- Email: [email protected]
[6] High-performance liquid chromatography: Care of columns
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ever, by developing a standard CM-D train that contains many times as much CM-D as the quantity of protein in the samples, one should be able to make such shifts insignificant. In the work of Francina et al. 16 mentioned above, microgram quantities of hemoglobin were applied to DE-81 paper strips for separation by ampholytes. Presumably, thin-layer chromatography of other proteins on other thin-layer media is feasible, since spacers can be easily applied. However, procedures for detecting enzyme activity must take into account the fact that much of the enzyme in a displaced band is not immobilized on the adsorbent and can readily be washed away by the reagent solution. Also, the dyes that are used to stain proteins are bound by positively charged adsorbents, and produced intensely colored backgrounds. Where the objective is the separation of isoenzymes to provide information of clinical interest, the use of microcolumns from which individual forms of the enzyme can be displaced by carefully selected narrow-range spacers is more promising. The suppression of tailing and the control of band width characteristic of displacement are of importance in such applications.
[6] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y : Care of Columns
By
C. TIMOTHY
WEHR
The high-performance liquid chromatography (HPLC) columns now used in protein isolation and characterization represent a considerable investment by the user compared to the low-pressure supports they are replacing. Careful use should allow these columns to give satisfactory performance from several months to over a year. This review is meant to provide information on the maintenance and troubleshooting of commercial HPLC columns to enable the user to achieve maximum performance and column lifetime. In addition to the specific recommendations on routine operation and column repair described below, a troubleshooting guide for defective column performance is outlined in Table I. Since the column is an integral part of a larger system, symptoms caused by pump, injector, or detector conditions may be misdiagnosed as column failure and are included in the table. Only the chromatographic modes commonly used in protein or peptide separations (size exclusion, ion exchange, reMETHODS IN ENZYMOLOOY, VOL. 104
Copyright © 1984by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182004-1
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versed phase) are considered; information on the use of adsorption or normal-phase HPLC columns has been presented. 1-3 Column Installation High-performance liquid chromatography columns consist of microparticulate (3- to 50-/xm particle diameter) materials based on silica or organic polymers (resins) most commonly packed at high pressure into stainless steel tubing. Although most HPLC packings have the high mechanical strength needed to withstand the pressure drops of 150-5000 psi encountered at typical flow rates, column beds will not tolerate undue mechanical shock. For this reason, columns should be handled carefully during installation and removal and should be stored so as to avoid shocks that might cause disruption of the packed bed. The majority of commercially available HPLC columns are "universal," that is, they may be installed on any HPLC pump or injector either directly or with a simple adaptor. However, when fitting a new column to an existing system, care must be taken to ensure that the connections between injector and column do not introduce dead volume. Inlet nut and ferrule must be mated with the column terminator, unions and connectors should be zero- or lowdead volume type, and connecting tubing should be of low internal diameter (-0.3 mm). Connections should be sufficiently tight to prevent leaks (approximately finger-tight plus a quarter-turn). If overtightening is required to prevent leakage, it suggests a mismatch between terminator and inlet nut or ferrule that can be remedied only by replacement. Fitting mismatch can result (in addition to leakage) in band broadening or carryover due to excessive dead volume or unswept voids. Since there is to date no standardization of terminators and fittings among manufacturers, it is recommended that, in cases where columns from different sources are used on the same HPLC system, separate transfer lines be fabricated for each column type using fittings supplied by the column manufacturer. Most commercial HPLC columns are shipped containing an organic solvent designated in the column installation instructions. If this solvent is not compatible with the mobile phase solvent, the column must be washed with an intermediate solvent prior to use. For example, reversed-phase columns shipped in methanol or acetonitrile should be washed with water before introduction of buffers or salts. Columns shipped in hexane (e.g., L. R. Snyder and J. J. Kirkland, "Introduction to Modem Liquid Chromatography," 2nd ed., pp. 782-823. Wiley, New York, 1979. 2F. M. Rabel, J. Chromatogr. Sci. 18, 394 (1980). 3D. J. Runser, "Maintaining and TroubleshootingHPLC Systems," pp. 69-90. Wiley, New York, 1981.
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nitrile or amino columns) must be washed with propanol or tetrahydrofuran before introduction of methanol, acetonitrile, or aqueous solvents. When a new column is installed, it should not be connected to the detector until several milliliters of wash solvent or mobile-phase solvent have passed through the column to prevent residual packing solvent or silica fines from entering the detector. The trend in HPLC continues to be toward the use of high efficiency columns packed with supports of 5-/zm particle diameter or less. These columns may be of standard 25- or 30-cm lengths for difficult separations requiring very high efficiency. Short columns 4-15 cm in length and packed with 5-/zm or sub-5-/zm materials may be operated at normal flow rates (-1.0 ml/min) to achieve moderate efficiency and low solvent consumption or at high flow rates (2-5 ml/min) to obtain very short analysis times. The user should be aware that as column efficiency increases and column length decreases, the extra-column contributions to band broadening will become more significant. Use of these columns demands that extra-column dead volume be kept to a minimum by reducing the length of transfer lines, using small inner diameter (i.d.) tubing, and installing appropriate guard columns. With microbore HPLC columns of 2-mm i.d. or less, extra-column effects are sufficiently severe that a conventional liquid chromatograph would require extensive modifications, including use of low-volume injectors, microbore transfer lines, and detectors with micro flow cells (<5/xl) and rapid time constants (<100 msec). Column Testing A newly purchased HPLC column should always be tested upon receipt using the manufacturer's suggested test compounds and separation conditions. Manufacturers generally test columns to minimize batch-tobatch variations in stationary-phase characteristics and to verify that individual columns meet performance specifications; test compounds are selected that effectively probe such stationary-phase characteristics as bed efficiency, selectivity, free silanol content, and contamination by trace metals. Test compounds are usually small molecules selected for their stability, low toxicity, and availability in the user's laboratory. In some cases, column packings designed expressly for chromatography of proteins may be batch-tested by the manufacturer with a protein test mixture to check selectivity or recovery. The user should test a column upon receipt to verify that the column meets the published specifications and has not been damaged in shipment; in some cases, failure to test a column within a given time period may void the manufacturer's warranty. Variances of up to 10% from the manufacturer's test results should not be
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considered indicative of a defective column since they may reflect extracolumn effects, different flow accuracy, or small differences in solvent composition. In addition to confirming column integrity, testing a new column provides a baseline for monitoring column performance during use. In some cases, it may be advisable also to test a new column with compounds more chemically related to the user's samples. Since interactions of polypeptides with HPLC stationary phases are in some respects different from those of small molecules,4 it is unlikely that a manufacturer's test results will adequately probe such column characteristics as protein selectivity and recovery. A test sample made up of standard proteins or peptides may be more relevant in establishing a performance baseline and monitoring column aging. Column Operation
Sample and Mobile-Phase Preparation The most common cause of early column failure is lack of care in solvent selection and preparation and inadequate cleanup of biological samples. Solvents may contain dissolved impurities that can be retained on the stationary phase and gradually alter the phase characteristics or elute as spurious or "ghost" peaks. Undissolved particulates in the mobile phase can plug system hydraulic components or the column head, resulting in excessive operating pressure. The first step in avoiding these problems is to use solvents of high purity (HPLC grade or spectroscopic grade) and filter them prior to use through an appropriate micropore (0.22 or 0.45/zm) filter. Where pumps with ball-and-seat inlet check valves are used, solvents must be thoroughly degassed prior to use to prevent loss of pump prime by cavitation. Degassing is also advisable when using stationary phases susceptible to oxidation, e.g., amino phases, or when using low UV detection (<--200 nm), where absorbance by dissolved oxygen can give rise to noisy or drifting baselines. Degassing mobile-phase solvents will minimize bubble formation due to outgassing in the detector cell, although this can be prevented by creating 20-100 psi resistance on the outlet side of the detector with a restrictor or with about 3 meters of 0.3 mm i.d. tubing. Since peptides and protein fragments may not always contain chromophores absorbing at the commonly used detection wavelengths (254 and 280 nm), low UV detection in the 205- to 220-nm range is often used. Many of the mobile-phase modifiers (alcohols, organic acids) employed in pep4 G. Vanecek and F. E. Regnier, Anal. Biochem. 121, 156 (1982).
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tide chromatography have UV cutoffs in this spectral range, and some baseline offset in gradient elution must be expected. Solvent impurities, however, become a severe problem at these wavelengths, giving rise to noisy baselines or spurious peaks that may be incorrectly diagnosed as column or pump disorders. Water is the most notorious offender, often containing trace organic impurities that become concentrated on reversed-phase columns and appear as spurious peaks during gradient elution. This can even be a problem with commercial HPLC-grade water whose optical purity is specified at longer wavelengths, e.g., 254 nm. Trace impurities are best removed by passing the water or aqueous mobile phase through an activated charcoal or reversed-phase column prior to use. The same procedure can be used to remove UV-absorbing impurities from mobile-phase additives such as buffer salts and ion pair agents. Solvent impurities are also a problem in ion-exchange chromatography employing phosphate gradient elution. Phosphate salts contain UVabsorbing impurities that cause severe baseline offset at high detector sensitivity. This problem is compounded by the tendency of these impurities to collect on the column and elute at high ionic strength; thus, the baseline offset increases with column use. Solvent purification techniques have been devised using recrystallization and ion-exchange cleanup 5 or passage through a chelating resin, 6 but none of them completely eliminates the difficulty. The best remedy is periodic stripping of adsorbed impurities from the column (see Column Repair, below). Where impurities in the weak (A) solvent in gradient elution can be removed by passage through an appropriate adsorbent bed, a stripper column packed with the adsorbent material can be placed in-line on the outlet side of the " A " pump in a multipump gradient HPLC system. However, the breakthrough volume of the stripper column must be accurately determined to avoid elution of impurities during an analysis. Where mixtures of an aqueous buffer and an organic solvent are used, as in reversed-phase chromatography, the user must be careful to avoid precipitation of buffer salts in the system. Premixed solvents should be filtered through a micropore filter. If aqueous-organic mixtures are proportioned from separate reservoirs by the HPLC pumping system, precipitation may occur in hydraulic components leading to reduced seal life or component failure. If precipitation occurs in the column bed, column failure is a certainty. The surest means for avoiding buffer-organic solvent incompatibility is to use as the strong eluent a premixed combination of buffer and organic modifier filtered prior to use. Neither the column nor 5 H. W. Shmukler, J. Chromatogr. Sci. 8, 581 (1970). 6 G. Karkas and G. Germerhausen, J. Chromatogr. 214, 267 (1981).
