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RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 2 Advances in the HPLC Column Packing Design Even though HPLC column technology is considered to be somewhat mature, new developments continue. Improvements in packing-material design, bonded-phase chemistry, column construction and formats have occurred. Users now have a better understanding of the advantages and limitations of silica- based materials and do not attempt to use them under conditions that may shorten their lifetime or decreas
  RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 2 Advances in the HPLC Column Packing Design Even though HPLC column technology is considered to besomewhat mature, new developments continue. Improvementsin packing-material design, bonded-phase chemistry, columnconstruction and formats have occurred. Users now have abetter understanding of the advantages and limitations of silica-based materials and do not attempt to use them underconditions that may shorten their lifetime or decrease theirperformance. In addition, new phases have extended operatingpH ranges (high and low) providing more versatility. In thisarticle, I will update developments in packing morphology andparticle design. Instead of trying to cover the entire domain of HPLC column development, I will focus on a few key areas. Improvements in Porous Packings Porous packings have been in favour throughout the history of HPLC. The transition from large porous particles and pellicularmaterials to small porous particles occurred in the early 1970s when microparticulate silica gel (  10 µm d  p ) came on thescene and appropriate packing methods were developed.Irregularly shaped microparticulate packings were in voguethroughout the 1970s until spherical materials were developedand perfected. The spherical packings could be packed morehomogeneously than their irregular predecessors, gave betterefficiencies and could be manufactured in higher purity.Indeed, the so-called Type B silica that was low in trace-metalcontent became the standard in the early 1990s and now mostcommercial silica-based analytical HPLC packing materials areof this higher level of purity. Trace metals in silica gel causeinteractions with certain compounds and can affect the acidity of residual silanols. 1 One aim of HPLC packings early in the game was to achievethe best efficiency possible and consequently betterchromatographic resolution. To better understand the variousapproaches employed to improve column efficiency, let’s briefly discuss the morphology of a porous packing material such assilica gel or alumina.Diffusive pores dominate a typical porous packing (Figure 1(a))and the major surface area of the particle is contained withinthese pores. A reduction in particle size improves bothinterparticle and intraparticle mass transfer. In a porous particle,solutes transfer from the moving mobile phase outside of theparticles into the stagnant mobile phase within the pores tointeract with the stationary phase. Following this interaction,the solute molecules must diffuse out of the particle andcontinue their journey down the column. Such a mass transferoccurs many thousands or even millions of times as thedifferential separation process proceeds and the solute is elutedfrom the column. While the solute spends its time in the diffusivepores, the mobile phase in which it was located srcinally movesdown the column ahead of the solute. This slow rate of masstransfer into and out of the porous particles is a major source of band broadening in HPLC. The use of smaller particles shortensthe path length of this diffusion process, improves mass transferand provides better efficiency. Manufacturers can now producesmall diameter particles with fairly narrow particle-sizedistributions down to 1.5 µm average diameter, although 3–3.5 µm and 5 µm particles are still the norm. Advances in HPLC Column Packing Design Ronald E. Majors, Agilent Technologies, Wilmington, Delaware, USA. Throughpore(a)(c)(d)1.5 um0.25 µ m5 µ m(b)Diffusive poreThin porous layer Figure 1: Flow characteristics and design of packing particlesin HPLC. (a) totally porous particle; (b) perfusion packing; (c) non-porous silica (NPS) or non-porous resin (NPR); (d) Poroshell particle. Majors However, congruent with this improvement in efficiency was adecrease in column permeability; that is, an increase in columnbackpressure. The increase in pressure is proportional to theinverse of the particle diameter squared. Thus, halving particlediameter will increase the column head pressure by a factor of four. Although pumps can provide the necessary increase inpressure output at normal