JANUARY 2003 LCGC NORTH AMERICA VOLUME 21 NUMBER 1 19 eversed-phase chromatography is by far the most widely used technique in high performance liquid chro- matography (HPLC) (1). It is popular because it is applicable to most nonpolar analytes and to many ionizable and ionic compounds. Most of the stationary phases used in reversed-phase chromatography are hydrophobic in nature; therefore, analytes are separated by their degree of hydropho- bic interaction with t
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  JANUARY 2003 LCGC NORTH AMERICA VOLUME 21 NUMBER 1 19 eversed-phase chromatography is by far the most widely used techniquein high performance liquid chro-matography (HPLC) (1). It is popularbecause it is applicable to most nonpolaranalytes and to many ionizable and ioniccompounds. Most of the stationary phasesused in reversed-phase chromatography arehydrophobic in nature; therefore, analytesare separated by their degree of hydropho-bic interaction with the stationary phaseand matrices with hydrophobic compo-nents also can be retained in a similar manner.Table I lists the most popular stationary phases usually bonded to silica gel (1).Phase subspecies — such as mixed phases(for example, phenyl–hexyl), endcappedand nonendcapped varieties, and polar-embedded phases — also exist within thesebonded silicas. Various other packing mate-rials have been used in reversed-phase chro-matography, including polymers, polymer-coated silicas and aluminas, inorganic–organic hybrids, coated zirconia, andgraphitized carbon. Each type of phase hasits own advantages and disadvantages.Reversed-phase chromatography columnsare used in various applications with a widevariety of mobile phases and additives.Some of these techniques use additives thatcan change or modify the surfaces of thepacking material. Sometimes, these addi-tives themselves may contaminate the sur-face or bonded phase. As with hydrophobic bonded phases, the surface of silica-gel packings has otherchemical features. Residual silanols are present on the surface of all silica-gelbonded-phase packings. Figure 1 depictsthe different types of silanols that can bepresent (2). Being weakly acidic in charac-ter, these silanols can interact with certainanalytes and matrix components, particu-larly with basic compounds. Because thep K  a  of the silanol is roughly 4.5, ionizationcan occur at intermediate pH values, andthus the possibility of electrostatic interac-tions with cationic species exists. The olderType A silicas can contain high concentra-tions of metal ions (sometimes 100 ppm ormore) that impart even greater acidity tothe silica surface and also can interact withmetal chelating or scavenging compounds This month’s “ColumnWatch” looks at practicalways to return acontaminated column to— or close to — itssrcinal state. Ron Majorsalso discusses cleaningprocedures for bonded-silica and other types ofreversed-phase columns. The Cleaning andRegeneration of Reversed-Phase HPLC Columns R Ronald E. Majors Column Watch Editor  Column  WatchColumn RelativePhaseUsage (%) C18 (octadecylsilane)39C8 (octyl)26Cyanopropyl † 14.5Phenyl12C4 (butyl)3.7Hydrophobic interaction1.8C2 (ethyl)1.1C1 (methyl)0.8Other0.8Polymers0.5 * Adapted from reference 1 and normalized. † Includes normal-phase usage, because reversed-phase versus normal-phase chromatography usewas not queried. Table I: Relative use of stationary phasesin HPLC * Figure 1: Types of silanols on the surface ofsilica gel (2).OHDecreased acidityFreesilanolGeminalsilanolsAssociatedsilanolsInternal metal(most acidic)Surface metalOHOHOHOHSiSiSiOHSiM  M  Si  20 LCGC NORTH AMERICA VOLUME 21 NUMBER 1 JANUARY 2003 pounds that have lesser retention, such assalts, usually will be eluted from the col-umn at the void volume. These undesiredinterferences might be observed by a detec-tor and appear as chromatographic peaks,blobs, baseline upsets, or even negativepeaks. If sample matrix components areretained strongly on the column and if themobile-phase solvent composition itself never becomes strong enough to elutethem, these adsorbed or absorbed com-pounds will accumulate, usually at the headof the column, after many injections. Thisbehavior often is observed with isocraticconditions. Sample compounds that are of intermediate retention can be eluted slowly and appear as wide peaks, baseline distur-bances, or baseline drift.Sometimes the sorbed sample compo-nents build up to levels high enough thatthey begin to act as a new stationary phase. Analytes can interact with these impuritiesthat