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the HPLC system should be stored while containing buffers, and when a system is converted from one application to another, e.g., from ion-exchange to reversed-phase chromatography, the entire system should be flushed with water before solvent and column are changed. Column failure may arise from the gradual accumulation of particulate material originating from wear of the system seals or microbial growth in the mobile-phase solvents. This can be avoided by installation of an inline filter either before the injector or between injector and column; the latter arrangement will protect against particulates originating from the sample or from wear of the injector seal. In-line filters, available from several manufacturers, have easily replaceable frits in the 0.5- to 2-tzm pore size range and add only a minimal amount of dead volume to the system. Microparticulate precolumns and guard columns also serve as effective solvent filters. A number of mobile-phase additives may cause column failure by irreversibly changing the stationary-phase characteristics or by accelerating column degradation. Ion-pairing agents or detergents that have bulky hydrophobic groups (e.g., camphorsulfonic acid, sodium dodecyl sulfate) may irreversibly partition into the stationary phase of steric exclusion and reversed-phase columns, changing the phase chemistry and reducing apparent pore volume. In applications requiring the uses of these components, it is recommended that the column be dedicated for this use. Anionic detergents cannot be used as mobile-phase components with anion-exchange columns. Cationic alkylamines used as ion-pairing agents or competing bases for silanol complexation (e.g., triethylamine, tetramethylammonium salts) tend to accelerate dissolution of the silica support in silica-based columns when used at neutral pH. It is recommended that these agents not be used at a mobile-phase pH of 6 or greater and that they be flushed from the column immediately afterward. The use of halide salts with stainless steel HPLC pumping systems and columns has long been a point of concern in protein purification, particularly since many ion-exchange procedures developed on carbohydrate-based gels employ chloride eluents as high as 1-2 M. Actually, the 316 stainless steel used in most HPLC components is reasonably resistant to chloride at neutral pH, and component lifetime should not be significantly reduced if such solutions are flushed from the system after use. Chloride at acidic pH should n o t be used in HPLC systems. If the investigator is uncertain about the solvent compatibility of a particular instrument or component, the manufacturer should be consulted. It is generally recommended that samples injected onto HPLC columns be as free as possible of contaminating material to minimize interferences in detection and to prevent unnecessary adsorption of sam-
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pie components on the column. This often is not possible when HPLC is used for purification of polypeptides from biological fluids or extracts. However, samples containing significant amounts of lipids should be extracted with a nonpolar solvent (ether, hexane) prior to injection. Sizeexclusion HPLC provides a rapid means of removing high-molecularweight material before injection onto ion-exchange or reversed-phase columns. Samples should always be filtered to remove particulates, and guard columns should always be used to prevent strongly retained components from reaching the analytical column. Chromatographic resolution will be affected by sample volume and sample concentration. In steric exclusion chromatography, sample volume should be less than 1% of the column permeation volume (e.g., a sample volume of about 100 ~1 for a standard 8-mm x 25-cm analytical column), and sample loads of between 1 and 5 mg can typically be applied. In ion-exchange and reversed-phase chromatography using gradient elution, the sample must be applied in a weak solvent (typically the initial or A solvent); here sample volume is not critical and several milliliters or more can be applied to an analytical column if necessary. Because of the high capacity of ion-exchange and reversed-phase HPLC supports, relatively large sample loads, 5-15 mg total protein or greater, can be injected, and often analytical columns can be used for semipreparative applications if gradient elution is employed. When such columns are operated isocratically, resolution will be more sensitive to sample volume, sample concentration, and sample matrix effects, particularly for low k' compounds. Guard Columns
Guard columns are used to protect the analytical column from particulate material or strongly retained contaminants in the sample matrix; they are by far the most effective means of preserving and extending the life of the analytical column. Guard columns are placed between the injector and analytical column and are discarded or repacked often to ensure that contaminants are not eluted. The frequency of replacement will vary depending on column capacity, solvent strength, sample type, and sample load; typically, the life of a guard column is between 10 and 50 injections. Selection of the appropriate guard column packing and column configuration must take into account two considerations. First, the packing should be similar in chemical structure to the analytical stationary phase so that the selectivity of the system is not altered. This is not always possible, since not all manufacturers supply guard column analogs of analytical packings and, when they are available, phase characteristics of the guard
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material may not be identical to the analytical packing. If a low-capacity guard material is used, selectivity mismatch is not as critical as with a high-capacity material. In steric exclusion chromatography, mismatches in guard column pore diameter and pore volume can affect protein elution profiles. The second consideration in selecting a guard column is loss of efficiency; ideally, this should not exceed 10% in order to preserve overall resolution. This is an important point when short, high-efficiency analytical columns are used, since extra-column contributions to band broadening are more significant. Here, particular care should be taken if a highcapacity microparticulate guard column is used. A high-capacity guard column should have a height equivalent of a theoretical plate (HETP) value similar to that of the analytical column. For example, coupling of a 3-cm guard column packed with 10-/~m material to a 15-cm, 5-/~m analytical column can result in a 30 to 40% loss in efficiency. Two types of guard column packings are currently available: pellicular materials and fully porous microparticulate packings. Pellicular packings consist of solid-core beads (usually 37-40/zm in diameter) with the stationary phase bonded to or polymerized on the surface as a "pellicle." Because of their large particle size, pellicular packings are easily packed in dry form by the user without any special equipment; replacement cost for material in a 4 × 50 mm guard column is about $4. Since the surface area of a peUicular packing is small compared to a porous microparticle, capacity is quite low. Thus, such columns must be repacked frequently, but their effect on selectivity is not large. Two manufacturers (Scientific Systems, Inc., State College, PA, and Upchurch Scientific, Bremerton, WA) have introduced low-volume guard columns that couple directly to the analytical column; these can be packed with pellicular materials and used with 5-/xm and sub-5-~m analytical columns to achieve low efficiency losses of 5% or less. The need for frequent replacement should be offset by the ease of repacking. Microparticulate guard columns are packed with the same material used in analytical columns and are designed for situations in which high efficiency is required. Because they must be slurry-packed, they are usually obtained from the manufacturer as prepacked columns or cartridges. Brownlee Laboratories (Santa Clara, CA) offers prepacked disposable guard cartridges that fit into a reusable holder, and they are available with a variety of 5- and 10-/zm reversed-phase, ion-exchange, and stericexclusion packings. Since microparticulate guard columns are of high capacity, a close selectivity match with the analytical column is important, and dead volume in connectors and transfer lines must be minimized to reduce extra-column band broadening. When an exact selectivity match is not possible, a guard column support that is less retentive than
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the analytical packing should be used. In some cases, saturated microparticulate guard columns can be regenerated off-line by washing with a series of strong solvents. Guard columns can also be used for the concentration or cleanup of samples. In a typical application, the guard column replaces the sample loop in a six-port rotary injection valve. The sample is introduced through the injection port and concentrated on the guard column; poorly retained contaminants may be eluted with a weak solvent, followed by valve rotation and introduction of the sample onto the analytical column with a stronger solvent. Saturator Columns A major limitation of silica-based HPLC supports is the instability of silica under alkaline conditions. Ion-exchange and size-exclusion chromatography of native proteins usually requires the use of mobile phases in a pH range of 7 to 8, conditions that reduce the lifetime of silica columns. Strong anion-exchange (SAX) columns with bonded quaternary ammonium phases are particularly vulnerable to high pH. Column life can be extended by saturating the mobile phase with dissolved silica to suppress degradation of the analytical support. This can be done by adding silica to the mobile phase during preparation or, more commonly, by installing an in-line saturator or solvent-conditioning precolumn before the injector, which is packed with porous silica. Large-particle (30-50/xm) silica is generally used for ease of packing and to reduce system pressure. Although it has been reported that use of a saturator may extend column life more than 10-fold, 7 lifetime extensions of 2- to 5-fold are more typical using a 4 x 300 mm precolumn. The precolumn should be maintained at the same temperature as the analytical column and should be topped off periodically to replace dissolved packing. Use of a saturator precolumn carries several disadvantages. First, a silica-saturated mobile phase may precipitate if allowed to stand in the system; the saturator column should be removed and the HPLC flushed prior to shutdown. Second, dissolution of the silica packing can generat~ fines that, if passed through the exit frit, can plug the injector, transfer lines, or analytical column. It is recommended that a small-pore (0.5-/.~m) frit be installed in the precolumn outlet terminator. Third, voids created by dissolution of the precolumn packing will increase the dead volume of the system, leading to poor reproducibility in gradient elution. The precolumn should be inspected and repacked often to prevent this. Fourth, the precolumn can act as a trap, concentrating solvent impurities that later elute when a stronger solvent is intro7 j. G. Atwood, G. J. Schmidt, and W. Slavin, J. Chrornatogr. 171, 109 (1979).
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duced. The analytical column should be removed initially during solvent changeover to prevent its contamination. Finally, the presence of silicates in isolated sample components may alter biological activity or interfere with subsequent chemical characterization.
Gradient Elution Gradient elution is employed in the vast majority of HPLC applications in protein isolation and characterization. Most commercial gradient HPLC systems have sufficiently high solvent proportioning precision to provide retention time reproducibility of a few percent or better in gradient elution. However, to achieve this level of performance, the user must make sure that the column is reequilibrated at initial conditions at the beginning of each analysis and that the gradient elution protocol including column regeneration and equilibration is exactly repeated for each trial. When solvents of widely different strengths are used, the column should be regenerated with a reverse gradient to initial conditions, followed by equilibration with 5-10 column volumes of the initial solvent. Regeneration and equilibration can be done at elevated flow rates to reduce turnaround time. In ion-exchange chromatography, column equilibration can require up to 50 column volumes or more if initial and final solvents differ in pH. It is not uncommon that ion-exchange columns, even when stored in the initial solvent, will exhibit retention time variations in the first analysis of the day and stabilize over successive runs. Operation of reversed-phase columns with ion-pairing agents or buffers will also require extended equilibration periods. For maximum reproducibility, the concentration of the ion-pairing agent should be kept constant across the solvent gradient.
Operation Limits Column supports and stationary phases differ in their limitations to pressure, flow rate, temperature, and pH. This information should be supplied in installation instructions delivered with the column and should be reviewed by the user to ensure that the column warranty is not voided. Some column packings are incompatible with certain mobile phases--for example, amine phases react with carbonyl groups; therefore, aldehydes or ketones cannot be used as mobile-phase modifiers. In cases where the user is unsure of incompatibility with an unusual solvent, the manufacturer should be contacted.
