A Practical User’s Guide
`
`Marvin C. McMaster
`
`FRESENIUS KABI 1022-0001
`
`

`
`
`
`Marvin C. McMaster, Ph.D.
`2070 Cordoba Drive
`Florissant, MO 63033
`
`Technical illustrations by Christopher A. McMaster.
`
`This book is printed on acid-free paper.
`
`Library of Congress Cataloging-in-Publication Data
`
`McMaster, Marvin C.
`HPLC, a practical user’s guide / Marvin C. McMaster.
`p.
`cm.
`Includes bibliographical references and index.
`ISBN 1-5608 l-636-8 (acid-free)
`1. High performance liquid chromatography 1. Title.
`QD79.C454M36
`1994
`543’.0894—dc2O
`
`93-42139
`CIP
`
`© 1994 VCH Publishers, Inc.
`
`This work is subject to copyright.
`All rights reserved, whether the whole or part of the material is concerned, specifically those of
`translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying
`machine or similar means, and storage in data banks.
`Registered names, trademarks, etc., used in this book, even when not specifically marked as such,
`are not to be considered unprotected by law.
`
`Printed in the United States of America
`
`ISBN l-56081-636-8 VCH Publishers
`
`Printing History:
`10
`9
`8
`7
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`Published jointly by
`
`VCH Publishers, Inc.
`220 East 23rd Street
`New York, New York 10010
`
`VCH Verlagsgesellschaft mbH
`P.O. Box 10 ll 61
`69451 Weinheim, Germany
`
`VCH Publishers (UK) Ltd.
`8 Wellington Court
`Cambridge CB1 lHZ
`United Kingdom
`
`FRESENIUS KABI 1022-0002
`
`1-‘
`
`

`
`Contents
`
`Part I: An HPLC Primer
`
`1
`
`1. Why HPLC? Advantages and Disadvantages
`1.1 How Does It Work?
`4
`
`3
`
`1.2 How Else Could I Get My Separation?
`
`10
`
`2. Selecting an HPLC System 15
`
`2.1 What Do I Look for in a System?
`2.2 From Whom Do I Buy It?
`17
`2.3 What Will It Cost?
`18
`2.4 Columns
`20
`
`15
`
`3. Running Your Chromatograph
`
`25
`
`25
`3.1 Setup and Start-up
`3.2 Sample Preparation and Column Calibration
`3.3 Your First Chromatogram
`37
`
`35
`
`Part II: HPLC Optimization
`
`43
`
`4. Separation Models
`
`45
`
`4.1 Partition
`
`45
`
`4.2 Ion-Exchange Chromatography
`IX
`
`56
`
`FRESENIUS KABI 1022-0003
`
`

`
`7 ”"’"""l
`
`HPLC: A PRACTICAL USER’S GUIDE
`
`4.3 Size-Exclusion Chromatography
`4.4 Affinity Chromatography
`59
`
`57
`
`. Column Preparation
`
`61
`
`5.1 Column Variations
`
`61
`
`5.2 Packing Materials and Hardware
`5.3 Column Selection
`66
`
`64
`
`. Column Aging, Diagnosis, and Healing
`
`71
`
`6.1 Packing Degradation——Bonded-Phase Loss
`6.2 Dissolving Packing Material—End Voids
`6.3 Bound Material
`76
`6.4 Pressure Increases
`79
`
`73
`74
`
`80
`6.5 Column Channeling—Center Voids
`6.6 Normal Phase, Ion Exchange, and Size Columns
`
`Modifications of Partition Chromatography
`7.1 Reverse Phase
`85
`89
`7.2 Acidic-Phase Silica
`7.3 Partition Mode Selection
`7.4 Hydrophilic Separations
`
`89
`90
`
`82
`
`85
`
`“Nonpartition” Chromatography
`
`93
`
`8.1 Ion Exchange
`8.2 Size Exclusion
`
`93
`95
`
`8.3 Aflinity Chromatography
`
`97
`
`Hardware Specifics
`
`99
`
`99
`
`9.1 System Protection
`9.2 Pumping
`102
`9.3 Injectors and Autosamplers
`9.4 Detectors
`109
`9.5 Fraction Collectors
`
`115
`
`109
`
`9.6 Data Collection and Processing
`
`1 16
`
`10.
`
`Troubleshooting and Optimization
`
`117
`
`10.1 Hardware and Too1s——System Pacification
`10.2 Reverse Order Diagnosis
`121
`10.3 Introduction to Data Acquisition
`
`123
`
`117
`
`FRESENIUS KABI 1022-0004
`
`

