A Practical User's Guide
Contents
1. High pressure liquid solid chromatography HPLC is rapidly becoming the method of choice for separations and analysis in many fields Almost anything that can be dissolved can be separated on some type of HPLC column However with this versatility comes the necessity to think about the separa tion desired and the best way to achieve it HPLC is not now and probably never will be a turn key push button type of operation Many dedicated system in a box packages are sold for specific separations but all of these still offer wide possibilities for separation Changing the column and the flow rate lets you change the separation and the amount of sample you can inject This is not the worst thing in the world for it does create great opportunity for the chromatographer and a great deal of job security for the instrument operator Fortunately controlling separations is not nearly as complicated as much of the literature may make it seem My aim is to cut through much of the detail and theory to make this a usable technique for you The separation models I present are those that have proven useful to me in predicting separations I make no claim for their accuracy except that they work There are many excel lent texts on the market in the technical literature and on the Internet con tinuously updated and revised that present the history and the current theory of chromatography separations This book was written to fill a need hopefully your2. Generations come and go but you can always count on Wiley to provide you the knowledge you need when and where you need it an VE tll WILLIAM J PESCE PETER BOOTH WILEY PRESIDENT AND CHIEF EXECUTIVE OFFICER CHAIRMAN OF THE BOARD HPLC A Practical User s Guide SECOND EDITION Marvin C McMaster WILEY INTERSCIENCE A John Wiley amp Sons Inc Publication Copyright 2007 by John Wiley amp Sons Inc All right reserved Published by John Wiley amp Sons Inc Hoboken New Jersey Published simultaneously in Canada No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 978 750 8400 fax 978 750 4470 or on the web at www copyright com Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 201 748 6011 fax 201 748 6008 or online at http www wiley com go permission Limit of Liability Disclaimer of Warranty While the publisher and author have used their best efforts in preparin
3. HPLC A Practical User s Guide SECOND EDITION Marvin C McMaster WILEY INTERSCIENCE A John Wiley amp Sons Inc Publication HPLC 1807 WILEY 2007 a BICENTENNIAL TVINN3ANA gt 18 THE WILEY BICENTENNIAL KNOWLEDGE FOR GENERATIONS ach generation has its unique needs and aspirations When Charles Wiley first opened his small printing shop in lower Manhattan in 1807 it was a generation of boundless potential searching for an identity And we were there helping to define a new American literary tradition Over half a century later in the midst ofthe Second Industrial Revolution it was a generation focused on building the future Once again we were there supplying the critical scientific technical and engineering knowledge that helped frame the world Throughout the 20th Century and into the new millennium nations began to reach out beyond their own borders and a new international community was born Wiley was there expanding its operations around the world to enable a global exchange of ideas opinions and know how For 200 years Wiley has been an integral part of each generation s journey enabling the flow of information and understanding necessary to meet their needs and fulfill their aspirations Today bold new technologies are changing the way we live and learn Wiley will be there providing you the must have knowledge you need to imagine new worlds new possibilities and new opportunities
4. Marvin C McMaster Florissant MO HPLC PRIMER ADVANTAGES AND DISADVANTAGES OF HPLC The first things we need to understand are how an HPLC system works its best applications and advantages over other ways of separating compounds and other techniques that might compliment or even replace it Is there a faster easier cheaper or more sensitive method of achieving your results The answer is yes no maybe It really depends on what you are trying to achieve HPLC s virtue lies in its versatility I have used it to separate compounds of molecular weights from 54 to 450 000 Daltons Amounts of material to be detected can vary from picograms and nanograms analytical scale to micro grams and milligrams semi preparative scale to multigrams preparative scale There are no requirements for volatile compounds or derivatives Aqueous samples can be run directly after a simple filtration Compounds with a wide polarity range can be analyzed in a single run Thermally labile com pounds can be run I had one customer whose company made explosives for primers Her first job of the day was to explode samples of the previous day run with a rifle Her second job was to carry out an HPLC analysis of that day s run An HPLC offers a combination of speed reproducibility and sensitivity Typical runs take from 10 to 30min but long gradients might consume 1 to 2hrs I have seen 15 to 30 sec stat runs on 3 um columns in hospital lab