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Column Storage For short periods, e.g., overnight, it is best to store silica-based columns with an organic solvent or mixtures of an organic solvent and water. Columns should not be stored with buffer or salt solutions; if it is necessary to leave buffer in the column for equilibration overnight, a low flow of about O. 1 ml per minute should be maintained to prevent precipitation within the column or within hydraulic components of the system. For long-term storage, silica-based columns should be flushed with water if they have been used with buffers or salts and then filled with an organic solvent (methanol or acetonitrile). Since stationary phases can act as substrates for microbial growth, aqueous solvents are not recommended for storage. Column terminators should be tightly capped as drying may cause changes in bed geometry, and the storage solvent should be indicated on a label attached to the column. Columns based on polystyrene resins or hydrophilic organic polymers should be stored with solvents recommended by the manufacturers. Columns should be kept in a place in which they will not be exposed to potential shock or extremes of temperature. It is good practice to maintain a log in which column use and performance are detailed. This should include the type of column and its serial number, along with initial test data; the history of its analytical use, including sample type, mobile-phase conditions, and operator; test data from periodic performance checks; details on column repair.
Troubleshooting the Column The most common problems encountered in the operation of HPLC columns are outlined in Table I, along with suggested diagnoses and treatments. Several of these problems occur frequently in chromatography of proteins and peptides, particularly in the reversed-phase mode. Spurious or "ghost" peaks can arise from mobile-phase impurities, from sample carryover in the injector or transfer lines, or from elution of adsorbed sample components in subsequent runs. Mobile-phase impurities are a common source of ghost peaks in gradient elution when detection in the low UV is used, and they typically originate from the water. This can be diagnosed by pumping water through the column for varying time periods, followed by blank gradients. An increase in ghost peak height as a function of the volume of water used is indicative of impure water. Mobile-phase additives (ion-pair agents, buffers, salts) can be checked individually in the same manner. Spurious peaks arising from
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TABLE I TROUBLESHOOTINGHPLC COLUMNS Circumstances
Rapid increase in pressure
Gradual increase in pressure
Reduced pressure
Fluctuating pressure
Pressure increasing; all peaks including solvent front affected Efficiency also decreasing, solvent front unaffected
Column pressure decreasing, solvent front unaffected Retention time varies with injection volume Large sample Gradient elution
Gradient elution or proportioned solvents
Possible cause
Remedy
A. Abnormal operating pressure Plug in detector, column, Working back from detecinjector, or chromatotor to pump, break each graph fitting to localize source of resistance. If localized to column, flush or replace frit. Otherwise, backflush or replace plugged component 1. Plugged inlet frit or 1. Replace frit; remove column head top of column bed and repack 2. Plugged outlet frit 2. Replace frit Leak or defective check Tighten fittings; with valve volatile solvents, slow leaks can be detected by cold fitting; check pump seals and check valves Defective hydraulic system Check inlet and outlet check valves, pulse damper, etc. B. Decreasing retention time Flow rate too high Check flow volumetric rate; repair or reset flow Losing stationary phase 1. Guard column is consumed 2. Replace inlet frit and top off 3. Replace column Column temperature inControl column temperacreasing ture Injection solvent decreasing column activity Column overload Column recondition incomplete Regeneration not reproducible Increase in percentage organic modifier
Use starting mobile phase as injection solvent Use smaller sample Use longer reconditioning Use more care in reconditioning program Control solvent composition, prepare new mixture, check performance of solvent proportioning system
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TABLE I (continued) Circumstances
Possible cause
Prolonged use
Column contaminated
Spiking sample with authentic knowns gives different Tr than standards alone.
Components in sample or injection solvent are altering chromatography (matrix effect)
Ion pair or ion exchange chromatography
Concentration of ion pair reagent too low; buffer pH or ionic strength incorrect Sample changing with concentration
Retention time of some (not all) peaks changes with sample amount
Pressure lower than normal, solvent front also affected Backpressure increasing, flow rate OK, poor peak shape, solvent front unaffected Liquid found or cold fitting; flow rate lower than expected
Reversed-phase chromatography
Gradient elution or isocratic with proportioned solvents
C. Increasing retention time Flow rate too low
Remedy 1. Flush with strong solvent 2. Replace precolumn 3. Replace analytical column I. Prepare standard in same controlled matrix 2. Remove matrix interference by sample cleanup 3. Use internal standard technique Increase concentration of ion pair agent; check buffer composition Dilute or concentrate sample to minimize effects; change sample solvent
Check volumetric flow rate; reset flow or repair
Column contaminated
1. Replace precolumn 2. Replace inlet filter
Leak nr defective check valve
Tighten fittings; slow leaks of volatile solvents can be detected by cold fitting. Inspect check valve Control solvent composition
Decrease in concentration of nonpolar solvent by evaporation or incorrect preparation One solvent not flowing owing to: 1. Empty reservoir 2. Plugged inlet frit 3. Air bubble in line 4. Inlet valve plugged or damaged 5. Programming error
1. 2. 3. 4.
Fill reservoir Clean frit Purge line Consult manual
5. Check program
(continued)
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TABLE I (continued) Circumstances
Possible cause
Widely varying environmental temperature Gradient elution
Column temperature change Variations in column regeneration program
Spiking sample with authentic knowns gives different retention time than standards alone
Components in sample or injection solvent are altering the chromatography (matrix effects)
Column pressure rising
Column temperature decreasing Tubing starting to plug
Ion-pair or ion-exchange chromatography
Concentration of ion pair reagent too high; buffer pH or ionic strength incorrect
Trend observed over several runs Retention times unchanged
Slow buildup of contaminants on column Column efficiency decreasing
Gradient elution or proportioned solvents
Efficiency unchanged, retention or selectivity changed
Retention time shorter
Flow too high
Gradient elution
Regeneration program inadequate Change in solvent composition
Remedy Control column temperature Use more care in reproducing reconditioning program 1. Prepare standards in same matrix 2. Remove matrix interference by sample cleanup 3. Use internal standard technique Control column temperature Working from detector to pump, break each fitting and determine if there is excessive resistance to flow anywhere but in the column; if found, investigate and repair Reduce concentration of ion pair agent; change buffer composition
D. Decreasing resolution
Changed or rechanged solvent reservoir recently Large sample load
Column overload
See Part B above Check column head for void; refill if necessary; replace column or precolumn Pump malfunction; confirm that each pump is delivering correct amount of solvent Check volumetric flow rate; reset flow or repair Lengthen regeneration Use more care in preparing eluent Dilute sample
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TABLE I (continued) Circumstances
Possible cause
All peaks affected
E. Peak tailing Void at top of column
All peaks affected
Poorly packed column
All peaks affected
Frits contaminated or partially plugged Adsorbed contaminants on column giving mixed mechanism separations
Most or all peaks affected
Reversed-phase chromatography; only a few peaks affected One peak affected
Acidic or basic samples interacting with silanol groups Unresolved minor component
Large sample load
Column overloaded
One peak affected
Sample decomposing on column
Reversed-phase ion-pair chromatography
Slow reaction kinetics
Ion-exchange chromatography
Secondary adsorption effects
Chromatography of surfactants All peaks affected, particularly early eluting peaks
Micelle formation Dead volume introduced in instrument
Remedy
1. Fill in void with suitable packing or filler 2. Avoid high flow rates, excessive pressures, high mobile phase pH 3. Replace column Confirm by comparison with good column. Repair column or replace column Replace fittings or frits 1. Flush with strong solvent 2. Use precolumn; replace as required Add competing base, e.g,, 0.1% (CH3)4NC1 Improve resolution: longer column, different mobile phase May be acceptable; decrease sample size 1. Decrease column temperature 2. Decompose sample to stable product 3. Change mobile phase 4. Change column packing 1. Use column with monolayer coverage 2. Increase concentration of ion pairing agent 1. Increase column temperature 2. Add low concentration of organic modifier such as isopropyl alcohol to solvent Use more polar mobile phase Remove dead volume, inspect fittings, use 0.010-in. tubing, keep lines short
(contmued)
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CHROMATOGRAPHY
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TABLE I (continued) Circumstances
Possible cause
Remedy
F. Fronting peaks All peaks affected
Poorly packed column
All peaks affected
G. Double or split peaks Void or channel in column bed
All peaks affected
Inlet frit plugged or contaminated
Reversed-phase gradient elution
Impurities in water
Gradient or isocratic elution Gradient or isocratic elution
Sample carryover
Concentrated or large sample
Detector overloaded
Near Vp (size exclusion chromatography) or solvent front (other modes)
1. Refractive index of injection solvent different than in mobile phase 2. Temperature fluctuation associated with injection
Replace or repack column 1. Check for void at top of column, fill if found 2. Replace column Replace or clean frit
H. Ghost peaks
Elution of sample components on column
1. Confirm by pumping varying volumes of aqueous solvent prior to gradient 2. Use HPLC grade water 3. Clean up water by passage over reversedphase support Flush injector with strong solvent 1. Repeat elution program with blank injections 2. Flush column with strong solvent
I. Truncated peak I. Decrease sample size 2. Use shorter path length cell 3. Use different wavelength
J. Negative peaks 1. Use same solvent for sample and mobile phase 2. Water jacket detector flow cell and column
K. Drifting baseline 1. Late eluting components 2. Temperature fluctuation
I. Flush column with strong solvent 2. Control ambient temperature or detector flow cell temperature
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TABLE I (continued) Circumstances
Possible cause 3. Dissolved oxygen in mobile phase (low UV detection) 4. Washout of previous solvent in pump or column 5. Residual immiscible solvent in pump or column 6. Selective evaporation of volatile mobile-phase component
Remedy 3. Degas mobile phase
4. Purge entire system with fresh solvent 5. Wash system with commonly miscible solvent, e.g., propanol 6. Cap solvent reservoirs
L. Baseline noise
1. Bubbles in detector cell
2. Leakage of packing material from column 3. Dirty or misaligned detector cell 4. Failing detector lamp 5. Defective pulse damper 6. Poor instrument grounding, faulty recorder connections
1. Degas solvents, install restrictor on detector outlet 2. Replace outlet terminator frit 3. Clean or realign cell according to manufacturer's instructions 4. Replace lamp 5. Replace damper 6. Provide adequate grounding; clean or replace connections
unswept volume between injector and column can be diagnosed by thoroughly flushing (in the worst cases, backflushing) transfer lines and injector; improperly seated tubing and ferrules on injector loops or transfer lines are often the cause. Occasionally, proteins that fail to elute quantitatively by the end of a gradient will appear as ghost peaks at the same elution position in subsequent blank gradients, with peak height decreasing progressively. In such instances the column can be washed between runs by injecting a volume (up to several milliliters) of a strong solvent between analyses. Tailing peaks and split peaks occur frequently with biological materials in HPLC. Peak tailing can arise from sample overload, a poorly packed or degraded column, buildup of adsorbed contaminants on the stationary phase, or extra-column effects. In reversed-phase chromatography of polypeptides, tailing is often a sign of interaction between resid-
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ual silanol groups on the stationary phase and basic amino acid side chains. The effect can be minimized by operating at acidic pH to suppress silanol ionization, by adding a competing base such as triethylamine or a tetramethylammonium salt to the mobile phase, and by using a column with high surface coverage that has been end-capped (undergone secondary silanization to reduce the number of residual free silanols.) Split peaks or doublets arise by the formation of multiple flow paths through the column, usually by settling or degradation of the column bed to form voids or channels. Peak doublets can also occur as sample contaminants partially plug the inlet flit or column head, creating partial flow resistance. The effect may be accompanied by a gradual rise in operating pressure and can be cured by frit replacement or, as a last resort, removing and repacking the top millimeter of the column bed. Column Repair Careful column operation will prolong column lifetime to a year or longer. However, column performance will degrade as strongly adsorbed sample components accumulate on the stationary phase and as a void is formed by the gradual dissolution of the support. Often column life can be extended for old or abused columns by simple operations such as washing and backflushing, frit replacement, and bed repair.