contribute to the separation mecha-nism. Retention times can shift, and tailing can occur. If sufficient contaminationoccurs, the column backpressure can buildup to intolerably high levels, which over-pressures the pump and can cause a columnto settle and create a void, depending upon where the blockage occurs. Washing Bonded-Silica Columns The keys to rejuvenating a contaminatedHPLC column are knowing the nature of the contaminants and finding an appropri-ate solvent that will remove them. Whencontamination results from the accumula-tion of strongly retained substances fromrepeated injections, a simple washing process to strip these contaminants oftencan restore column performance. Some-times, after isocratic operation, flushing a column with 20 column volumes of 90–100% solvent B (the stronger solvent ina binary reversed-phase system) can removethe contaminants. (Table II lists the col-umn volumes for various sizes of HPLCcolumns, so readers can easily determine a flush volume for a particular column.) Forexample, lipids can be removed by washing a column with nonaqueous solvents such asmethanol, acetonitrile, or tetrahydrofuran.If you are using a buffered aqueous mobilephase, do not jump immediately to thestrong solvent. An abrupt change to highorganic solvent content could result inbuffer precipitation in the HPLC flow sys-tem, which could cause even bigger prob-lems such as plugged frits, plugged con-necting tubing, pump seal failure, a scratched piston, or injection valve rotor(3). Residual silanols are more bothersomeon nonendcapped bonded silicas and onshort-chain-bonded phases such as C2 orC4 phases.Users must be aware of the surface char-acteristics of their particular stationary phases and of possible analyte–surfaceinteractions, so they can take into accountpossible matrix interactions when they aredeveloping and using reversed-phase meth-ods. For example, very hydrophobic samplematrices such as corn oil, highly aromaticmaterials, and waxes can stick to reversed-phase packing surfaces and change theircharacteristics. Biological fluids containing proteinaceous materials can adsorb onpacking surfaces. Despite analysts’ bestattempts to protect HPLC columns fromforeign substances, eventually certain analyte–matrix combinations can affect stationary phases detrimentally. After a column is contaminated, its chro-matographic performance can be differentfrom that of an uncontaminated column.Contaminated columns can exhibit back-pressure problems. A contaminatedreversed-phase column must be cleaned andregenerated to return it to its srcinal oper-ating condition. This installment of “Col-umn Watch” will discuss practical ways toreturn a column to or nearly to its srcinalstate. Because bonded-silica columns arethe most popular, I will focus on them. Atthe end, I will discuss cleaning proceduresfor other types of reversed-phase columns. What Causes Contaminant Buildupin Reversed-Phase Columns? Usually, sample matrices contain com-pounds that are of no interest to analysts.Salts, lipids, fatty compounds, humic acids,hydrophobic proteins, and other biologicalcompounds are a few of the possible sub-stances that can come in contact with anHPLC column during its use. These mate-rials can have lesser or greater retentionthan the analytes of interest. Those com-failure. Instead, flush the column with a buffer-free mobile phase (that is, replace thebuffer with water). After flushing with5–10 column volumes, the stronger solventthen can be passed through the column.Occasionally, the strong solvent compo-nent of a mobile phase is insufficient toremove the column contaminants. A stronger solvent or series of solvents will benecessary to clean the column. If the con-taminants are nonbiological, then users canpass one or more additional organic sol-vents through the column to remove theundesired compounds. The solvents andsolvent combinations that can be used arenumerous. Visit one or more column man-ufacturers’ web sites to see various recom-mended solvent systems.Generally, all washing approaches follow a similar pattern. The wash solvents usedare increased in their solvent strength, oftenending with a solvent that could be very nonpolar (for example, ethyl acetate or evena hydrocarbon), which helps to solubilizenonpolar substances such as lipids and oils.It is important to ensure that each solventin the series is miscible with the next sol-vent. At the conclusion of the wash cycle,go backwards through an intermediately miscible solvent before returning to thesrcinal mobile-phase system. For