5. Finally used correctly HPLC is a great tool for rapid reaction monitoring either in glassware or in large production kettles I started my analytical career with a HPLC system cast off by the Analytical Department and a 15 min train ing course by another plant monitoring chemist He gave me an existing HPLC procedure for my compound and turned me loose The next day I was getting research information I could see starting material disappear intermediates form and both final product and by products appear It was like having a window on my reaction flask through which I could observe the chemistry of the ongoing synthesis Later I used the same technique to monitor a produc tion run in a 6000 gallon reactor The sampling technique was different but the HPLC analysis was identical Versatility however brings with it challenge An HPLC is easily assembled and easily run but to achieve optimum separation the operator needs to understand the system its columns and the chemistry of the compounds being separated This will require a little work and a little thought but the skills required do offer a certain job security I don t want to leave you with the impression that I feel that HPLC is the perfect analytical system The basic system is rather expensive compared with some analytical tools columns are expensive with a relatively short operating life solvents are expensive and disposal of used solvent is becoming a real headache Other te
6. differences in polarity size shape charge specific affinity for a site stereo and optical iso merism Columns exist to separate mixtures of small organic acid present in the Krebs cycle to mixtures of macromolecules such as antibody proteins and DNA restriction fragments Fatty acids can be separated based on the number of carbons atoms in the chains or a combination of carbon number and degree of unsaturation Electrochemical detectors exist that detect separations at the picogram range for rat brain catecholamines Liquid crystal compounds are routinely purified commercially at 50g per injection The typical injection however is of 20 uL of solvent containing 10 50 ng of sample Typical runs are made at 1 2mL min and take 5 15 min isocratic or Ihr gradient 1 2 OTHER WAYS TO MAKE MY SEPARATION Obvious there are many other analytical tools in the laboratory that could be used to make a specific separation Other techniques may offer higher 10 ADVANTAGES AND DISADVANTAGES OF HPLC sensitivity less expensive equipment different modes of separation or faster and dirty tools for cleaning a sample before injection into the HPLC Often a difficult separation can only be achieved by combining these tools in a sequential analysis or purification I ll try to summarize what I know about these tools their strengths and drawbacks 1 2 1 FPLC Fast Protein Liquid Chromatography FPLC is a close cousin of the HPLC optimized to run biolog
7. required about the way I teach First I have learned that repetition is a powerful tool not a sign of incipient senility as many people have hinted Second I have found in lecturing that few people can stand more than 45 minutes of technical material at one sitting However I have also learned that carefully applied humor can sometimes act as a mental change of pace Properly applied it allows us to continue with the work at hand So occa sionally I will tiptoe around the lab bench I do not apologize for it but I thought you ought to know The instrument itself is the most effective teacher Think logically about the system and the chemistry and physics occurring inside the column You will be surprised how well you will be able to predict and control your separation Remember HPLC is a versatile powerful but basically simple separation tool It is a time machine that can speed your research and thereby allow you to do many things not possible with slower techniques It is both an analytical and a preparative machine When I finish I hope you will have the confidence to run your instrument make your own mistakes and be able to find your own solutions PREFACE xiii Your HPLC success depends on three things 1 The suitability of the equipment you buy 2 Your ability to keep it up and running or find someone to service it and 3 The support you receive starting out in new directions or in solving prob lems that come up