Frit Cleaning and Replacement To remove a column flit, tightly secure the column tube and carefully remove the top terminator fitting, being careful not to disturb the column bed. Replaceable frits may sometimes remain in the terminator body; they can usually be dislodged with the tip of a spatula blade or by passing a 20or 24-gauge wire through the terminator inlet. Although replaceable stainless steel frits may sometimes be cleaned by immersing them in a sonic bath containing 3-6 N nitric acid, it is easier simply to install a new flit. When refitting the terminator, be sure the new flit is aligned and that surfaces are free of packing material to permit proper seating. Where the terminator ferrule has been distorted, it may be difficult to achieve a leakfree connection with the new flit; installation of double flits will sometimes allow a tight seal to be formed. Terminators with pressed-in flits can be cleaned by pumping a nitric acid solution through the terminator. If this procedure is ineffective (as indicated by continued high backpressure during flushing), the terminator can be installed on an empty guard column and pumped in the reverse direction to backflush the frit. The analytical column should be capped
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with a spare terminator during frit cleaning to prevent drying or disruption of the bed.
Column Backflushing When a column exhibits excessive operating pressure due to partial plugging of the inlet frit or column head, it is often possible to dislodge the material by reversing the direction of flow and backflushing with the mobile phase or a wash solvent; this is a simple remedy when a column begins to show a gradual rise in pressure, and it avoids the hazards of opening a terminator. However, several precautions should be observed. First, the pressure rise must not be accompanied by a loss in efficiency. Efficiency loss is indicative of voids in the bed, and backflushing may disturb the bed so as to prevent repair by topping off. Second, not all manufacturers recommend backflushing of columns; the user should refer to the column installation instructions or contact the manufacturer. Third, the column should be disconnected from the detector during backflushing to prevent contaminants or particulates from entering the flow cell. Occasionally, increasing pressure can arise from blockage of the outlet terminator, particularly when fines are generated by degradation of silica supports during operation at high pH, high temperature, or with cationic ion-pair agents. Under such conditions, backflushing will produce only a partial or transient reduction in pressure, and the outlet terminator frit should be replaced.
Repair of Voids and Bed Irregularities Symptoms such as peak broadening, peak tailing, or split peaks suggest that a void or channel may have been formed in the column bed. If these are observed with a new column upon receipt or during the first few injections of normal operation, it indicates a damaged or poorly packed column, which should be returned. Voids that develop with extended use can be repaired by topping off; original performance will probably not be recovered, but the improvement in resolution is often acceptable, particularly in gradient elution. After removal of the inlet terminator, the column bed should be level and flush with the face of the column tube. A light gray or brown discoloration on the bed surface is normal after extended use, but dark discoloration indicates a contaminated bed, which should be removed and repacked. Even small depressions in the bed can result in significant band broadening and should be filled; large voids of a centimeter or more probably cannot be repaired successfully. Before filling the void, the bed
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should be leveled using a square blade; a small flat-tip laboratory spatula trimmed to the column internal diameter works well. The void can be filled with glass beads, a microparticulate packing, or a pellicular guard column packing. Glass beads are not recommended because, unless they have been completely silanized, they will adsorb proteins. If a microparticulate material is used, it should be prepared as a slurry in an organic solvent and added dropwise to the column, allowing excess solvent to permeate the bed. Since microparticulate packings are high capacity, the stationary phase should be the same as that of the column to prevent selectivity changes. In most cases, a pellicular packing will serve as the best repair material since loss in efficiency due to a poorly repacked void is not as significant with a low-capacity packing. Once the void is filled, the bed surface should be leveled flush with the face of the tubing, excess packing material removed from the tubing surface, and the terminator replaced. After the repaired column has been tested, the terminator should be removed and the bed checked for settling. If settling has occurred, the top-off procedure should be repeated.
Column Washing With extended use, the buildup of strongly retained material such as lipids or basic and hydrophobic proteins will cause increased operating pressure and altered chromatographic behavior of an HPLC column. Often column performance can be recovered by stripping adsorbed material with one or a series of strong solvents. The key in rejuvenating a contaminated column lies in knowing the nature of the contaminants and an appropriate strong solvent. Lipids can be removed by washing with nonpolar solvents such as methanol, acetonitrile, or tetrahydrofuran. There is no solvent or series of solvents that will universally strip all adsorbed proteins from HPLC stationary phases, but Table II lists several strong eluents or solubilizing agents that have been used in specific instances for stripping proteins from HPLC columns. Some columns, particularly those based on organic polymer supports rather than silica, are not compatible with these solvents, and the user should contact the manufacturer regarding solvent compatibility. In most cases, neat organic solvents such as acetonitrile or methanol are not strong eluents in protein chromatography and, therefore, are not effective stripping solvents; 50:50 mixtures of organic solvents with a buffer, organic acid, or ion-pairing agent serve as better stripping agents for reversed-phase and size-exclusion columns. It has been observed s that repeated gradients followed by retrogradients 8 F. E. Regnier, this series, Vol. 91, p. 165. See also this volume [8].
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TABLE II WASH SOLVENTS FOR HPLC COLUMNSa Solvent Reversed-phase and size-exclusion columns Acetic acid Trifluoroacetic acid 0.1% Aqueous trifluoroacetic acid/propanol b TEAP~/propanoP Aqueous urea or guanidine Aqueous sodium chloride, sodium phosphate, sodium sulfate DMSO-water' Ion-exchange columns Acetic acid Phosphoric acid Aqueous sodium chloride, sodium phosphate, or sodium sulfate
Composition
1% in water 1% in water 40:60 40:60 5-8 M 0.5-1 M 50 : 50 1% in water 1% in water 1-2 M
Consult manufacturer for column compatibility. b High viscosity solvent; pump at reduced flow rate to prevent overpressure. c Triethylamine-phosphoric acid; adjust 0.25 N phosphoric acid to pH 2.5 with triethylamine.
between aqueous trifluoroacetic acid (TFA) and TFA-propanol can be effective in regenerating contaminated HPLC columns. Although detergents such as sodium dodecyl sulfate and Triton are good protein solvents, they tend to be strongly retained on HPLC stationary phases and may irreversibly change column characteristics; detergent cleanup should be used as a last resort and be followed by extensive washing with water and methanol. Several precautions should be observed during column cleanup. If the initial wash solvent is not compatible or miscible with the mobile phase, the column should be flushed with an intermediate solvent. Similarly, sets of wash solvents used in series must be compatible or, if not, interspersed with a mutually compatible flushing solvent. For example, after washing with high salt, urea, or guanidine, the column m u s t be purged with 5-10 volumes of water before the introduction of any organic solvent. Similarly, if nonpolar solvents such as hexane or methylene chloride are used to strip lipids from a reversed-phase column, a propanol purge is necessary before the introduction of aqueous solvents. To avoid shocking the column bed, it is advisable to introduce wash solvents with gradients over 5-10 column volumes. Viscous solvents such as dimethyl sulfoxide
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(DMSO)-water, methanol-water, and propanol-water mixtures should be pumped at reduced flow rates to prevent high pressures. Successful regeneration of a contaminated column can be a time-consuming process. With microprocessor-controlled HPLC systems, multistep washing sequences can be programmed and run automatically overnight. Alternatively, column regeneration can be carried out off-line with an inexpensive low-pressure pump at reduced flow rates.
[7] H i g h - P e r f o r m a n c e Size-Exclusion C h r o m a t o g r a p h y
By KLAUS UNDER Since 1980 the classical gel filtration technique employing soft and semirigid organic gels for protein characterization and purification has received progressively greater competition from high-performance sizeexclusion chromatography (HPSEC). The breakthrough of HPSEC is associated with the development of highly efficient buffer-compatible columns operating at elevated back pressures. The columns are packed with microparticulate organic-based or silica-based particles of tailormade graduated pore size and hydrophilic surface composition. Highresolution separation of proteins on these columns is attained by adjusting appropriate operating conditions. The proteins elute in the sequence of decreasing molecular weight and size. In its simplest version, the retention of the proteins in HPSEC is considered to be a selective permeation of biopolymeric solutes through the pores of the particles of the column bed; smaller proteins should penetrate a larger portion of the pore volume of the packing and hence will be retarded longer than larger proteins. Two limiting cases arise: (1) proteins so large that they are excluded from the pores will be eluted with a volume equal to the interparticle volume of the column, V0; (2) small proteins that totally permeate the specific pore volume of the packing, Vi, will elute with Ve = V0 + Vi. The total accessible volume is then equal to Vt = V0 + Vi. The intraparticle volume of the column is termed Vi. Between these two limits, a linear relationship is established between the logarithm of molecular weight (M) of the protein and its elution volume, Ve. This retention mechanism is distinctly different from other modes of column liquid chromatography, such as adsorption and ion exchange, and it entails several advantages. Proteins are eluted quickly with relatively narrow bands. A predictable volume interMETHODS IN ENZYMOLOGY, VOL. 104
Copyright © 1984 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-182004-1
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ever, by developing a standard CM-D train that contains many times as much CM-D as the quantity of protein in the samples, one should be able to make such shifts insignificant. In the work of Francina et al. 16 mentioned above, microgram quantities of hemoglobin were applied to DE-81 paper strips for separation by ampholytes. Presumably, thin-layer chromatography of other proteins on other thin-layer media is feasible, since spacers can be easily applied. However, procedures for detecting enzyme activity must take into account the fact that much of the enzyme in a displaced band is not immobilized on the adsorbent and can readily be washed away by the reagent solution. Also, the dyes that are used to stain proteins are bound by positively charged adsorbents, and produced intensely colored backgrounds. Where the objective is the separation of isoenzymes to provide information of clinical interest, the use of microcolumns from which individual forms of the enzyme can be displaced by carefully selected narrow-range spacers is more promising. The suppression of tailing and the control of band width characteristic of displacement are of importance in such applications.
