particle Size (30,000) 120 Resolution µm x 30 cm 5 µm x 15 cm 3 µm x 9 cm 1 µm x 3 cm Analysis Time [min]

63 Performance Characteristics of a 5 µm and a 3.5 µm Symmetry Column Analysis Time [min] Plate-Count µm, 3.9 mm x 150 mm 3.5 µm, 4.6 mm x 100 mm 2000 L/dp~30,000 B. A. Alden, U. D. Neue Flow-Rate [ml/min]

64 Enhancing Sensitivity Sensitivity can be enhanced by: increasing efficiency decreasing asymmetry reducing column diameter using shorter columns decreasing detector noise using more sensitive detection modes decreasing capacity factor

65 Solvent Consumption -- Column Length Flow rate: 1 ml/min Column Length Elution Time Solvent % Savings 15 cm 6.5 min 6.5 ml cm 4.5 min 4.5 ml 31% 5 cm 2.5 min 2.5 ml 62%

66 Solvent Consumption -- Column Diameter Linear Velocity: cm/sec Column Length: 15 cm Elution Time: 8 min Column Diameter Flow Rate Solvent % Savings 4.6 mm 1.4 ml/min 11.2 ml mm 1.0 ml/min 8.0mL 28% 3.0 mm 0.6 ml/min 4.8 ml 57% 2.1 mm 0.3 ml/min 2.4 ml 79%

67 Instrument Optimization Needed for Narrow Bore and Microbore Columns Instrument Bandspread: 25µl Areas to optimize: * Detector Flow Cell * Injector Sample Loop * 0.005" Tubing * Perfect Connections * Detector Time Constant < 0.2 (normally > 80µl)

68 Performance Monitoring To perform a measurement: - disconnect column from system - connect injector directly to detector Parameter Flow Rate Chart Speed Detector Sensitivity Time Constant Setting 1.0 ml/min 20 cm/min AUFS 0.2 seconds or less dilute test mixture 1 to 10 in mobile phase inject 2 to 5 µl of this solution

69 Performance Monitoring Using 5 sigma efficiency method, measure the peak width at 4.4% of peak height Convert to microliters using the following equation: ( ) 2cm ( )( ) ( ) 1min 1 ml 1000µL = PW 20 cm min. ml 100 (µl) where: 1min/20cm = chart speed 1 ml/min = flow rate 1000 µl/ml = volume correction factor Typical LC System should be 100µL +/- 30µL Microbore System should be no greater than 20µL

70 ...because Chromatography is still a Science SM Troubleshooting Common HPLC Problems Course Abstract: This course covers all commonly encountered HPLC problems and is designed for both the users and the developers of HPLC methods. It will assist the chromatographer in spotting potential problems during the operation of their HPLC and in eliminating instrument downtime. Covered subjects include: Key examples are shown, and each attendee will receive a complete set of the slides in hardcopy form to keep for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. problem prevention (column installation, equilibration, protection, storage) performance monitoring (instrument band spreading performance, column efficiency performance) Duration: 2 hours Order Course # PSL examples and troubleshooting of common problems 1998 Phase Separations

71 SM Troubleshooting Common HPLC Problems

72 Course Outline * Column Maintenance Information * Problem Sources * Column Installation and Equilibration * Column Use * Performance Monitoring * Column Protection * Column Storage * Troubleshooting of Column Problems - Peak Shape Problems - Retention Time Problems ^ Hydrophobic Collapse - Miscellaneous Problems

73 Troubleshooting Problem CHEMISTRY COLUMN/GUARD SOLVENT SAMPLE HARDWARE PUMP INJECTOR DETECTOR INTEGRATOR Always do the easiest thing first: 1. Stop flow 2. Remove the column

74 Installation and Equilibration Waters Spherisorb Parker Style other Waters Columns Waters

75 Extra-Column Band Spreading Column Connection

76 Extra-Column Band Spreading.009".040".020" note the differences of the inner diameter of this tubing

77 Performance Monitoring Effect of Connecting Tubing on System Bandspreading.009".020".040" sample band dispersion inside tubing

78 Extra-Column Band Spreading The Observed Bandwidth (TOT) * Sum of the Bandspreading Contributions - Column (COL) - Extra-Column (EC) Instrument components TOT COL EC σ 2 = σ 2 + σ 2