`
`CONTENTS
`
`-
`
`Xi
`
`Part III: HPLC Utilization
`
`127
`
`11. Preparative Chromatography
`
`129
`
`11.1 Analytical Preparative
`11.2 Semipreparative
`131
`11.3 “True” Preparative
`
`130
`
`131
`
`12. Sample Preparation and Methods Development
`
`135
`
`12.1 Sample Preparation
`12.2 Methods Development
`12.3 Gradient Development
`
`135
`
`143
`148
`
`13. Application Logic: Separations Overview 151
`
`151
`13.1 Fat-Soluble Vitamins, Steroids, and Lipids
`152
`13.2 Water-Soluble Vitamins, Carbohydrates, and Acids
`13.3 DNA Family: Nucleic Acids, Nucleosides/Nucleotides, and RNA/
`DNA
`152
`
`13.4 Protein Family: Amino Acids, Peptides, and Proteins
`13.5 Clinical Drug Monitoring, Toxicology, and Forensics
`13.6 Environmental and Reaction Monitoring
`155
`13.7 Application Trends
`156
`
`154
`155
`
`14. Automation: Separations and Data Acquisition
`
`159
`
`159
`14.1 Analog-to-Digital Interfacing
`161
`14.2 Digital Information Exchange
`14.3 HPLC System Control and Automation
`14.4 Data Collection and Interpretation
`162
`° 14.5 Automated Methods Development
`164
`14.6 Data Exportation to the Real World
`168
`
`161
`
`15. Where Do We Go from Here?
`
`171
`
`15.1 Microsystems and Superdetectors
`15.2 LC/MS Primer
`172
`15.3 New Column Directions
`
`181
`
`171
`
`15.4 Dedicated Systems: Pushbutton Clinical and the Personal HPLC
`182
`
`Appendixes
`
`183
`
`A. Personal Separation Guide
`B. A Glossary of HPLC Terms
`
`185
`187
`
`FRESENIUS KABI 1022-0005
`
`

`
`xii
`
`HPLC: A PRACTICAL USER’S GUIDE
`
`C. HPLC Troubleshooting Quick Reference
`D. Laboratory Experiments
`197
`197
`Laboratory l—System Start-up and Column QC
`Laboratory 2—Sample Preparation and Method Development
`199
`
`193
`
`Laboratory 3—Column and Solvent Switching and Pacification
`200
`E. Selected Reference List
`
`203
`
`Index
`
`205
`
`A
`FRESENIUS KABI 1022-0006
`
`

`
`
`
`CHAPTER
`
`3
`
`Running Your Chromatograph
`
`This chapter is designed to help you get your HPLC up and running. We will
`walk through making tubing fittings, putting the hardware together, preparing
`solvents and sample, initialization of the column, making an injection, and,
`then, getting information from the chromatogram produced. Let us begin with
`connecting the hardware and work our way toward acquiring information.
`
`3.1 Setup and Start-up
`
`When your chromatograph arrives someone will have to put it together. If you
`bought it as a system, a service representative from the company may do this
`for you. No matter who will put it together, you should immediately unpack it
`and check for missing components and for shipping damage.
`If you bought only components or ifyou are inheriting a system from some-
`one else, you will have to put it together yourself. More than likely, you will
`need, at a minimum, a 10-ft coil each.of0.010-in. (ten-thousandths) and 0.020-
`in. (twenty-thousandths) tubing, compression fittings appropriate to your sys-
`tem, cables to connect detectors to recorder/integrators and pumps to control-
`ler, and tools. Our model will be a simple, isocratic system: a single pump, a
`flush valve, an injector, a C“, analytical column, a fixed wavelength UV detec-
`tor, and a recorder (Fig. 1.4). The first thing we need to do is to get the system
`plumbed up or connected with small internal diameter tubing. For now, check
`the columns to make sure they were shipped or were left with the ends capped.
`We will put them aside until later.
`
`25
`
`FRESENIUS KABI 1022-0007 I
`
`