[6] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y : Care of Columns
By
C. TIMOTHY
WEHR
The high-performance liquid chromatography (HPLC) columns now used in protein isolation and characterization represent a considerable investment by the user compared to the low-pressure supports they are replacing. Careful use should allow these columns to give satisfactory performance from several months to over a year. This review is meant to provide information on the maintenance and troubleshooting of commercial HPLC columns to enable the user to achieve maximum performance and column lifetime. In addition to the specific recommendations on routine operation and column repair described below, a troubleshooting guide for defective column performance is outlined in Table I. Since the column is an integral part of a larger system, symptoms caused by pump, injector, or detector conditions may be misdiagnosed as column failure and are included in the table. Only the chromatographic modes commonly used in protein or peptide separations (size exclusion, ion exchange, reMETHODS IN ENZYMOLOOY, VOL. 104
Copyright © 1984by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182004-1
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versed phase) are considered; information on the use of adsorption or normal-phase HPLC columns has been presented. 1-3 Column Installation High-performance liquid chromatography columns consist of microparticulate (3- to 50-/xm particle diameter) materials based on silica or organic polymers (resins) most commonly packed at high pressure into stainless steel tubing. Although most HPLC packings have the high mechanical strength needed to withstand the pressure drops of 150-5000 psi encountered at typical flow rates, column beds will not tolerate undue mechanical shock. For this reason, columns should be handled carefully during installation and removal and should be stored so as to avoid shocks that might cause disruption of the packed bed. The majority of commercially available HPLC columns are "universal," that is, they may be installed on any HPLC pump or injector either directly or with a simple adaptor. However, when fitting a new column to an existing system, care must be taken to ensure that the connections between injector and column do not introduce dead volume. Inlet nut and ferrule must be mated with the column terminator, unions and connectors should be zero- or lowdead volume type, and connecting tubing should be of low internal diameter (-0.3 mm). Connections should be sufficiently tight to prevent leaks (approximately finger-tight plus a quarter-turn). If overtightening is required to prevent leakage, it suggests a mismatch between terminator and inlet nut or ferrule that can be remedied only by replacement. Fitting mismatch can result (in addition to leakage) in band broadening or carryover due to excessive dead volume or unswept voids. Since there is to date no standardization of terminators and fittings among manufacturers, it is recommended that, in cases where columns from different sources are used on the same HPLC system, separate transfer lines be fabricated for each column type using fittings supplied by the column manufacturer. Most commercial HPLC columns are shipped containing an organic solvent designated in the column installation instructions. If this solvent is not compatible with the mobile phase solvent, the column must be washed with an intermediate solvent prior to use. For example, reversed-phase columns shipped in methanol or acetonitrile should be washed with water before introduction of buffers or salts. Columns shipped in hexane (e.g., L. R. Snyder and J. J. Kirkland, "Introduction to Modem Liquid Chromatography," 2nd ed., pp. 782-823. Wiley, New York, 1979. 2F. M. Rabel, J. Chromatogr. Sci. 18, 394 (1980). 3D. J. Runser, "Maintaining and TroubleshootingHPLC Systems," pp. 69-90. Wiley, New York, 1981.
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nitrile or amino columns) must be washed with propanol or tetrahydrofuran before introduction of methanol, acetonitrile, or aqueous solvents. When a new column is installed, it should not be connected to the detector until several milliliters of wash solvent or mobile-phase solvent have passed through the column to prevent residual packing solvent or silica fines from entering the detector. The trend in HPLC continues to be toward the use of high efficiency columns packed with supports of 5-/zm particle diameter or less. These columns may be of standard 25- or 30-cm lengths for difficult separations requiring very high efficiency. Short columns 4-15 cm in length and packed with 5-/zm or sub-5-/zm materials may be operated at normal flow rates (-1.0 ml/min) to achieve moderate efficiency and low solvent consumption or at high flow rates (2-5 ml/min) to obtain very short analysis times. The user should be aware that as column efficiency increases and column length decreases, the extra-column contributions to band broadening will become more significant. Use of these columns demands that extra-column dead volume be kept to a minimum by reducing the length of transfer lines, using small inner diameter (i.d.) tubing, and installing appropriate guard columns. With microbore HPLC columns of 2-mm i.d. or less, extra-column effects are sufficiently severe that a conventional liquid chromatograph would require extensive modifications, including use of low-volume injectors, microbore transfer lines, and detectors with micro flow cells (<5/xl) and rapid time constants (<100 msec). Column Testing A newly purchased HPLC column should always be tested upon receipt using the manufacturer's suggested test compounds and separation conditions. Manufacturers generally test columns to minimize batch-tobatch variations in stationary-phase characteristics and to verify that individual columns meet performance specifications; test compounds are selected that effectively probe such stationary-phase characteristics as bed efficiency, selectivity, free silanol content, and contamination by trace metals. Test compounds are usually small molecules selected for their stability, low toxicity, and availability in the user's laboratory. In some cases, column packings designed expressly for chromatography of proteins may be batch-tested by the manufacturer with a protein test mixture to check selectivity or recovery. The user should test a column upon receipt to verify that the column meets the published specifications and has not been damaged in shipment; in some cases, failure to test a column within a given time period may void the manufacturer's warranty. Variances of up to 10% from the manufacturer's test results should not be
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considered indicative of a defective column since they may reflect extracolumn effects, different flow accuracy, or small differences in solvent composition. In addition to confirming column integrity, testing a new column provides a baseline for monitoring column performance during use. In some cases, it may be advisable also to test a new column with compounds more chemically related to the user's samples. Since interactions of polypeptides with HPLC stationary phases are in some respects different from those of small molecules,4 it is unlikely that a manufacturer's test results will adequately probe such column characteristics as protein selectivity and recovery. A test sample made up of standard proteins or peptides may be more relevant in establishing a performance baseline and monitoring column aging. Column Operation
Sample and Mobile-Phase Preparation The most common cause of early column failure is lack of care in solvent selection and preparation and inadequate cleanup of biological samples. Solvents may contain dissolved impurities that can be retained on the stationary phase and gradually alter the phase characteristics or elute as spurious or "ghost" peaks. Undissolved particulates in the mobile phase can plug system hydraulic components or the column head, resulting in excessive operating pressure. The first step in avoiding these problems is to use solvents of high purity (HPLC grade or spectroscopic grade) and filter them prior to use through an appropriate micropore (0.22 or 0.45/zm) filter. Where pumps with ball-and-seat inlet check valves are used, solvents must be thoroughly degassed prior to use to prevent loss of pump prime by cavitation. Degassing is also advisable when using stationary phases susceptible to oxidation, e.g., amino phases, or when using low UV detection (<--200 nm), where absorbance by dissolved oxygen can give rise to noisy or drifting baselines. Degassing mobile-phase solvents will minimize bubble formation due to outgassing in the detector cell, although this can be prevented by creating 20-100 psi resistance on the outlet side of the detector with a restrictor or with about 3 meters of 0.3 mm i.d. tubing. Since peptides and protein fragments may not always contain chromophores absorbing at the commonly used detection wavelengths (254 and 280 nm), low UV detection in the 205- to 220-nm range is often used. Many of the mobile-phase modifiers (alcohols, organic acids) employed in pep4 G. Vanecek and F. E. Regnier, Anal. Biochem. 121, 156 (1982).
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tide chromatography have UV cutoffs in this spectral range, and some baseline offset in gradient elution must be expected. Solvent impurities, however, become a severe problem at these wavelengths, giving rise to noisy baselines or spurious peaks that may be incorrectly diagnosed as column or pump disorders. Water is the most notorious offender, often containing trace organic impurities that become concentrated on reversed-phase columns and appear as spurious peaks during gradient elution. This can even be a problem with commercial HPLC-grade water whose optical purity is specified at longer wavelengths, e.g., 254 nm. Trace impurities are best removed by passing the water or aqueous mobile phase through an activated charcoal or reversed-phase column prior to use. The same procedure can be used to remove UV-absorbing impurities from mobile-phase additives such as buffer salts and ion pair agents. Solvent impurities are also a problem in ion-exchange chromatography employing phosphate gradient elution. Phosphate salts contain UVabsorbing impurities that cause severe baseline offset at high detector sensitivity. This problem is compounded by the tendency of these impurities to collect on the column and elute at high ionic strength; thus, the baseline offset increases with column use. Solvent purification techniques have been devised using recrystallization and ion-exchange cleanup 5 or passage through a chelating resin, 6 but none of them completely eliminates the difficulty. The best remedy is periodic stripping of adsorbed impurities from the column (see Column Repair, below). Where impurities in the weak (A) solvent in gradient elution can be removed by passage through an appropriate adsorbent bed, a stripper column packed with the adsorbent material can be placed in-line on the outlet side of the " A " pump in a multipump gradient HPLC system. However, the breakthrough volume of the stripper column must be accurately determined to avoid elution of impurities during an analysis. Where mixtures of an aqueous buffer and an organic solvent are used, as in reversed-phase chromatography, the user must be careful to avoid precipitation of buffer salts in the system. Premixed solvents should be filtered through a micropore filter. If aqueous-organic mixtures are proportioned from separate reservoirs by the HPLC pumping system, precipitation may occur in hydraulic components leading to reduced seal life or component failure. If precipitation occurs in the column bed, column failure is a certainty. The surest means for avoiding buffer-organic solvent incompatibility is to use as the strong eluent a premixed combination of buffer and organic modifier filtered prior to use. Neither the column nor 5 H. W. Shmukler, J. Chromatogr. Sci. 8, 581 (1970). 6 G. Karkas and G. Germerhausen, J. Chromatogr. 214, 267 (1981).