example,isopropanol is an excellent solvent for thisintermediate step because it is miscible withorganic solvents such as hexane or methyl-ene chloride and also is miscible with aque-ous solutions. Because isopropanol is quiteviscous, make sure that the flow rate is nothigh enough to cause pump overpressure. Also, if using a UV detector, avoid using solvents that absorb in the ultravioletregion of the spectrum because it may require a great deal of washing to removeall of the absorbing solvent to get a stablebaseline. A recommended column washing systemfor a typical bonded-silica column and a mobile phase without buffer salts is to use Column SizeVoid Volume(mm  mm)(mL) 250  4.62.5150  4.61.5150  3.00.64150  2.10.2850  4.60.5030  4.60.3015  4.60.15 Table II: Column volumes of analyticalcolumns Some additives that can change or modify the surfaces of the packing material may contaminate the surface or bonded  phase.  22 LCGC NORTH AMERICA VOLUME 21 NUMBER 1 JANUARY 2003 ã 100% methanol, ã 100% acetonitrile, ã 75% acetonitrile–25% isopropanol, ã 100% isopropanol, ã 100% methylene chloride, and ã 100% hexane. When using methylene chloride orhexane, the column must be flushed withisopropanol before returning to an aqueousmobile phase because of solvent immisci-bility. A minimum of 10 column volumesof each wash solvent should be passedthrough a column. For 250 mm  4.6mm analytical columns, analysts can use a typical 1–2 mL/min HPLC flow rate. Toreturn to the srcinal mobile phase, chro-matographers usually can skip going through the entire series in reverse order.Using isopropanol as an intermediate sol-vent is recommended, followed by mobilephase without buffer, then finally with thestarting mobile-phase composition.Tetrahydrofuran is another popular solventthat can be used for cleaning contaminatedcolumns. If users suspect severe fouling,they can mix dimethyl sulfoxide (DMSO)or dimethylformamide mixed 50:50 with water and pass them at flow rates less than0.5 mL/min. The successful regenerationof a reversed-phase column can be a time-consuming process, and solvent washingscan be programmed into a gradient systemfor overnight operation. A question arises as to whether to reversethe HPLC column during the washing procedure. Because most of the strongly held contaminants usually are at the headof the column, reversing the column canshorten the migration distance that the sol-ubilized contaminants must travel to exitthe column. As far as the packed-bed sta-bility is concerned, most modern HPLCcolumns have been packed at a consider-ably higher pressure than the normal oper-ating pressure; therefore, their beds shouldnot be disturbed by the reversed flow.However, if a top frit is of a higher poros-ity than a bottom frit, this type of reversalcould be detrimental. For example, if thebottom frit is of 2-  m porosity, it usually is sufficient to contain column packing  with an average particle size of 5  m (witha  2-  m particle size distribution). How-ever, a manufacturer sometimes will put a larger-porosity frit at the top of the col-umn to prevent plugging with sample ormobile-phase particulates. If the porosity of this frit is larger than that of the smallestparticles in the particle-size distributioncurve, some of the packing material con-ceivably could pass through the frit and beremoved from the column, thereby creat-ing a void. If a column has an arrow torecommend the direction of flow, I wouldconsult the manual or instruction sheet,the manufacturer’s web site, or the techni-cal support group before reversing the col-umn to make sure that it is a safe practice. Whether you reverse the column or not, itis a good practice to disconnect the col-umn from the HPLC detector so that con-taminants or particulates lodged on the fritare not swept into the detector cell, wherethey can cause contamination.The frequency of cleaning fouledreversed-phase columns depends upon how much unretained material has beeninjected onto the column. Becausereversed-phase columns sometimes can withstand a great deal of contaminationbefore resolution loss or elution of extrane-ous compounds, users tend to wait untilthey observe some unusual behavior. How-ever, an increased buildup of contaminants will make it more difficult to clean the col-umn. For this reason, if you know that youare subjecting your reversed-phase columnsto dirty sample matrices, I recommendcleaning your columns on a regular basis.The more frequent the cleaning, the lessrigorous cleaning conditions you will need. Cleaning Protein Residues