`
`26
`
`HPLC: A PRACTICAL USER'S GUIDE
`
`3.1.1 Hardware Plumbing 101: Tubing and Fittings
`We will need é-in. stainless steel HPLC tubing with 0.020-in. i.d. going from
`the outlet check valve ofthe pump to the flush valve and on to the injector inlet.
`Three types of tubing are used in making HPLC fittings: 0.04, 0.02, and 0.01
`in. i.d.; the latter two types are easily confused. If you look at the ends of all
`three types, 0.04 in. looks like a sewer pipe, more hole than tube. Look at the
`tubing end; if you can see a very small hole and think that it is 0.01 in., it is
`probably 0.02 in. Ifyou look at the end ofthe tubing, and, at first glance, think
`it is a solid rod and then look again and can barely see the hole, it is 0.01-in.
`tubing. From the injector to the column and from the column on to the detec-
`tor we will use 4-in. pieces of this 0.010-in. tubing.
`It is critically important to understand this last‘ point. There are two tubing
`volumes that can dramatically affect the appearance of your separation: the
`ones coming from the injector to the column and from the column to the detec-
`tor flow cell. It is important to keep this volume as small as possible. The
`smaller the column diameter and the smaller the packing material diameter,
`the more effect these tubing volumes will have on the separation’s appearance
`(peak sharpness).
`A case in point is a troubleshooting experience that I had. We were visiting
`a customer who had just replaced a column in the system. The brand new col-
`umn was giving short, broad, overlapping peaks. It looked much worse than
`the discarded column, but retention times looked approximately correct. Since
`the customer was replacing a competitive column with one that we sold, I was
`very concerned. I asked her if she had connected it to the old tubing coming
`from the injector and she replied that the old one did not fit. She had used a
`piece of tubing out of the drawer that already had a fitting on it that would fit.
`This is always dangerous, since fittings need to be prepared where they will be
`used or they may not fit properly. They can open dead volumes that serve as
`mixing spaces. I had her remove the column and looked at the tubing. Not only
`was the end of the tubing protruding beyond the ferrule too short, the tubing
`was 0.04 in. i.d. This is like trying to do separations in a sewer pipe. We replaced
`it with 0.01-in. tubing, made new new fittings in the holes they were to connect
`with, and reconnected the column. The next run gave needle-sharp, baseline-
`resolved peaks!
`To make fittings we need to be able to cut stainless steel tubing. Do not cut
`tubing with wire cutters; that is an act of vandalism. Tubing is out like glass. It
`is scored around its circumference with a file or a microtubirig cutter. The best
`apparatus for this is called a Terry Tool and is available from many chroma-
`tography suppliers. If adjusted for the inner diameter of the tubing, it almost
`always gives cuts without burrs. If you do not have such a tool, score around
`the diameter with a file. Grasp the tube on both sides of the score with blunt
`nosed pliers and gently flex the piece to be discarded until the tubing separates.
`Scoring usually causes the tubing to flare at the cut. A flat file is used to smooth
`around the circumference. Then, the face of the cut is filed at alternating 90°
`angles until the hole appears as a dot directly in the center of a perfect circle.
`
`I
`FRESENIUS KABI 1022-0008
`
`

`
`RUNNING YOUR CHROMATOGRAPH
`
`27
`
`The ferrule should then slide easily onto the tubing. Be sure not to leave filings
`in the hole; connect the other end to the pumping system and use solvent pres-
`sure from the pump to wash them out.
`The tubing is connected to the pump’s outlet check valve by a compression
`fitting. The fitting is made up of two parts: a screw with a hex head and a conical
`shaped ferrule (Fig. 3. la). The top of the outlet valve housing has been drilled
`and treaded to accept the fitting.
`
`1
`-1-5 inch tubing
`
`F9i"'"Ul9
`
`Compression
`Screw
`
`Ferrule
`
`
`
`Compression
`Screw
`
`(a) Male fitting
`
`
`
`(b) Female fitting
`
`(C) Zero Dead Volume Union
`
`Compression
`
`
`
`I 0.01 inch tubing
`
`(d) Column Bridge
`
`Figure 3.]. Compression fittings. (a) Male fitting; (b) Female fitting; (c) Zero dead vol-
`ume union; (d) Column bridge.
`
`FRESENIUS KABI 1022-0009
`
`