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the HPLC system should be stored while containing buffers, and when a system is converted from one application to another, e.g., from ion-exchange to reversed-phase chromatography, the entire system should be flushed with water before solvent and column are changed. Column failure may arise from the gradual accumulation of particulate material originating from wear of the system seals or microbial growth in the mobile-phase solvents. This can be avoided by installation of an inline filter either before the injector or between injector and column; the latter arrangement will protect against particulates originating from the sample or from wear of the injector seal. In-line filters, available from several manufacturers, have easily replaceable frits in the 0.5- to 2-tzm pore size range and add only a minimal amount of dead volume to the system. Microparticulate precolumns and guard columns also serve as effective solvent filters. A number of mobile-phase additives may cause column failure by irreversibly changing the stationary-phase characteristics or by accelerating column degradation. Ion-pairing agents or detergents that have bulky hydrophobic groups (e.g., camphorsulfonic acid, sodium dodecyl sulfate) may irreversibly partition into the stationary phase of steric exclusion and reversed-phase columns, changing the phase chemistry and reducing apparent pore volume. In applications requiring the uses of these components, it is recommended that the column be dedicated for this use. Anionic detergents cannot be used as mobile-phase components with anion-exchange columns. Cationic alkylamines used as ion-pairing agents or competing bases for silanol complexation (e.g., triethylamine, tetramethylammonium salts) tend to accelerate dissolution of the silica support in silica-based columns when used at neutral pH. It is recommended that these agents not be used at a mobile-phase pH of 6 or greater and that they be flushed from the column immediately afterward. The use of halide salts with stainless steel HPLC pumping systems and columns has long been a point of concern in protein purification, particularly since many ion-exchange procedures developed on carbohydrate-based gels employ chloride eluents as high as 1-2 M. Actually, the 316 stainless steel used in most HPLC components is reasonably resistant to chloride at neutral pH, and component lifetime should not be significantly reduced if such solutions are flushed from the system after use. Chloride at acidic pH should n o t be used in HPLC systems. If the investigator is uncertain about the solvent compatibility of a particular instrument or component, the manufacturer should be consulted. It is generally recommended that samples injected onto HPLC columns be as free as possible of contaminating material to minimize interferences in detection and to prevent unnecessary adsorption of sam-
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pie components on the column. This often is not possible when HPLC is used for purification of polypeptides from biological fluids or extracts. However, samples containing significant amounts of lipids should be extracted with a nonpolar solvent (ether, hexane) prior to injection. Sizeexclusion HPLC provides a rapid means of removing high-molecularweight material before injection onto ion-exchange or reversed-phase columns. Samples should always be filtered to remove particulates, and guard columns should always be used to prevent strongly retained components from reaching the analytical column. Chromatographic resolution will be affected by sample volume and sample concentration. In steric exclusion chromatography, sample volume should be less than 1% of the column permeation volume (e.g., a sample volume of about 100 ~1 for a standard 8-mm x 25-cm analytical column), and sample loads of between 1 and 5 mg can typically be applied. In ion-exchange and reversed-phase chromatography using gradient elution, the sample must be applied in a weak solvent (typically the initial or A solvent); here sample volume is not critical and several milliliters or more can be applied to an analytical column if necessary. Because of the high capacity of ion-exchange and reversed-phase HPLC supports, relatively large sample loads, 5-15 mg total protein or greater, can be injected, and often analytical columns can be used for semipreparative applications if gradient elution is employed. When such columns are operated isocratically, resolution will be more sensitive to sample volume, sample concentration, and sample matrix effects, particularly for low k' compounds. Guard Columns
Guard columns are used to protect the analytical column from particulate material or strongly retained contaminants in the sample matrix; they are by far the most effective means of preserving and extending the life of the analytical column. Guard columns are placed between the injector and analytical column and are discarded or repacked often to ensure that contaminants are not eluted. The frequency of replacement will vary depending on column capacity, solvent strength, sample type, and sample load; typically, the life of a guard column is between 10 and 50 injections. Selection of the appropriate guard column packing and column configuration must take into account two considerations. First, the packing should be similar in chemical structure to the analytical stationary phase so that the selectivity of the system is not altered. This is not always possible, since not all manufacturers supply guard column analogs of analytical packings and, when they are available, phase characteristics of the guard
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material may not be identical to the analytical packing. If a low-capacity guard material is used, selectivity mismatch is not as critical as with a high-capacity material. In steric exclusion chromatography, mismatches in guard column pore diameter and pore volume can affect protein elution profiles. The second consideration in selecting a guard column is loss of efficiency; ideally, this should not exceed 10% in order to preserve overall resolution. This is an important point when short, high-efficiency analytical columns are used, since extra-column contributions to band broadening are more significant. Here, particular care should be taken if a highcapacity microparticulate guard column is used. A high-capacity guard column should have a height equivalent of a theoretical plate (HETP) value similar to that of the analytical column. For example, coupling of a 3-cm guard column packed with 10-/~m material to a 15-cm, 5-/~m analytical column can result in a 30 to 40% loss in efficiency. Two types of guard column packings are currently available: pellicular materials and fully porous microparticulate packings. Pellicular packings consist of solid-core beads (usually 37-40/zm in diameter) with the stationary phase bonded to or polymerized on the surface as a "pellicle." Because of their large particle size, pellicular packings are easily packed in dry form by the user without any special equipment; replacement cost for material in a 4 × 50 mm guard column is about $4. Since the surface area of a peUicular packing is small compared to a porous microparticle, capacity is quite low. Thus, such columns must be repacked frequently, but their effect on selectivity is not large. Two manufacturers (Scientific Systems, Inc., State College, PA, and Upchurch Scientific, Bremerton, WA) have introduced low-volume guard columns that couple directly to the analytical column; these can be packed with pellicular materials and used with 5-/xm and sub-5-~m analytical columns to achieve low efficiency losses of 5% or less. The need for frequent replacement should be offset by the ease of repacking. Microparticulate guard columns are packed with the same material used in analytical columns and are designed for situations in which high efficiency is required. Because they must be slurry-packed, they are usually obtained from the manufacturer as prepacked columns or cartridges. Brownlee Laboratories (Santa Clara, CA) offers prepacked disposable guard cartridges that fit into a reusable holder, and they are available with a variety of 5- and 10-/zm reversed-phase, ion-exchange, and stericexclusion packings. Since microparticulate guard columns are of high capacity, a close selectivity match with the analytical column is important, and dead volume in connectors and transfer lines must be minimized to reduce extra-column band broadening. When an exact selectivity match is not possible, a guard column support that is less retentive than
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the analytical packing should be used. In some cases, saturated microparticulate guard columns can be regenerated off-line by washing with a series of strong solvents. Guard columns can also be used for the concentration or cleanup of samples. In a typical application, the guard column replaces the sample loop in a six-port rotary injection valve. The sample is introduced through the injection port and concentrated on the guard column; poorly retained contaminants may be eluted with a weak solvent, followed by valve rotation and introduction of the sample onto the analytical column with a stronger solvent. Saturator Columns A major limitation of silica-based HPLC supports is the instability of silica under alkaline conditions. Ion-exchange and size-exclusion chromatography of native proteins usually requires the use of mobile phases in a pH range of 7 to 8, conditions that reduce the lifetime of silica columns. Strong anion-exchange (SAX) columns with bonded quaternary ammonium phases are particularly vulnerable to high pH. Column life can be extended by saturating the mobile phase with dissolved silica to suppress degradation of the analytical support. This can be done by adding silica to the mobile phase during preparation or, more commonly, by installing an in-line saturator or solvent-conditioning precolumn before the injector, which is packed with porous silica. Large-particle (30-50/xm) silica is generally used for ease of packing and to reduce system pressure. Although it has been reported that use of a saturator may extend column life more than 10-fold, 7 lifetime extensions of 2- to 5-fold are more typical using a 4 x 300 mm precolumn. The precolumn should be maintained at the same temperature as the analytical column and should be topped off periodically to replace dissolved packing. Use of a saturator precolumn carries several disadvantages. First, a silica-saturated mobile phase may precipitate if allowed to stand in the system; the saturator column should be removed and the HPLC flushed prior to shutdown. Second, dissolution of the silica packing can generat~ fines that, if passed through the exit frit, can plug the injector, transfer lines, or analytical column. It is recommended that a small-pore (0.5-/.~m) frit be installed in the precolumn outlet terminator. Third, voids created by dissolution of the precolumn packing will increase the dead volume of the system, leading to poor reproducibility in gradient elution. The precolumn should be inspected and repacked often to prevent this. Fourth, the precolumn can act as a trap, concentrating solvent impurities that later elute when a stronger solvent is intro7 j. G. Atwood, G. J. Schmidt, and W. Slavin, J. Chrornatogr. 171, 109 (1979).
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duced. The analytical column should be removed initially during solvent changeover to prevent its contamination. Finally, the presence of silicates in isolated sample components may alter biological activity or interfere with subsequent chemical characterization.
Gradient Elution Gradient elution is employed in the vast majority of HPLC applications in protein isolation and characterization. Most commercial gradient HPLC systems have sufficiently high solvent proportioning precision to provide retention time reproducibility of a few percent or better in gradient elution. However, to achieve this level of performance, the user must make sure that the column is reequilibrated at initial conditions at the beginning of each analysis and that the gradient elution protocol including column regeneration and equilibration is exactly repeated for each trial. When solvents of widely different strengths are used, the column should be regenerated with a reverse gradient to initial conditions, followed by equilibration with 5-10 column volumes of the initial solvent. Regeneration and equilibration can be done at elevated flow rates to reduce turnaround time. In ion-exchange chromatography, column equilibration can require up to 50 column volumes or more if initial and final solvents differ in pH. It is not uncommon that ion-exchange columns, even when stored in the initial solvent, will exhibit retention time variations in the first analysis of the day and stabilize over successive runs. Operation of reversed-phase columns with ion-pairing agents or buffers will also require extended equilibration periods. For maximum reproducibility, the concentration of the ion-pairing agent should be kept constant across the solvent gradient.
Operation Limits Column supports and stationary phases differ in their limitations to pressure, flow rate, temperature, and pH. This information should be supplied in installation instructions delivered with the column and should be reviewed by the user to ensure that the column warranty is not voided. Some column packings are incompatible with certain mobile phases--for example, amine phases react with carbonyl groups; therefore, aldehydes or ketones cannot be used as mobile-phase modifiers. In cases where the user is unsure of incompatibility with an unusual solvent, the manufacturer should be contacted.