8. 2006040640 Printed in the United States of America 10987654321 CONTENTS PREFACE xi HPLC PRIMER 1 1 Advantages and Disadvantages of HPLC 3 1 1 How It Works 4 1 2 1 1 1 A Separation Model of the Column 5 1 1 2 Basic Hardware A Quick First Look 7 1 1 3 Use of Solvent Gradients 8 1 1 4 Ranges of Compounds 9 Other Ways to Make My Separation 9 1 2 1 FPLC Fast Protein Liquid Chromatography 10 1 2 2 LC Traditional Liquid Chromatography 10 1 2 3 GLC Gas Liquid Chromatography 11 1 24 SFC Supercritical Fluid Chromatography 11 1 2 5 TLC Thin Layer Chromatography 12 1 2 6 EP Electrophoresis 12 1 2 7 CZE Capillary Zone Electrophoresis 13 2 Selecting an HPLC System 15 2 1 2 2 Characteristic Systems 16 2 1 1 Finding a Fit Detectors and Data Processing 16 2 1 2 System Models Gradient Versus Isocratic 16 2 1 3 Vendor Selection 17 2 1 4 Brand Names and Clones 17 2 1 5 Hardware Service Support 18 System Cost Estimates 19 2 2 1 Type I System QC Isocratic Cost 10 15 000 19 2 2 2 Type U System Research Gradient Cost 20 25 000 19 vi CONTENTS 2 2 3 Type HI System Automated Clinical Cost 25 35 000 20 2 2 4 Type IV System Automated Methods Cost 30 50 000 21 2 3 Columns 21 2 3 1 Sizes Analytical and Preparative 21 2 3 2 Separating Modes Selecting Only What You Need 22 2 3 3 Tips on Column Use 23 Running Your Chromatogr
9. alves can partially compensate for the lack of an autosampler 1 2 2 LC Traditional Liquid Chromatography LC is the predecessor of HPLC It uses slurry packed glass column filled with large diameter 35 60 um porous solid material Materials to be separated are dissolved in solvent and applied directly to the column head The mobile phase is placed in a reservoir above the column and gravity fed to the column to elute the sample bands Occasionally a stirred double chamber reservoir is used to generate linear solvent gradients and a peristaltic pump is used to feed solvent to the column head Packing materials generally made of silica gel alumina and agarose are available to allow separation by partition adsorp tion ion exchange size and affinity modes A useful LC modification is the quick clean up column The simplest of this is a capillary pipette plugged with glass wool and partially filled with packing material The dry packed column is wetted with solvent sample is applied and
10. aph 25 3 1 Set up and Start up 25 3 1 1 Hardware Plumbing 101 Tubing and Fittings 26 3 1 2 Connecting Components 28 3 1 3 Solvent Clean up 30 3 1 4 Water Purity Test 33 3 1 5 Start up System Flushing 34 3 1 6 Column Preparation and Equilibration 35 3 2 Sample Preparation and Column Calibration 36 3 2 1 Sample Clean up 36 3 2 2 Plate Counts 37 3 3 Your First Chromatogram 37 3 3 1 Reproducible Injection Techniques 38 3 3 2 Simple Scouting for a Mobile Phase 39 3 3 3 Examining the Chromatogram 40 3 3 4 Basic Calculations of Results 41 HPLC OPTIMIZATION 43 Separation Models 45 4 1 Partition 45 4 1 1 Separation Parameters 48 4 1 2 Efficiency Factor 49 4 1 3 Separation Chemistry Factor 53 4 2 Ion Exchange Chromatography 56 4 3 Size Exclusion Chromatography 57 4 4 Affinity Chromatography 59 Column Preparation 61 5 1 Column Variations 61 5 2 Packing Materials and Hardware 64 5 3 Column Selection 66 CONTENTS vii 6 Column Aging Diagnosis and Healing 73 6 1 Packing Degrading Bonded Phase Loss 74 6 2 Dissolved Packing Material End Voids 77 6 3 Bound Material 78 6 4 Pressure Increases 81 6 5 Column Channeling Center Voids 83 6 6 Normal Phase lon Exchange and Size Columns 84 6 7 Zirconium and Polymer Columns 86 7 Partition Chromatography Modifications 89 7 1 Reverse Phase and Hybrid Silica 89 7 1 1 Ionization Suppression 90 7 1 2 Ion Pai
11. as an equilibration concentration in each layer If we were to draw off the bottom layer and dry it to recover the red dye we would find it still contaminated with the other component the blue dye Repeated washings with fresh lower layer would eventually leave only insignificant amounts of contaminating red dye in the top layer but would also remove part of the desired blue compound Obviously we need a better technique to achieve a complete separation The HPLC column operates in a similar fashion The principle of like attracting like still holds In this case our nonpolar layer happens to be a moist very fine bonded phase solid packing material tightly packed in the column Polar solvent pumped through the column our mobile phase serves as the second immiscible phase If we dissolve our purple dye in the mobile phase then inject the solution into the flow from the pump to the column our two compounds will again partition between the two phases The more non ea Hexane NW ob If Figure 1 2 Separation model 1 separatory funnel HOW IT WORKS 7 polar blue dye will have a stronger partition affinity for the stationary phase The more polar red dye favors the mobile phase moves more rapidly down the column than the blue dye and emerges first from the column into the detector If we could see into the column we would see a purple disc move down the column gradually separating into a fast moving red dis