`
`28
`
`HPLC: A PRACTICAL USER’S GUIDE
`
`First the compression screw then the ferrule are pushed on to the tubing; the
`narrow end of the ferrule and the treads of the screw point toward the tubing’s
`end. The end of the tubing is pushed snugly into the threaded hole on the check
`valve. Slide the ferrule down the tube into the hole, followed by the compres-
`sion screw. Using your fingers, tighten the screw until it is as snug as possible;
`then use a wrench to tighten it another quarter turn. As the screw goes forward,
`it forces the ferrule against the thread and squeezes it down on to the tubing,
`forming a permanent male compression fitting. The fitting can be removed
`from the hole, but the ferrule will stay on the tubing. The tubing must be cut
`to remove the ferrule.
`
`It is important not to overtighten the fitting. It should be just tight enough
`to prevent leakage under pressure. Try it out. If it leaks, tighten it enough to
`stop the leak. By leaving compliance in the fitting, you will considerably
`increase its working life time. Many people overtighten fittings. If you work at
`it, it is even possible to shear the head off the fitting. But please, do not.
`There is a second basic type of compression fitting, the female fitting (Fig.
`3.1b), that you will see on occasion. Some column ends have a protruding,
`threaded connector tube and will require this type of fitting. This fitting is made
`from a threaded cap with a hole in the center. It slides over the tubing with its
`threads pointed toward the tubing end. A ferrule is added exactly as above and
`the tubing and the ferrule are inserted into the end of the protruding column
`tube with external threads. Tightening the compression cap again squeezes the
`ferrule into the tapered end of the tube and down onto the tubing, forming a
`permanent fitting. The third type of device for use with compression fittings is
`the zero dead volume union (Fig. 3.1c). A union allows you to connect two
`male connection fittings.
`You will find that stainless steel fittings will cause a number of headaches
`over your working career. An easier solution in many cases is the polymeric
`“finger tight” fittings sold by many suppliers such as Upchurch and SS1. These
`fittings slide over the tubing and are tightened like stainless steel fittings, but
`are not permanently “swagged” onto the tubing and can be reused. They are
`designed to give a better zero dead volume fitting, but they have pressure and
`solvent limits._ They are also more expensive, but only in the short run.
`
`3.1.2 Connecting Components
`
`New pumps are generally shipped with isopropanol or a similar solvent in the
`pump head and this will need to be washed out. Always try and determine the
`history of a pump before starting it up. Systems that have not been run for a
`while may have dried out. If buffer was left in the pump, it may have dried and
`crystallized. In any event, running a dry pump can damage seals, plungers, and
`check valves.
`
`First we will need to hook up the pump inlet line. This consists of a length
`of large-diameter Teflon® tubing with a combination sinker/filter pushed into
`one end and a compression fitting that will screw into the inlet fitting at the
`
`FRESENIUS KABI 1022-0016
`
`