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Column Storage For short periods, e.g., overnight, it is best to store silica-based columns with an organic solvent or mixtures of an organic solvent and water. Columns should not be stored with buffer or salt solutions; if it is necessary to leave buffer in the column for equilibration overnight, a low flow of about O. 1 ml per minute should be maintained to prevent precipitation within the column or within hydraulic components of the system. For long-term storage, silica-based columns should be flushed with water if they have been used with buffers or salts and then filled with an organic solvent (methanol or acetonitrile). Since stationary phases can act as substrates for microbial growth, aqueous solvents are not recommended for storage. Column terminators should be tightly capped as drying may cause changes in bed geometry, and the storage solvent should be indicated on a label attached to the column. Columns based on polystyrene resins or hydrophilic organic polymers should be stored with solvents recommended by the manufacturers. Columns should be kept in a place in which they will not be exposed to potential shock or extremes of temperature. It is good practice to maintain a log in which column use and performance are detailed. This should include the type of column and its serial number, along with initial test data; the history of its analytical use, including sample type, mobile-phase conditions, and operator; test data from periodic performance checks; details on column repair.
Troubleshooting the Column The most common problems encountered in the operation of HPLC columns are outlined in Table I, along with suggested diagnoses and treatments. Several of these problems occur frequently in chromatography of proteins and peptides, particularly in the reversed-phase mode. Spurious or "ghost" peaks can arise from mobile-phase impurities, from sample carryover in the injector or transfer lines, or from elution of adsorbed sample components in subsequent runs. Mobile-phase impurities are a common source of ghost peaks in gradient elution when detection in the low UV is used, and they typically originate from the water. This can be diagnosed by pumping water through the column for varying time periods, followed by blank gradients. An increase in ghost peak height as a function of the volume of water used is indicative of impure water. Mobile-phase additives (ion-pair agents, buffers, salts) can be checked individually in the same manner. Spurious peaks arising from
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TABLE I TROUBLESHOOTINGHPLC COLUMNS Circumstances
Rapid increase in pressure
Gradual increase in pressure
Reduced pressure
Fluctuating pressure
Pressure increasing; all peaks including solvent front affected Efficiency also decreasing, solvent front unaffected
Column pressure decreasing, solvent front unaffected Retention time varies with injection volume Large sample Gradient elution
Gradient elution or proportioned solvents
Possible cause
Remedy
A. Abnormal operating pressure Plug in detector, column, Working back from detecinjector, or chromatotor to pump, break each graph fitting to localize source of resistance. If localized to column, flush or replace frit. Otherwise, backflush or replace plugged component 1. Plugged inlet frit or 1. Replace frit; remove column head top of column bed and repack 2. Plugged outlet frit 2. Replace frit Leak or defective check Tighten fittings; with valve volatile solvents, slow leaks can be detected by cold fitting; check pump seals and check valves Defective hydraulic system Check inlet and outlet check valves, pulse damper, etc. B. Decreasing retention time Flow rate too high Check flow volumetric rate; repair or reset flow Losing stationary phase 1. Guard column is consumed 2. Replace inlet frit and top off 3. Replace column Column temperature inControl column temperacreasing ture Injection solvent decreasing column activity Column overload Column recondition incomplete Regeneration not reproducible Increase in percentage organic modifier
Use starting mobile phase as injection solvent Use smaller sample Use longer reconditioning Use more care in reconditioning program Control solvent composition, prepare new mixture, check performance of solvent proportioning system
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TABLE I (continued) Circumstances
Possible cause
Prolonged use
Column contaminated
Spiking sample with authentic knowns gives different Tr than standards alone.
Components in sample or injection solvent are altering chromatography (matrix effect)
Ion pair or ion exchange chromatography
Concentration of ion pair reagent too low; buffer pH or ionic strength incorrect Sample changing with concentration
Retention time of some (not all) peaks changes with sample amount
Pressure lower than normal, solvent front also affected Backpressure increasing, flow rate OK, poor peak shape, solvent front unaffected Liquid found or cold fitting; flow rate lower than expected
Reversed-phase chromatography
Gradient elution or isocratic with proportioned solvents
C. Increasing retention time Flow rate too low
Remedy 1. Flush with strong solvent 2. Replace precolumn 3. Replace analytical column I. Prepare standard in same controlled matrix 2. Remove matrix interference by sample cleanup 3. Use internal standard technique Increase concentration of ion pair agent; check buffer composition Dilute or concentrate sample to minimize effects; change sample solvent
Check volumetric flow rate; reset flow or repair
Column contaminated
1. Replace precolumn 2. Replace inlet filter
Leak nr defective check valve
Tighten fittings; slow leaks of volatile solvents can be detected by cold fitting. Inspect check valve Control solvent composition
Decrease in concentration of nonpolar solvent by evaporation or incorrect preparation One solvent not flowing owing to: 1. Empty reservoir 2. Plugged inlet frit 3. Air bubble in line 4. Inlet valve plugged or damaged 5. Programming error
1. 2. 3. 4.
Fill reservoir Clean frit Purge line Consult manual
5. Check program
(continued)
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TABLE I (continued) Circumstances
Possible cause
Widely varying environmental temperature Gradient elution
Column temperature change Variations in column regeneration program
Spiking sample with authentic knowns gives different retention time than standards alone
Components in sample or injection solvent are altering the chromatography (matrix effects)
Column pressure rising
Column temperature decreasing Tubing starting to plug
Ion-pair or ion-exchange chromatography
Concentration of ion pair reagent too high; buffer pH or ionic strength incorrect
Trend observed over several runs Retention times unchanged
Slow buildup of contaminants on column Column efficiency decreasing
Gradient elution or proportioned solvents
Efficiency unchanged, retention or selectivity changed
Retention time shorter
Flow too high
Gradient elution
Regeneration program inadequate Change in solvent composition
Remedy Control column temperature Use more care in reproducing reconditioning program 1. Prepare standards in same matrix 2. Remove matrix interference by sample cleanup 3. Use internal standard technique Control column temperature Working from detector to pump, break each fitting and determine if there is excessive resistance to flow anywhere but in the column; if found, investigate and repair Reduce concentration of ion pair agent; change buffer composition
D. Decreasing resolution
Changed or rechanged solvent reservoir recently Large sample load
Column overload
See Part B above Check column head for void; refill if necessary; replace column or precolumn Pump malfunction; confirm that each pump is delivering correct amount of solvent Check volumetric flow rate; reset flow or repair Lengthen regeneration Use more care in preparing eluent Dilute sample
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TABLE I (continued) Circumstances
Possible cause
All peaks affected
E. Peak tailing Void at top of column
All peaks affected
Poorly packed column
All peaks affected
Frits contaminated or partially plugged Adsorbed contaminants on column giving mixed mechanism separations
Most or all peaks affected
Reversed-phase chromatography; only a few peaks affected One peak affected
Acidic or basic samples interacting with silanol groups Unresolved minor component
Large sample load
Column overloaded
One peak affected
Sample decomposing on column
Reversed-phase ion-pair chromatography
Slow reaction kinetics
Ion-exchange chromatography
Secondary adsorption effects
Chromatography of surfactants All peaks affected, particularly early eluting peaks
Micelle formation Dead volume introduced in instrument
Remedy
1. Fill in void with suitable packing or filler 2. Avoid high flow rates, excessive pressures, high mobile phase pH 3. Replace column Confirm by comparison with good column. Repair column or replace column Replace fittings or frits 1. Flush with strong solvent 2. Use precolumn; replace as required Add competing base, e.g,, 0.1% (CH3)4NC1 Improve resolution: longer column, different mobile phase May be acceptable; decrease sample size 1. Decrease column temperature 2. Decompose sample to stable product 3. Change mobile phase 4. Change column packing 1. Use column with monolayer coverage 2. Increase concentration of ion pairing agent 1. Increase column temperature 2. Add low concentration of organic modifier such as isopropyl alcohol to solvent Use more polar mobile phase Remove dead volume, inspect fittings, use 0.010-in. tubing, keep lines short
(contmued)
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TABLE I (continued) Circumstances
Possible cause
Remedy
F. Fronting peaks All peaks affected
Poorly packed column
All peaks affected
G. Double or split peaks Void or channel in column bed
All peaks affected
Inlet frit plugged or contaminated
Reversed-phase gradient elution
Impurities in water
Gradient or isocratic elution Gradient or isocratic elution
Sample carryover
Concentrated or large sample
Detector overloaded
Near Vp (size exclusion chromatography) or solvent front (other modes)
1. Refractive index of injection solvent different than in mobile phase 2. Temperature fluctuation associated with injection
Replace or repack column 1. Check for void at top of column, fill if found 2. Replace column Replace or clean frit
H. Ghost peaks
Elution of sample components on column
1. Confirm by pumping varying volumes of aqueous solvent prior to gradient 2. Use HPLC grade water 3. Clean up water by passage over reversedphase support Flush injector with strong solvent 1. Repeat elution program with blank injections 2. Flush column with strong solvent
I. Truncated peak I. Decrease sample size 2. Use shorter path length cell 3. Use different wavelength
J. Negative peaks 1. Use same solvent for sample and mobile phase 2. Water jacket detector flow cell and column
K. Drifting baseline 1. Late eluting components 2. Temperature fluctuation
I. Flush column with strong solvent 2. Control ambient temperature or detector flow cell temperature
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TABLE I (continued) Circumstances
Possible cause 3. Dissolved oxygen in mobile phase (low UV detection) 4. Washout of previous solvent in pump or column 5. Residual immiscible solvent in pump or column 6. Selective evaporation of volatile mobile-phase component
Remedy 3. Degas mobile phase
4. Purge entire system with fresh solvent 5. Wash system with commonly miscible solvent, e.g., propanol 6. Cap solvent reservoirs
L. Baseline noise
1. Bubbles in detector cell
2. Leakage of packing material from column 3. Dirty or misaligned detector cell 4. Failing detector lamp 5. Defective pulse damper 6. Poor instrument grounding, faulty recorder connections
1. Degas solvents, install restrictor on detector outlet 2. Replace outlet terminator frit 3. Clean or realign cell according to manufacturer's instructions 4. Replace lamp 5. Replace damper 6. Provide adequate grounding; clean or replace connections
unswept volume between injector and column can be diagnosed by thoroughly flushing (in the worst cases, backflushing) transfer lines and injector; improperly seated tubing and ferrules on injector loops or transfer lines are often the cause. Occasionally, proteins that fail to elute quantitatively by the end of a gradient will appear as ghost peaks at the same elution position in subsequent blank gradients, with peak height decreasing progressively. In such instances the column can be washed between runs by injecting a volume (up to several milliliters) of a strong solvent between analyses. Tailing peaks and split peaks occur frequently with biological materials in HPLC. Peak tailing can arise from sample overload, a poorly packed or degraded column, buildup of adsorbed contaminants on the stationary phase, or extra-column effects. In reversed-phase chromatography of polypeptides, tailing is often a sign of interaction between resid-
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ual silanol groups on the stationary phase and basic amino acid side chains. The effect can be minimized by operating at acidic pH to suppress silanol ionization, by adding a competing base such as triethylamine or a tetramethylammonium salt to the mobile phase, and by using a column with high surface coverage that has been end-capped (undergone secondary silanization to reduce the number of residual free silanols.) Split peaks or doublets arise by the formation of multiple flow paths through the column, usually by settling or degradation of the column bed to form voids or channels. Peak doublets can also occur as sample contaminants partially plug the inlet flit or column head, creating partial flow resistance. The effect may be accompanied by a gradual rise in operating pressure and can be cured by frit replacement or, as a last resort, removing and repacking the top millimeter of the column bed. Column Repair Careful column operation will prolong column lifetime to a year or longer. However, column performance will degrade as strongly adsorbed sample components accumulate on the stationary phase and as a void is formed by the gradual dissolution of the support. Often column life can be extended for old or abused columns by simple operations such as washing and backflushing, frit replacement, and bed repair.