12. c followed by a slower moving blue disc Fig 1 3 1 1 2 Basic Hardware A Quick First Look The simplest HPLC system is made up of a high pressure solvent pump an injector a column a detector and a data recorder Fig 1 4 Note The high pressures referred to are of the order of 2000 6000 psi Since we are working with liquids instead of gases high pressures do not pose an explosion hazard Leaks occur on overpressurizing the worse problems to be expected are drips streams and puddles Solvent mobile phase from a solvent reservoir is pulled up the solvent inlet line into the pump head through a one way check valve Pressurized in Time T2 T3 T4 De El 610 Y ea A C18 Packing Figure 1 3 Separation model 2 HPLC column Detector Reservoir Pump Injector Column Recorder Figure 1 4 An isocratic HPLC system 8 ADVANTAGES AND DISADVANTAGES OF HPLC the pump head the mobile phase is driven by the pump against the column back pressure through a second check valve into the line leading to the sample injector The pressurized mobile phase passes through the injector and into the column where it equilibrates with the stationary phase and then exits to the detector flow cell and out to the waste collector The sample dissolved in mobile phase or a similar solvent is first loaded into the sample loop and then injected by turning a handle sw
13. chniques offer more sensitivity of detection or improved separation for certain types of compounds i e volatiles by GLC large charged molecules by electrophoresis Nothing else that I know of however offers the laboratory the wide range of separating modes the combination of qualitative and quantitative separation and the basic versatility of the HPLC system 1 1 HOW IT WORKS The HPLC separation is achieved by injecting the sample dissolved in solvent into a stream of solvent being pumped into a column packed with a solid sep HOW IT WORKS 5 arating material The interaction is a liquid solid separation It occurs when a mixture of compounds dissolved in a solvent can either stay in the solvent or adhere to the packing material in the column The choice is not a simple one since compounds have an affinity for both the solvent and the packing On a reverse phase column separation occurs because each compound has different partition rates between the solvent and the packing material Left alone each compound would reach its own equilibrium concentration in the solvent and on the solid support However we upset conditions by pumping fresh solvent down the column The result is that components with the highest affinity for the column packing stick the longest and wash out last This dif ferential washout or elution of compounds is the basis for the HPLC separa tion The separated or partially separated discs of each component dissolve
14. d in solvent move down the column slowly moving farther apart and elute in turn from the column into the detector flow cell These separated compounds appear in the detector as peaks that rise and fall when the detector signal is sent to a recorder or computer This peak data can be used either to quanti tate with standard calibration the amounts of each material present or to control the collection of purified material in a fraction collector 1 1 1 A Separation Model of the Column Since the real work in an HPLC system occurs in the column it has been called the heart of the system The typical column is a heavy walled stainless steel tube 25 cm long with a 3 5 mm i d equipped with large column compression fittings at either end Fig 1 1 Immediately adjacent to the end of the column held in place by the column fittings is a porous stainless steel disc filter called a frit The frit serves two purposes It keeps injection sample particulate matter above a certain size from entering the packed column bed At the outlet end of the column it also serves as a bed support to keep the column material from being pumped into the tubing connecting out to the detector flow cell Each column end fitting is drilled out to accept a zero dead volume compression fitting which allows the column to be connected to tubing coming from the injector and going out to the detector Outlet end fitting I
15. e could switch to a more polar mobile phase such as methanol to make compounds retain longer and have more time to separate We can achieve much the same effect by adding a known percentage of water which is very polar to our starting acetonitrile mobile phase step gradient We could also start with a mobile phase con taining a large percentage of water to make nonpolar compounds stick tightly to the top of the column and then gradually increase the amount of acetoni trile to wash them off solvent gradient By changing either the initial amount of acetonitrile the final amount of acetonitrile or the rate of change of ace tonitrile addition we can modify the separation achieved Separation of very complex mixtures can be carried out using solvent gradients There are however penalties to be paid in using gradients More costly equipment is required solvent changes need to be done slowly enough to be reproducible and the column must be re equilibrated before making the next injection Iso cratic separations made with constant solvent compositions can generally be run in 5 15 min True analytical gradients require run times of around 1 hr with about a 15 min re equilibration But some separations can only be made with a gradient We will discuss gradient development in a later section 1 1 4 Ranges of Compounds Almost any compound that can be retained by a column can be separated by a column HPLC separations have been achieved based on