`
`RUNNING YOUR CHROMATOGRAPH
`
`29
`
`bottom of the pump head on the other end. Drop the sinker into the solvent
`reservoir and screw the other end into the inlet check valve housing.
`The next step is to use compression fittings to hook the pump outlet check
`valve to the flush valve with a length of 0.02-in.-i.d. tubing. A flush valve is a
`small needle valve used to prime the pump by diverting solvent away from the
`column when rapidly flushing the pump to atmospheric pressure. Open the
`valve and the line is vented to the atmosphere. This removes back pressure
`from the column, a major obstacle when trying to push solvent into a plumbed
`system.
`From the flush valve we can connect with fittings and 0.02-in. tubing onto
`the injector inlet port. The back of the injector usually has ports for an inlet
`and an outlet line, two ports for the injection loop, and a couple of wash ports.
`If a sample loop is not in place, connect it, then make a short piece of 0.0 1-in.-
`i.d. tubing with fittings to be used in connecting the column. Use the column
`end to prepare the compression fitting that will fit into it. At the outlet end of
`the column, hook up with compression fittings a piece of 0.01-in. tubing that
`connects to the detector flow cell inlet line. When this is done remove and recap
`the column and set it aside.
`Next we are going to create a very useful tool for working with the HPLC
`system. I call it a “column bridge” (Fig. 3.1d). It bridges over the place in the
`system where we would normally connect the column. It is very valuable for
`running, diagnosing, and cleaning a “columnless system.” It is made up of a 5-
`ft piece of 0.01-in. tubing with a male compression fitting on each end screwed
`into zero dead volume unions (female/female). Our column bridge now has
`two ends simulating the end fittings on the column.
`Connect one end of our column bridge to the tubing from the injector outlet;
`the other end is connected to the line leading to the detector flow cell. We have
`one more line to connect to complete our fluidics. A piece of 0.02-in. tubing
`can be fitted to the detector flow cell outlet port to carry waste solvent to a con-
`tainer. In some systems, this line will be replaced with small-diameter Teflon®
`tubing.
`In either case, the line should end in a backpressure regulator, an adjustable
`flow resistance device designed to keep about 40-70 psi backpressure on the
`flow cell to prevent bubble formation that will interfere with the detector signal.
`Air present in the solvent is forced into solution during the pressurization in
`the pump. The column acts as a depressurizer. By the time our flow stream
`reaches the detector cell, the only pressure in the system is provided by the out-
`let line. If this is too low, bubbles can form in the flow cell and break loose,
`resulting in sharp spikes in the baseline. The backpressure regulator prevents
`this from happening.
`The final connections are electrical. A power cable needs to be connected to
`the pump. Check the manuals to see if fuses need to be installed and do so if
`required. Finally, connect the 0- to 10-mV analog signal connectors on the
`back of the detector to similar posts on the strip chart recorder. Connect red to
`red, black to black. If a third ground wire is present in the cable connect it only
`
`FRESENIUS KABI 1022-0011
`
`

`
`30
`
`HPLC: A PRACTICAL USER’S GUIDE
`
`at one end, either the detector or the recorder end. (Note: The ground wire con-
`nects to the cable shield, which is wrapped around the other two wires in the
`cable. If no ground is connected, no shielding of the signal occurs. If both ends
`of a ground are connected, the shield becomes an antenna; this is worse than
`no shield at all.)
`Now our system is ready to run. We will need to prepare solvent, flush out
`each component, then connect, flush out, and equilibrate the column before
`we are ready to make our first injection of standard.
`
`3.1.3 Solvent Cleanup
`
`Before we tackle the column, let us look at how to prepare solvents for our sys-
`tem. I have found that 90% ofall system problems turn out to be column prob-
`lems. Many of these can be traced to the solvents used, especially water.
`Organic solvents for HPLC are generally very good. There are four rules to
`remember: always use HPLC grade solvents, buy from a reliable supplier, filter
`your solvents, and check them periodically with your HPLC. Most manufac-
`turers do both GLC and HPLC quality control on their solvents; some do a
`better job than others. The best way to find good solvents is to talk to other
`chromatographers.
`Even the best solvents need to be filtered. I have received solvents, from what
`I considered to be the best manufacturer of that time, that left black residue on
`a 0.54-,u.1‘l’l filter. There is a second reason to filter solvents. Vacuum filtration
`through a 0.54-um filter on a sintered glass support is an excellent way to do a
`rough degassing of your solvents. Because of their filter and check valve
`arrangements, some pumps cavitate and have problems running solvents con-
`taining dissolved gases.
`There are numerous filter types available for solvent filtration. Cellulose ace-
`tate filters should be used with aqueous samples with less than 10% organic sol-
`vents. With much more organic in the solvent, the filter will begin to dissolve
`and contaminate your sample. Teflon® filters are used for organic solvent with
`less than 75% water. The two types are easily told apart; the Teflon® tends to
`wrinkle very easily, while the cellulose is more rigid. If you are using the Tef-
`lon® with high percentages of water in the solvent, wet the filter first with the
`pure organic solvent, then with the aqueous solvent before beginning filtration.
`Ifyou fail to do this it will take hours to filter a liter of25% acetonitrile in water.
`'5 Recently, nylon filters for solvent filtration have appeared that can be used with
`either aqueous or organic solvents. They work very well as a universal filter,
`but use with very acidic or basic solutions should be avoided.
`If you are still having problems after vacuum filtration, try placing the fil-
`trate in an ultrasonication bath for 15 min (organic solvents) or 35 min (aque-
`ous solvents). Ultrasonic baths large enough to accept a 1-liter flask are in com-
`mon use in biochemistry laboratories and are very suitable for HPLC solvent
`degassing. Stay away from the insertion probe type of sonicator; they throw
`solvent and simply make a mess. Ultrasonication is much better than heating
`
`i
`
`
`
`FRESENIUS KABI 1022-0012
`
`