Frit Cleaning and Replacement To remove a column flit, tightly secure the column tube and carefully remove the top terminator fitting, being careful not to disturb the column bed. Replaceable frits may sometimes remain in the terminator body; they can usually be dislodged with the tip of a spatula blade or by passing a 20or 24-gauge wire through the terminator inlet. Although replaceable stainless steel frits may sometimes be cleaned by immersing them in a sonic bath containing 3-6 N nitric acid, it is easier simply to install a new flit. When refitting the terminator, be sure the new flit is aligned and that surfaces are free of packing material to permit proper seating. Where the terminator ferrule has been distorted, it may be difficult to achieve a leakfree connection with the new flit; installation of double flits will sometimes allow a tight seal to be formed. Terminators with pressed-in flits can be cleaned by pumping a nitric acid solution through the terminator. If this procedure is ineffective (as indicated by continued high backpressure during flushing), the terminator can be installed on an empty guard column and pumped in the reverse direction to backflush the frit. The analytical column should be capped
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with a spare terminator during frit cleaning to prevent drying or disruption of the bed.
Column Backflushing When a column exhibits excessive operating pressure due to partial plugging of the inlet frit or column head, it is often possible to dislodge the material by reversing the direction of flow and backflushing with the mobile phase or a wash solvent; this is a simple remedy when a column begins to show a gradual rise in pressure, and it avoids the hazards of opening a terminator. However, several precautions should be observed. First, the pressure rise must not be accompanied by a loss in efficiency. Efficiency loss is indicative of voids in the bed, and backflushing may disturb the bed so as to prevent repair by topping off. Second, not all manufacturers recommend backflushing of columns; the user should refer to the column installation instructions or contact the manufacturer. Third, the column should be disconnected from the detector during backflushing to prevent contaminants or particulates from entering the flow cell. Occasionally, increasing pressure can arise from blockage of the outlet terminator, particularly when fines are generated by degradation of silica supports during operation at high pH, high temperature, or with cationic ion-pair agents. Under such conditions, backflushing will produce only a partial or transient reduction in pressure, and the outlet terminator frit should be replaced.
Repair of Voids and Bed Irregularities Symptoms such as peak broadening, peak tailing, or split peaks suggest that a void or channel may have been formed in the column bed. If these are observed with a new column upon receipt or during the first few injections of normal operation, it indicates a damaged or poorly packed column, which should be returned. Voids that develop with extended use can be repaired by topping off; original performance will probably not be recovered, but the improvement in resolution is often acceptable, particularly in gradient elution. After removal of the inlet terminator, the column bed should be level and flush with the face of the column tube. A light gray or brown discoloration on the bed surface is normal after extended use, but dark discoloration indicates a contaminated bed, which should be removed and repacked. Even small depressions in the bed can result in significant band broadening and should be filled; large voids of a centimeter or more probably cannot be repaired successfully. Before filling the void, the bed
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should be leveled using a square blade; a small flat-tip laboratory spatula trimmed to the column internal diameter works well. The void can be filled with glass beads, a microparticulate packing, or a pellicular guard column packing. Glass beads are not recommended because, unless they have been completely silanized, they will adsorb proteins. If a microparticulate material is used, it should be prepared as a slurry in an organic solvent and added dropwise to the column, allowing excess solvent to permeate the bed. Since microparticulate packings are high capacity, the stationary phase should be the same as that of the column to prevent selectivity changes. In most cases, a pellicular packing will serve as the best repair material since loss in efficiency due to a poorly repacked void is not as significant with a low-capacity packing. Once the void is filled, the bed surface should be leveled flush with the face of the tubing, excess packing material removed from the tubing surface, and the terminator replaced. After the repaired column has been tested, the terminator should be removed and the bed checked for settling. If settling has occurred, the top-off procedure should be repeated.
Column Washing With extended use, the buildup of strongly retained material such as lipids or basic and hydrophobic proteins will cause increased operating pressure and altered chromatographic behavior of an HPLC column. Often column performance can be recovered by stripping adsorbed material with one or a series of strong solvents. The key in rejuvenating a contaminated column lies in knowing the nature of the contaminants and an appropriate strong solvent. Lipids can be removed by washing with nonpolar solvents such as methanol, acetonitrile, or tetrahydrofuran. There is no solvent or series of solvents that will universally strip all adsorbed proteins from HPLC stationary phases, but Table II lists several strong eluents or solubilizing agents that have been used in specific instances for stripping proteins from HPLC columns. Some columns, particularly those based on organic polymer supports rather than silica, are not compatible with these solvents, and the user should contact the manufacturer regarding solvent compatibility. In most cases, neat organic solvents such as acetonitrile or methanol are not strong eluents in protein chromatography and, therefore, are not effective stripping solvents; 50:50 mixtures of organic solvents with a buffer, organic acid, or ion-pairing agent serve as better stripping agents for reversed-phase and size-exclusion columns. It has been observed s that repeated gradients followed by retrogradients 8 F. E. Regnier, this series, Vol. 91, p. 165. See also this volume [8].
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TABLE II WASH SOLVENTS FOR HPLC COLUMNSa Solvent Reversed-phase and size-exclusion columns Acetic acid Trifluoroacetic acid 0.1% Aqueous trifluoroacetic acid/propanol b TEAP~/propanoP Aqueous urea or guanidine Aqueous sodium chloride, sodium phosphate, sodium sulfate DMSO-water' Ion-exchange columns Acetic acid Phosphoric acid Aqueous sodium chloride, sodium phosphate, or sodium sulfate
Composition
1% in water 1% in water 40:60 40:60 5-8 M 0.5-1 M 50 : 50 1% in water 1% in water 1-2 M
Consult manufacturer for column compatibility. b High viscosity solvent; pump at reduced flow rate to prevent overpressure. c Triethylamine-phosphoric acid; adjust 0.25 N phosphoric acid to pH 2.5 with triethylamine.
between aqueous trifluoroacetic acid (TFA) and TFA-propanol can be effective in regenerating contaminated HPLC columns. Although detergents such as sodium dodecyl sulfate and Triton are good protein solvents, they tend to be strongly retained on HPLC stationary phases and may irreversibly change column characteristics; detergent cleanup should be used as a last resort and be followed by extensive washing with water and methanol. Several precautions should be observed during column cleanup. If the initial wash solvent is not compatible or miscible with the mobile phase, the column should be flushed with an intermediate solvent. Similarly, sets of wash solvents used in series must be compatible or, if not, interspersed with a mutually compatible flushing solvent. For example, after washing with high salt, urea, or guanidine, the column m u s t be purged with 5-10 volumes of water before the introduction of any organic solvent. Similarly, if nonpolar solvents such as hexane or methylene chloride are used to strip lipids from a reversed-phase column, a propanol purge is necessary before the introduction of aqueous solvents. To avoid shocking the column bed, it is advisable to introduce wash solvents with gradients over 5-10 column volumes. Viscous solvents such as dimethyl sulfoxide
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(DMSO)-water, methanol-water, and propanol-water mixtures should be pumped at reduced flow rates to prevent high pressures. Successful regeneration of a contaminated column can be a time-consuming process. With microprocessor-controlled HPLC systems, multistep washing sequences can be programmed and run automatically overnight. Alternatively, column regeneration can be carried out off-line with an inexpensive low-pressure pump at reduced flow rates.
[7] H i g h - P e r f o r m a n c e Size-Exclusion C h r o m a t o g r a p h y
By KLAUS UNDER Since 1980 the classical gel filtration technique employing soft and semirigid organic gels for protein characterization and purification has received progressively greater competition from high-performance sizeexclusion chromatography (HPSEC). The breakthrough of HPSEC is associated with the development of highly efficient buffer-compatible columns operating at elevated back pressures. The columns are packed with microparticulate organic-based or silica-based particles of tailormade graduated pore size and hydrophilic surface composition. Highresolution separation of proteins on these columns is attained by adjusting appropriate operating conditions. The proteins elute in the sequence of decreasing molecular weight and size. In its simplest version, the retention of the proteins in HPSEC is considered to be a selective permeation of biopolymeric solutes through the pores of the particles of the column bed; smaller proteins should penetrate a larger portion of the pore volume of the packing and hence will be retarded longer than larger proteins. Two limiting cases arise: (1) proteins so large that they are excluded from the pores will be eluted with a volume equal to the interparticle volume of the column, V0; (2) small proteins that totally permeate the specific pore volume of the packing, Vi, will elute with Ve = V0 + Vi. The total accessible volume is then equal to Vt = V0 + Vi. The intraparticle volume of the column is termed Vi. Between these two limits, a linear relationship is established between the logarithm of molecular weight (M) of the protein and its elution volume, Ve. This retention mechanism is distinctly different from other modes of column liquid chromatography, such as adsorption and ion exchange, and it entails several advantages. Proteins are eluted quickly with relatively narrow bands. A predictable volume interMETHODS IN ENZYMOLOGY, VOL. 104
Copyright © 1984 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-182004-1