16. evelopment 12 1 12 2 12 3 Sample Preparation 143 12 1 1 Deproteination 144 12 1 2 Extraction and Concentration 145 12 1 3 SFE Cartridge Column Preparations 145 12 1 4 Extracting Encapsulated Compounds 147 12 1 5 SFE Trace Enrichment and Windowing 148 12 1 6 Derivatives 151 Methods Development 151 12 2 1 Standards Development 152 12 2 2 Samples Development 154 Gradient Development 156 Application Logics Separations Overview 13 1 13 2 13 3 13 4 13 5 13 6 13 7 13 8 Fat Soluble Vitamins Steroid and Lipids 159 Water Soluble Vitamins Carbohydrates and Acids 160 Nucleomics 161 Proteomics 162 Clinical and Forensic Drug Monitoring 163 Pharmaceutical Drug Development 164 Environmental and Reaction Monitoring 164 Application Trends 165 125 135 137 143 159 CONTENTS ix 14 Automation 167 15 16 14 1 14 2 14 3 14 4 14 5 14 6 Analog to Digital Interfacing 167 Digital Information Exchange 169 HPLC System Control and Automation 169 Data Collection and Interpretation 170 14 4 1 Preinjection Baseline Setting 171 14 4 2 Peak Detection and Integration 171 14 4 3 Quantitation Internal External Standards 172 Automated Methods Development 172 14 5 1 Automated Isocratic Development 173 14 5 2 Hinge Point Gradient Development 176 Data Exportation to the Real World 177 14 6 1 Word Processors ASC DOC RTF WS WP Forma
17. g this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages For general information on our other products and services or for technical support please contact our Customer Care Department within the United States at 800 762 2974 outside the United States at 317 572 3993 or fax 317 572 4002 Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at www wiley com Library of Congress Cataloging in Publication Data McMaster Marvin C HPLC a practical user s guide Marvin C McMaster 2nd ed p cm Includes bibliographical references and index ISBN 13 978 0 471 75401 5 cloth ISBN 10 0 471 75401 3 cloth 1 High performance liquid chromatography 1 Title QD79 C454M36 2007 543 84 de22
18. he material in the book Part II shows you how to make the best use of the common columns and how to keep them up and running Chapter 6 on column healing should pay for the book in itself It discusses the various pieces of HPLC equipment how they go together to form systems and how to systematically troubleshoot system problems We will take a look at the newest innovations and improve ments in column technology and how to put these to work in your research New detectors are emerging to make possible analysis of compounds and quantities that previously were not detectable Finally in Part III we will talk about putting the system to work on real world applications We will look at systematic methods development both manual and automated and the logic behind many of the separations that others have made We will discuss how to interface the HPLC system to com puters and robotic workstations I will also give you my best guesses as to the direction in which HPLC columns systems detectors and liquid chromato graphy mass spectrometer LC MS systems will be going It is important to give credit where it is due Christopher Alan McMaster created many of the illustrations in this text before he died of the ravages of muscular dystrophy six years ago I supplied hand drawn sketches of the illus trations I used on boards in my classes Chris turned them into art on his Macintosh His collaborative efforts are greatly missed A brief note is
19. ical macromole cules on pressure fragile agarose or polymeric monobead based columns It uses the same basic system components but with inert fluid surfaces i e Teflon titanium and glass and is designed to operate at no more than 700 psi Inert surfaces are necessary since many of the resolving buffers contain high concentrations of halide salts that attack and corrode stainless steel sur faces Glass columns are available packed with a variety of microporous high resolution packings size partition ion exchange and affinity modes A two pump solvent gradient controller injector valve filter variable detector and a fraction collector complete the usual system The primary separation modes are strong anion exchange or size separation rather than reverse phase partition as in HPLC FPLC advantages include excellent performance and lifetimes for the monobead columns inert construction against the very high salt concentra tions often used in protein chromatography capability to run all columns types traditionally selected by protein chemist availability of smart automated injec tion and solvent selection valves and very simple system programming Dis advantages include lack of capability to run high pressure reverse phase columns lack of a variable detector designed for the system and lack of a true autosampler HPLC components have been adapted to solve the first two problems but have proved to be poor compromises The automated v