`
`RUNNING YOUR CHROMATOGRAPH
`
`31
`
`for removing gases from mixed solvents. There is much less chance of frac-
`tional distillation with solvent compositional change when placing mixtures in
`an ultrasonic bath. One manufacturer actually designed an HPLC system that
`was designed to remove dissolved gas by heating under a partial vacuum. Obvi-
`ously they never used rotary vacuum flash evaporators in their laboratories, at
`least not intentionally!
`Other techniques recommended for solvent degassing involve bubbling
`gases (nitrogen or helium) through the solvent. Helium sparging is partially
`effective, but expensive when used continuously. It is required in some low-
`pressure mixing gradient systems as will be described later. The only other time
`I use any of these degassing techniques is in deoxygenating solvent for use with
`amine or anionic-exchange columns, which tend to oxidize (see Fig. 6.3).
`"3 Water is the major offender for column contamination problems. I have
`diagnosed many problems, which customers initially blamed on detectors or
`pumps or injectors, that turned out to be due to water impurities. Complex
`gradient separations are especially susceptible to water contamination effects.
`In one case, a customer was running a PTH amino acid separation, a com-
`plex gradient run on a reverse-phase column. He would wash his column with
`acetonitrile, then water, and run standards. Everything looked fine. Five or /six
`injections later his unknown results began to look weird. He ran his standards
`again only to find the last two compounds were gone. He blamed the problem
`on the detector. I said it looked like bad water. He exploded, and told me that
`his water was triple distilled and good enough for enzyme reactions. It was good
`enough for HPLC, he said. Over the following 6 months we replaced every
`component in that system. Eventually, the customer borrowed HPLC grade
`water from another institution, and washed his column with acetonitrile, then
`with water. The problem disappeared and never came back—until he went
`back to his own water. lfilgnpglar impurities codistilling with the water were
`accumulating at the head ofthe column and retaining the late runners in the
`column.
`While HPLC grade water is commercially available, I have found it to be
`expensive and to have limited shelf life. The best technique for purifying water
`seems to be to pass it though a bed of either reverse-phase packing material or
`of activated charcoal, as in a Milli-Q system. Even triple distillation tends to
`codistil volatile impurities unless done using a fractionation apparatus.
`I have used an HPLC and an analytical C18 column at 1.0 ml/min overnight
`to purify a liter ofdistilled water for the next day’s demonstration run. The next
`morning, I simply washed the column with acetonitrile, then with water, equil-
`ibrated with mobile phase, and ran my separation. It might be a good idea to
`reserve a column strictly for water purification.
`An even better solution is to use vacuum filtration through a bed of reverse-
`Dhase packing. Numerous small Cm SFE cartridges are available that are used
`for sample cleanup and for trace enrichment. They are a tremendous boon to
`the chromatographer for sample preparation, but also can be of help in water
`cleanup. These SFE cartridges are a dry pack ofCu, packing and must be wetted
`
`FRESENIUS KABI 1022-0013
`
`