from Bonded-Silica Reversed-Phase Columns If biological materials such as plasma orserum build up on a reversed-phase col-umn, chromatographers must use a some- what different cleaning process. In mostcases, neat organic solvents such as acetoni-trile or methanol do not dissolve peptidesand proteins and are ineffective for clean-ing reversed-phase columns. However,mixtures of organic solvents with buffer,acids, and sometimes, ion-pairing reagentscan be effective. Initially, flushing a col-umn with mobile phase that has a some- what higher percentage of the stronger sol-vent (solvent B) should be attempted.Freiser and co-workers (4) found thatrepeated up-and-down gradients betweenaqueous trifluoroacetic acid and trifluo-roacetic acid–propanol can regenerate con-taminated reversed-phase columns. Bhad- waj and Day (5) suggested that a plug injection of 100  L of trifluoroethanol in a 250 mm  4.6 mm column could work. If these procedures fail, the strong eluents orsolubilizing agents recommended by Cunico and colleagues (6) can be used tostrip proteins (see Table III). Before flush-ing columns with these solvents, however,consult the column manual or the manu-facturer to ensure that these solvents arecompatible with the packing material. Silica-based columns usually are compati-ble but organic polymer–based columnscan swell or shrink with certain solventcombinations, and the performance couldbe affected. As with the previous solvent series,ensure that the sets of solvents in Table IIIused in series are miscible. Propanol is a good intermediate flush solvent. A mini-mum of 20 column volumes should beused for each solvent system. Because someof the solvent systems are quite viscous, theflushing flow rates should be adjustedaccordingly to ensure that no overpressur-ing occurs. After cleaning a column withthe guanidine or urea reagents, use a mini-mum of 40–50 column volumes of HPLC-grade water to flush the column.For reversed-phase columns, it has beeninadvisable to use detergents such assodium dodecyl sulfate (SDS) and Triton,because these compounds apparently are SolventComposition Acetic acid1% in waterTrifluoroacetic acid1% in water0.1% Trifluoroacetic acid–propanol40:60 (v/v) (viscous; use reduced flow rate)TEA–propanol40:60 (v/v) (adjust 0.25 N phosphoric acid to pH 2.5 with triethylamine before mixing)Aqueous urea or guanidine5–8 M (adjusted to pH 6–8)Aqueous sodium chloride, sodium 0.5–1.0 M (sodium phosphate pH 7.0)phosphate, or sodium sulfateDMSO–water or dimethylformamide–water50:50 (v/v) * Adapted from reference 6. Table III: Wash solvents for removing proteinaceous material from HPLC reversed-phasecolumns *  24 LCGC NORTH AMERICA VOLUME 21 NUMBER 1 JANUARY 2003 adsorbed strongly on bonded-silica packing and are difficult to remove. Using deter-gents can affect the surface of the packing and change its characteristics. However, a study by the Separations Group found thata column contaminated by a protecting group and scavenger products from a pep-tide synthesis could be cleaned by injecting 500  L of 1% SDS solution into themobile phase flowing at 1 mL/min (7). If followed by a gradient from 5% to 95%acetonitrile with 0.1% (v/v) trifluoroaceticacid and equilibration at the starting con-ditions, the polypeptide separation wasrestored. Special Techniques for CleaningBonded-Silica Reversed-PhaseColumns Sometimes, washing with organic solventscan fail to remove the column contami-nants. This situation is particularly true if metallic ions are sorbed to the silica orbonded phase. A chelating reagent such as0.05 M ethylenediaminetetraacetic acid(EDTA) can be flushed through a column.The EDTA complexes with many metallicspecies and solubilizes them. After treat-ment with an EDTA solution, analysts can wash the column thoroughly with water. If the sample matrix contains ionizable com-pounds, a change of pH could put theminto an un-ionized form, and they couldbe flushed from the column with water–organic solvent mixtures. For example, a strongly basic matrix component some-times can be removed by adjusting the pHto less than 3, at which the protonatedamine becomes more water soluble. Acidicmatrix components can removed by adjust-ing the pH to a higher value — greaterthan the p K  a  — approximately pH 8 or 9,at which the acids are in their ionizedform. However, be cautious with silica-based columns because they can be dam-aged by long-term exposure to high pHlevels (8).To control bacterial growth that couldbe present in a buffer system or in columnsleft unattended in aqueous