20. inging the sample loop into the pressurized mobile phase stream Fresh solvent pumped through the injector sample loop washes the sample onto the column head and down the column The separated bands in the effluent from the column pass through the column exit line into the detector flow cell The detector reads concentration changes as changes in signal voltage This change in voltage with time passed out to the recorder or computer over the signal cable and is traced on paper as a chromatogram allowing fractions to be detected as rising and falling peaks There are always two outputs from a detector one electrical and one liquid The electrical signal is sent to the recorder for display and quantitation ana lytical mode The liquid flow from the detector flow cell consists of concen tration bands in the mobile phase The liquid output from nondestructive detectors can be collected and concentrated to recover the separated materi als preparative mode It is very important to remember that HPLC is both an analytical and a preparative tool Often the preparative capabilities of the HPLC are over looked While normal analytical injections contain picogram to nanogram quantities HPLCs have been used to separate as much as 11b in a single injec tion obviously by a candidate for the Guinness Book of World Records Typical preparative runs inject 1 3 g to purify standard samples To be effective the detector must be capable of responding to c
21. need It was designed to help the beginning as well as the experienced chromatographer in using an HPLC system as a tool Twenty five years in HPLC first as a user then in field sales and application support for HPLC manufacturers and finally working as a teacher and consultant has shown me that the average user wants an instru ment that will solve problems not create new ones I will be sharing with you my experience gained through using my own instrument through troubleshooting customer s separations and from field demos the tricks of the trade I hope they will help you do better more rapid separations and methods development Many of the suggestions are based on tips and ideas from friends and customers I apologize for not giving them credit but the list is long and my memory is short It has been said that pla giarism is stealing ideas from one person and research is borrowing from many This book has been heavily researched and I would like to thank the many xi xii PREFACE who have helped with that research I hope I have returned more than I borrowed I have divided this guide into three parts The first part should give you enough information to get your system up and running When you have fin ished reading it put the book down and shoot some samples You know enough now to use the instruments without hurting them or yourself When you have your feet wet not literally I hope come back and we will take another run at t
22. nlet end fitting IT AA co me Packing material in solvent IE Frit Column ferrule Inlet column Frit filter bed support ferrule Figure 1 1 HPLC column design 6 ADVANTAGES AND DISADVANTAGES OF HPLC The most common HPLC separation mode is based on separating by dif ferences in compound polarity A good model for this partition familiar to most first year chemistry students is the separation that takes place in a sep aratory funnel using immiscible liquids such as water and hexane The water very polar has an affinity for polar compounds The lighter hexane very non polar separates from the water and rises to the top in the separating funnel as a distinct upper layer If you now add a purple dye made up of two com ponents a polar red compound and a nonpolar blue compound and stopper and shake up the contents of the funnel a separation will be achieved Fig 1 2 The polar solvent attracts the more polar red compound forming a red lower layer The blue nonpolar dye is excluded from the polar phase and dis solves in the relatively nonpolar upper hexane layer To finish the separation we simply remove the stopper open the separatory funnel s stopcock and draw off the aqueous layer containing the red dye and evaporate the solvent The blue dye can be recovered in turn by drawing off the hexane layer The problem with working with separatory funnels is that the separation is generally not complete Each component h