`
`32
`
`HPLC: A PRACTICAL USER’S GUIDE
`
`before use with organic solvent, then with water, or an aqueous solution. You
`wash first with 2 ml of methanol or acetonitrile and then with 2 ml of water
`before applying sample. If you forget and try to pass water or an aqueous solu-
`tion through them, you will get high resistance and nonpolars will not stick.
`SFE cartridges contain from 0.5 to 1 .0 g ofpacking and will hold approximately
`25-50 mg of nonpolar impurities. If care is taken not to break their bed, they
`can be washed with acetonitrile and water for reuse. Eventually, long eluting
`impurities will build up and the SFE must be discarded. I have used them about
`six times, cleaning about a liter of single distilled water on each pass. If larger
`quantities of water are required, there are commercially available vacuum car-
`tridge systems using large-pore, reverse-phase packing designed to purify gal-
`lons of water at a time.
`The most common choice for large laboratories is mixed-bed, activated
`charcoal and ion-exchange systems that produce water on demand. These sys-
`tems usually have a couple of ion-exchange cartridges and one activated char-
`coal filter in series. They work very well, but I prefer to have the charcoal as the
`last filter in the purification bank. After all, we are trying to remove organics. I
`find that the ion-exchange resins break down after about 6 months and begin
`to appear in the water. The system uses an ion conductivity sensor as an indi-
`cator of water purity, but water that passes this test often is still unsuitable for
`HPLC use.
`
`3.1.4 Water Purity Test
`
`The final step is to check the purity of the solvents. Again I have found the C“,
`column to be an excellent tool for this purpose. Select either 254 nm or the UV
`wavelength you will be using for the chromatogram. Wash the column with
`acetonitrile until a flat UV baseline is established and then pump water though
`the column at 1.0 ml/min for 30 min. This allows nonpolar impurities to accu-
`mulate on the column. The final step is to switch back to acetonitrile. I prefer
`to do this by running a gradient to 100% acetonitrile over 20 min. If no peaks
`appear after 5 min at final conditions, the water is good. The chromatogram
`(Fig. 3.2) gives you an idea of the expected baseline appearance.
`Peaks that appear during the first acetonitrile washout are ignored as impu-
`rities already on the column. Watch the baseline on switching to water. At 254
`nm, the baseline should gradually elevate. If instead it drops, you may have
`impurities in your acetonitrile. Ifthe baseline makes a very sharp step up before
`leveling off, you may have a large amount of polar impurities in the water.
`Polar impurities probably will not bother you on reverse-phase columns, but
`might have some long-term accumulation effects. Peaks appearing during the
`acetonitrile gradient come from nonpolar impurities in the water that accu-
`mulated on the column and are now eluting.
`I have done this with water from a Milli-Q system in need of regeneration.
`Even though their indicator glow light shows no evidence of charged material
`being released from the ion exchanger, peaks that will affect reverse-phase chro-
`
`FRESENIUS KABI 1022-0034
`
`

`
`RUNNING YOUR CHROMATOGRAPH
`
`100% AN
`
`33
`
`AN
`
`50%
`40%
`
`20%
`
`/ 100%80%
`
`/‘
`
`'
`
`100% H20
`
`
`
`Non-Polar‘ Organics in
`Water
`
`Polar Organics in Water“
`
`UV Absorbance in AN
`
`30
`
`6D
`
`90
`
`Figure 3.2. Water purity test.
`
`matography show up at around the 70% acetonitrile portion of the gradient
`run.
`
`If your water passes this test at the wavelength you will be using for your
`chromatography, you are ready to use it to equilibrate the column. The next
`step is to flush out the dry system and prepare to add the column.
`
`3.1.5 Start-up System Flushing
`
`Fill the solvent reservoir with degassed, filtered solvent by pouring it down the
`wall of the flask to avoid remixing air into it. I usually start pumps up with 40-
`50% methanol in water. Even if the pump was shut down and allowed to stand
`and dry out in buffer, there is a good chance this will clear it. It is also a good
`idea to loosen the compression fitting holding the tubing in the outlet check
`valve at the top of the pump head to relieve any system backpressure. This is
`an especially important step to use if the column is still connected. When run-
`ning with a column bridge, as we are, it is less important.
`The first step is to ensure that the pump is primed. This may mean pushing
`solvent from an inlet manifold valve through the inlet valve and into the pump-
`ing chamber. A few pumps on the market, like the old Waters M6000, use
`spring-loaded check valves, so you may have to really work to get solvent into
`the chamber. With other pumps, you open a flush valve and use a large priming
`syringe to pull solvent through the pumphead. The next step is either to turn
`the pump flow to maximum speed or use the priming function of the pump,
`which does the same thing.
`
`FRESENIUS KABI 1022-0015
`
`