buffer, chro-matographers can use common householdbleach diluted 1:10 or 1:20. Run at least50 column volumes followed by another50 column volumes of HPLC-grade water.Do not run the bleach through the detec-tor, because it could attack the flow cell.To prevent bacterial growth in the solventreservoir, use just enough buffer for theday’s use and store unused buffer in therefrigerator, use 0.1% sodium azide in thebuffer, and don’t let the column sit inbuffer solution for long periods of time without any flow.Chromatographers frequently have dis-cussed the effect of the ion-pairing reagentson the stationary phase for columns usedfor ion-pairing chromatography. Appar-ently, ion-pairing reagents such as octane-sulfonic acid (used for cations) andtetraalkylammonium bromide (used foranions) strongly sorb on the surfaces of bonded-silica columns at certain concen-trations of organic modifier. The columnsbecome contaminated and cannot beregenerated to their srcinal state, and thestory goes that any column used for ion-pairing work should be dedicated to thattechnique and never used again for regularreversed-phase chromatography.Bidlingmeyer (9) disagrees with this generality and feels that the aggressive pHvalues used for the ion-pairing coupling actually can change the nature of somecolumns by either hydrolysis of the bondedphase or endcapping silane under acidicconditions (pH 1–3) or by silica dissolu-tion at higher pH values (pH 7–8). Toremove sulfonic acid ion-pairing reagents,he recommends first washing the column(minimum of 20 column volumes) withthe same mobile phase without the ion-pairing reagent and then washing withmobile phase without the buffer (methanolmight be a better organic solvent than ace-tonitrile in this wash step; for very long–chain ion-pairing reagents, use tetrahydro-furan). Apparently, sulfonic acid ion-pairing reagents and amine ion-pairing reagents exhibit different behaviors.Bidlingmeyer and co-workers (10) demon-strated that when using a C18 column with mobile-phase concentrations greaterthan 70% methanol, SDS, which is a long-chain anion-pairing agent, is not adsorbedonto the stationary phase. This finding agrees with the Separations Group work (7).Bonded-silica monolith columns such as Chromolith columns (Merck KGaA,Darmstadt, Germany) should be treated asany other silica-based columns. Regeneration for Polymeric Columns Polymeric columns used to separate biolog-ical molecules also can become contami-nated or require sanitization. The chemicalstability of polymeric materials generally isconsidered one of their strengths. In fact,many manufacturers recommend washing their columns with 1.0 M nitric acid or1.0 M sodium hydroxide. Certain reversed-phase polymeric columns such as thosepacked with poly(styrene–divinylbenzene)(PS–DVB) beads and polymeric monolithssuch as CIM RP-SDVB disks (BIA Separa-tions, Ljublijana, Slovenia) and Swiftcolumns (Isco, Lincoln, Nebraska) can withstand a wide range of pH values (usu-ally pH 1–13 or sometimes pH 0–14), butusers should exercise some care when wash-ing these columns with harsh organic sol-vents. Depending upon their degree of cross-linking, swelling or shrinking canoccur when the columns are exposed tosome organic solvents. Highly cross-linkedpolymers with greater than 8–10% cross-linking usually have good mechanical sta-bility with minimal shrinking in aqueoussolvents and minimal swelling in organicsolvents. Before washing a polymer column with a series of solvents, however, it is a good practice to consult the column’s man-ual or contact the technical support groupof the column’s manufacturer. According to BIA Separations (11), userscan regenerate a polymer-based monolithiccolumn made of PS–DVB by  ã  washing the column with 10 columnvolumes of 0.1% trifluoroacetic acid in2-propanol at one-half of the working flow rate, ã  washing the column with at least 5 col-umn volumes of 100% mobile phase Bat one-half of the working flow rate, and ã re-equilibrating the column with at least10 column volumes of 100% mobilephase A at the working flow rate.If a methacrylate-based monolith withbutyl or ethyl chemistry is cleaned, precipi-tated protein can be removed by flushing the column in a reversed direction with 10column volumes each of 1.0 M sodiumhydroxide, water, 20% ethanol solution,and the working buffer (12). For more The keys torejuvenating acontaminated HPLC column areknowing the natureof the contaminantsand finding anappropriate solvent that will removethem.
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