79 Performance Monitoring To perform a Band Spread measurement: - disconnect column from system - connect injector directly to detector Parameter Flow Rate Chart Speed Detector Sensitivity Time Constant Setting 1.0 ml/min 20 cm/min AUFS 0.2 seconds or less dilute test mixture 1 to 10 in mobile phase inject 2 to 5 µl of this solution

80 Performance Monitoring Using 5 sigma efficiency method, measure the peak width at 4.4% of peak height Convert to microliters using the following equation: ( )( 1min )( 1 ml ) ( 1000µL ) = 2cm PW 20 cm min. ml 100 (µl) where: 1min/20cm = chart speed 1 ml/min = flow rate 1000 µl/ml = volume correction factor Typical LC System should be 100µL +/- 30µL Microbore System should be no greater than 20µL

81 Column Protection Extension of column lifetime with Guard Column using a mixture of sulfa drugs as the sample A. Initial injection on Symmetry C 8 Sentry guard column B. After 550 injections on same Sentry guard column C. New Sentry Guard column for injection 551 on analytical column

82 Column Collapse voids - high back pressure, distorted and/or double peaks

83 Extra Column Effects Isocratic LC - Time Constant Differences left is 0.1 secs right is 10 secs note the noisy baseline on left chromatogram

84 Hydrophobic Collapse Chromatographers have observed complete loss of retention when working with low organic mobile phases. Vo? 40 min 40 min When we investigated this phenomenon we found that retention times were stable for over 20 hrs (77 injections) using 100% aqueous mobile phase. However, when flow was stopped, then restarted, retention was lost.. This observation suggested that the mobile phase is extruded from the pores when pressure is released from the column.

85 Retention Time Reproducibility Solvent Composition - Hydrophobic Collapse Temperature ph Ion Pairing Ionic Strength Extraneous Peaks Gradient Control

86 ph - Control

87 ...because Chromatography is still a Science SM Purchasing High Performance Chromatographic Supplies Course Abstract: This course is designed specifically for Purchasing Professionals to enhance their understanding of the technology of Chromatography as it relates to improving their ability to effectively purchase these supplies while meeting corporate goals, such as improved customer satisfaction, and reduced purchasing costs through vendor consolidation. Key topics include: Chromatography technology / terminology (with a focus on HPLC) [includes live demonstration] End user performance needs Regulatory considerations Manufacturer / Supplier considerations Key purchasing specifications This full day course will provide you with the technical information you will need to intelligently discuss product specifications and performance, as well as outline critical issues in the development of an improved purchasing program for your chromatography supplies. Specific examples will be given showing how to achieve significant benefits by organizing your chromatography purchasing needs in product/ performance/ vendor priority. A complete booklet of the course presentation materials will be furnished for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. Duration: 7 hours Order Course # PSL Phase Separations

88 Purchasing High Performance Chromatographic Supplies A Course For Purchasing Professionals

89 Purchasing High Performance Chromatographic Supplies Topics * Chromatography Technology Backgrounder * Terminology * Hands-On Chromatographic Experiments * HPLC Supplies * GC Supplies * End-User Performance Needs * Manufacturer / Supplier Considerations * Regulatory Considerations * Key Purchasing Specifications

90 Purchasing High Performance Chromatographic Supplies Chromatography Technology Backgrounder Solvent HPLC Column Data Injector Pump Detector Waste

91 Purchasing High Performance Chromatographic Supplies Chromatography Terminology * Sample - The Original Representative Material Which Is To Be Analyzed -- Also Called The Sample Matrix * Analyte(s) - A Specific Compound(s) Contained In The Sample Which Is(Are) To Be Separated And Analyzed * Compound - Pure Chemical Component In A Sample, Also Called An Analyte Or Solute

92 Purchasing High Performance Chromatographic Supplies Chromatography Terminology * How Does A Chromatographic Column Work? Sample Band Analyte Bands

93 Purchasing High Performance Chromatographic Supplies Chromatography Terminology * How Does A Chromatographic Column Work? WHY Do They Separate? Yellow Likes The Mobile Phase And Goes As Fast As It Does - And Comes Out First Red Likes The Stationary Phase Somewhat, And Slows Down Blue Likes The Stationary Phase Best, And Slows Down The Most