`
`34
`
`HPLC: A PRACTICAL USER'S GUIDE
`
`As soon as the pump begins to pump solvent by itself, tighten the outlet com-
`pression fitting and drop the flow rate to about 1 ml/min. The pump is ready
`to run and should be allowed to pump into a breaker for a few minutes to wash
`out any machining oils, if new, or soluble residues or dissolved buffers if old.
`Before we move on, let us talk about shutting down a pump. The pump seal
`around the plunger is lubricated by the contents of the pumping chamber.
`There is always a microevaporation through this seal/plunger combination,
`whether the pump is running or not. Buffers and other mobile phases contain-
`ing dissolved solids should not be left in a pump when it is to be turned off
`overnight. This evaporation causes crystallization on the sapphire plunger and
`can result in either plunger breakage or seal damage on starting up the pump.
`Solvents containing dissolved solids should always be washed out before the
`pump is shut down. I prefer to wash out and leave a pump in 25-50% metha-
`nol/water to prevent bacteria growth in the fluidics system.
`Occasionally, I have had to leave buffer in a pump overnight. When I do that
`I leave the pump running slowly (0.1 ml/min) and leave enough solvent in the
`reservoir so that it can run all night. This has an additional value of washing
`the column overnight. Ifthe column is clean and does not require further wash-
`ing, you can throw the detector outlet into your inlet reservoir and recycle the
`solvent, ensuring that you will not run out.
`Now we can move past the flush valve to the next major system component,
`the injector. Whichever position you find the injector handle in, leave it there!
`Never turn the handle on a dry injector. The injector seal is hardened Teflon®
`facing a metal surface and can tear if not lubricated with solvent. Once solvent
`is flowing through the injector to lubricate the seal, turn the handle to the inject
`position so that the sample loop is washed. Watch the pressure gauge on the
`pump; a plugged sample loop will cause the pressure to jump. If this happens
`go to the troubleshooting section in Appendix C.
`
`3.1.6 Column Preparation and Equilibration
`The next step is to hook up the column. Stop the pump flow. I assume you have
`a C18 colpmn compatible with 40% methanol/water (otherwise, select a solvent
`appropriate for your column). Disconnect the column bridge, remove the col-
`umn fittings from both ends ofthe stored column, and connect the inlet end to
`the line coming from the injector. The inlet end of a column is almost always
`marked; check for an arrow or a tag pointing in the direction of flow. I have
`always preferred to hook up a column with some solvent running. Turn the
`flow rate on the pump to 0.2 ml/min. Fill the end of the column with solvent
`and screw in the compression fitting at the end ofthe injector line. Place a bea-
`ker at the outlet end ofthe column to catch washout solvent. Flush the column
`with start—up solvent ifit is anold column that might have been stored in buffer.
`(This is a very bad technique, but you never know ifyou were not the last per-
`son to use the column! It is a good idea to label a column with the last solvent
`used before you store it.)
`
`FRESENIUS KABI 1022-001.6
`
`

`
`RUNNING YOUR CHROMATOGRAPH
`
`35
`
`Next, change the solvent in the reservoir to 70% acetonitrile in water, turn
`the pump on, and flush it with the new solvent. Turn the flow rate up to 1.0
`ml/min while catching the column effluent in a beaker. Check back up line for
`leaks; if you see any, tighten the appropriate fittings until the leaks just stop.
`You will always have leaks! If you do not you are probably overtightening your
`fittings. Leaks are messy, but are probably a sign of successful technique (leaks,
`not streams).
`Check the pump pressure. The pump pressure gauge and the baseline trace
`are the two major tools fo