23. oncentra tion changes in all of the compounds of interest with sensitivity sufficient to measure the component present in the smallest concentration There are a variety of HPLC detectors Not all detectors will see every component sepa rated by the column The most commonly used detector is the variable ultra violet UV absorption detector which seems to have the best combination of compound detectability and sensitivity Generally the more sensitive the detector the more specific it is and the more compounds it will miss Detec tors can be used in series to gain more information while maintaining sensi tivity for detection of minor components 1 1 3 Use of Solvent Gradients Solvent gradients are used to modify the separations achieved in the column We could change the separation by changing the polarity of either the column or the mobile phase Generally it is easier faster and cheaper to change the character of the solvent OTHER WAYS TO MAKE MY SEPARATION 9 The key to changing the separation is to change the difference in polarity between the column packing and the mobile phase Making the solvent polar ity more like the column polarity lets compounds elute more rapidly Increas ing the difference in polarities between column and mobile phase makes compounds stick tighter and come off later The effects are more dramatic with compounds that have polarities similar to the column On a nonpolar column running in acetonitrile w
24. ora tories Retention times on the same column run to run should reproduce by 1 Two columns of the same type from the same manufacturer should give 5 or better retention time reproduction on the same standard set While the HPLC can be used in a variety of research and production operations there are a few places where it really shines Because it can run HPLC A Practical User s Guide Second Edition by Marvin C McMaster Copyright O 2007 by John Wiley amp Sons Inc 4 ADVANTAGES AND DISADVANTAGES OF HPLC underivatized mixtures it is a great tool for separating and analyzing crude mixtures with minimum sample preparation I began my HPLC career analyz ing herbicide production runs as a method of trouble shooting product yield problems HPLC was routinely used in the quality control lab to fingerprint batches of final product using a similar analysis I have helped my customers run tissue extracts agricultural run off waters urine and blood samples with minimum clean up These samples obviously are not very good for columns whose performance degrades rapidly under these conditions Columns can usually be restored with vigorous washing but an ounce of prevention is gener ally more effective than a pound of cure and also much more time effective Standards purification is another role in which the HPLC excels It is fairly easy to purify microgram to milligram quantities of standard compounds using the typical laboratory system
25. ring 91 7 1 3 Organic Modifiers 92 7 1 4 Chelation 92 72 Acidic Phase Silica 93 7 3 Reverse Phase Zirconium 93 7 4 Partition Mode Selection 94 8 Nonpartition Chromatography 95 8 1 Ion Exchange 96 8 1 1 Cationic Weak and Strong 96 8 1 2 Anionic Weak and Strong 97 8 2 Size Exclusion 98 8 2 1 Organic Soluble Samples 98 8 2 2 Hydrophilic Protein Separation 99 8 3 Affinity Chromatography 101 8 3 1 Column Packing Modification 102 8 3 2 Chelation and Optically Active Columns 103 9 Hardware Specifics 105 9 1 System Protection 105 9 1 1 Filters Guard Columns and Saturation Columns 106 9 1 2 Inert Surfaces and Connections 107 9 2 Pumping 108 9 2 1 High and Low Pressure Mixing Controllers 109 9 2 2 Checking Gradient Performance 112 9 3 Injectors and Autosamplers 113 9 4 Detectors 116 9 4 1 Mass Dependent Detectors 116 9 4 2 Absorptive Detectors 119 9 43 Specific Detectors 122 viii 10 11 12 13 CONTENTS 9 5 Fraction Collectors 123 9 6 Data Collection and Processing 123 Troubleshooting and Optimization 10 1 10 2 10 3 10 4 Hardware and Tools System Pacification 125 Reverse Order Diagnosis 129 Introduction to Data Acquisition 132 Solvent Conservation 133 HPLC UTILIZATION Preparative Chromatography 11 1 11 2 11 3 Analytical Preparative 138 Semipreparative 139 True Preparative 139 Sample Preparation and Methods D
26. ts 177 14 6 2 Spread Sheets DIF WK XLS Formats 178 14 6 3 Databases DB2 Format 178 14 6 4 Graphics PCX TIFF JPG Formats 178 14 6 5 Chromatographic Files Metafiles and NetCDF 178 Recent Advances in LC MS Separations 181 15 1 15 2 15 3 15 4 15 5 A LC MS Primer 181 15 1 1 Quadrupole MS and Mass Selection 183 15 1 2 Other Types of MS Analyzers for LC MS 185 15 1 3 LC MS Interfaces 187 15 1 4 LC MS Computer Control and Data Processing 189 Microflow Chromatography 191 Ultrafast HPLC Systems 192 Chip HPLC Systems 192 Standardized LC MS in Drug Design 193 New Directions in HPLC 195 16 1 16 2 16 3 16 4 16 5 16 6 Temperature Controlled Chromatography 195 Ultrafast Chromatography 196 Monolith Capillary Columns 196 Micro Parallel HPLC Systems 197 Two Dimensional HPLC Systems 197 The Portable LC MS 198 x CONTENTS APPENDICES 201 APPENDIX A Personal Separations Guide 203 APPENDIX B FAQs for HPLC Systems and Columns 205 APPENDIX C Tables of Solvents and Volatile Buffers 211 APPENDIX D Glossary of HPLC Terms 213 APPENDIX E HPLC Troubleshooting Quick Reference 221 APPENDIX F HPLC Laboratory Experiments 227 Laboratory 1 System Start up and Column Quality Control 227 Laboratory 2 Sample Preparation and Methods Development 229 Laboratory 3 Column and Solvent Switching and Pacification 231 Appendix G Selected Reference List 233 INDEX 235 PREFACE
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