94 Purchasing High Performance Chromatographic Supplies Chromatography Terminology * How Do You Get Peaks? Detector Sample Band Sample Peak Detector Cell

95 Purchasing High Performance Chromatographic Supplies Chromatography Terminology How Do You Identify Which Compounds Are Which When You Have A Series Of Peaks On You Chromatogram??? I Know From The Peaks' Retention Time Determined By Pure Standards Time

96 Purchasing High Performance Chromatographic Supplies Chromatography Terminology How Do You Know How Much Is Present In The Sample??? I Know From The Peaks' Area And Height Time

97 Purchasing High Performance Chromatographic Supplies Chromatography Terminology Look What Happens When It Rains Right After I WAX My Car! [Non-Polar Surface] Water Beads Up Polar Water DISLIKES Non-Polar Surface

98 Purchasing High Performance Chromatographic Supplies Chromatography Terminology * Solid Phase Extraction (SPE) Chemical Technique Using Column Chromatography In A Small, Single Use Disposable Cartridge Format. Can Perform Chemical Separation, Concentration And Fractionation Used With Vacuum Stations, Syringes And Automated Sample Processors

99 Purchasing High Performance Chromatographic Supplies Chromatography Terminology Big Ones Calibration Curve Molecular Weight Distribution "Finger Print" Mol. Wt. Retention Time

100 Purchasing High Performance Chromatography Supplies End User Performance Needs Stages Of A "Method" The Success Of The Method Is Critical: - Reputation Of The Researcher - Reduce Cost And Time To Market For A New Product If The Method Is Not Reproducible (Has To Be Redeveloped And Revalidated -- Can Be Incredibly Costly)

101 Purchasing High Performance Chromatography Supplies Manufacturer/Supplier Considerations Primary Manufacturer Column RePacker Specialized Distributor YOU 3rd Party Supplies Distributor Secondary Manufacturer/ Column Packer Column Packer Sourcing HPLC Columns

102 Purchasing High Performance Chromatography Supplies Manufacturer/Supplier Considerations * Manufacturers -- Some Names: Primary: Secondary: Alltech Bio-Rad GL Science Hamilton Hypersil Macherey-Nagel E. Merck Akso Nobel Polymer Labs Rockland HP Showa Denko Separations Group Toso Haas Waters Whatman YMC BrownLee Jones Keystone Phenomenex Supelco Packer: Bischoff Beckman Chrompack HPLC Technology HP Isolation Tech. Metachem Micra Resolution Systems Capital Hichrom Mac-Mod PE + Many Others

103 Purchasing High Performance Chromatography Supplies Manufacturer/Supplier Considerations Secondary Manufacturer/Packer Distributor Primary Packer/Technical Distributor End User Distributor Packer/ Repacker End User

104 Purchasing High Performance Chromatography Supplies Key Purchasing Considerations Brands Supplied By Different Vendors Brand # Vendors # Columns Hypersil Inertsil 5 40 Kromasil 5 75 LiChrospher 3 8 Nucleosil Waters Spherisorb 8 80

105 ...because Chromatography is still a Science SM Understanding Reversed-Phase HPLC Separations Effect of Silica Type/Activity Course Abstract: This course is designed for those individuals developing new reversed-phase HPLC methods. Key topics include: Resolution Theory Controlling Chromatographic Parameters Silica Particle Technology Examples are given, and a complete set of handouts is provided for future reference. A Certificate of Achievement will be given to all attendees, which documents your participation in this training program. Duration: 1.5 hours Order Course # PSL Bonding Process Packing Material Comparison Reproducibility Concerns 1998 Phase Separations

106 HPLC Method Development & Troubleshooting Understanding Reversed-Phase Separations 'The Effect of the Silica Chemistry' Phase Separations Customer Education Programme

107 Review of Chromatographic Terminology RETENTION, k' = (V 1 -V 0 )/V 0 SELECTIVITY, α = k' 2 /k' 1 PLATE COUNT, N = 16(V/W) 2

108 Resolution Resolution is a numerical measure of the separation of two compounds and is a function of N, k' and α. This equation is only valid for isocratic separations. Selectivity, retention and peak widths may be further enhanced by using gradients. R s =( N ). (α-1). k' 2 4 α (k' 2 +1)

109 k', N, α How They Control Resolution