flow-rates (e.g., 1–3 mL/min) withcommonly used solvents (e.g., water, methanol, acetonitrile,hexane, etc.), users found that columns were not particularly stable when run near to pump pressure limits as high as 450 bar. Column efficiency, H or HETP (height equivalent to atheoretical plate), is proportional to d  px , where x isapproximately 1.6–1.9. Resolution is proportional to N 1/2 .Thus, if one uses smaller particles packed into shorter columnsof the same internal diameter, the loss in resolution does notfall off as rapidly as the efficiency improves. A current trend inHPLC for high-throughput separations is to use shortercolumns with smaller particles (3 or 3.5 µm particles in 4.6 mm i.d. by 20–50 mm length), rather than longer columns with larger particles (5 µm particles in 4.6 mm i.d.  150–250 mm lengths). Because separation time is proportionalto length, shortening the column results in faster separations.Figure 2 provides an example of the time saved when usingsmaller particles ( d  p  5, 3.3, 1.8 µm) in shorter columns (L  250, 100 and 30 mm, respectively), compared with moretraditional HPLC analytical columns. The flow-rate on allcolumns is the same except for Figure 2(d) in which the flow isincreased to 2 mL/min to illustrate a possible further decreasethe separation time by increasing the flow-rate. Compared withthe separation on a conventional 4.6 mm  250 mm column,the separation time was reduced 15-fold (a little over 2 min). Although short columns with small particles provide rapidseparations, the column plate number (efficiency) is notincreased. Thus, complex, multicomponent samples cannotbe separated on these columns. To increase plate count over100000 requires particles in long columns, but at theexpense of greatly increased column pressure. The firstdemonstration of ultrahigh-pressure HPLC separations wasby Bidlingmeyer and co-workers 2,3 in 1969 with submicronparticles packed into long, thick columns. However, thequality of the packings was not equivalent to today’smaterials, and more recent studies by Jorgenson andcoworkers 4,5 from the University of North Carolina involvedsmall particles (down to 1 µm) with ultra high pressure.Conventional pumps cannot handle these columns so specialhigh-pressure pumps capable of pressures in excess of 5000bar (75000 psi) are required. However, such small-particlecolumns have the capability of generating a quarter of amillion plates in less than an hour! The ultrahigh-pressurechromatograph can also be used for gradient elution. Todirect users on how to employ these systems routinely, MiltonLee and coworkers 6 from Brigham Young University inProvo, Utah, USA have devoted their attention to solvingsome of the practical concerns of ultrahigh-pressure systems.Particular attention has been paid to the designs of injection valves with respect to injection reproducibility, injection time,maximum operating pressure, sample amount injected andthe valve’s impact on system efficiency. These workers havealso used supercritical carbon dioxide as a packing solvent.Until commercial HPLC systems are available that can handlethese ultrahigh-pressure columns, they will be mostly used inacademic research laboratories. Perfusion Packings Perfusion packings were developed by Afeyan and co-workers 7–9 and commercialized by Perseptive Biosystems(Cambridge, Massachusetts, USA, now part of AppliedBiosystems) in the late 1980s that gave improvedchromatographic performance, particularly for largermolecules. A simplified pictorial representation of a perfusionpacking is shown in Figure 1(b). Compared with the porouspackings, the perfusion packings consists of two different typesof pores: diffusive pore and through pores. The diffusive poresare the same type present in the porous particles and providethe sorption capacity. The through-pores allow mobile phase topass through the packing itself thereby increasing the rate of mass transfer in the mobile phase. Instead of predominantly flowing around the particles, a portion of the mobile phaseflows through the particle allowing the solute to spend lesstime undergoing the mass transfer process and giving narrowerpeaks. The process is actually a combination of diffusion andconvection.Commercial perfusion packings are polymeric particles largerthan those typically used in HPLC packings, with the smallestaverage particle size being ~12 µm. However, when compared Peaks: 1  estradiol, 2  ethylestradiol, 3  dienestrol, 4  norethindrone. 051015202530Time (min)051015202530 0.5112342.521.5 Rs (1,2)   3.14.6   30 mm, 1.8 µ m2 mL/min2.09Rs (1,2)   3.74.6   100 mm, 3.5 µ m12.71 1234 051015202530Rs (1,2)   3.34.6   100 mm, 1.8 µ m1 mL/min4.15 1234 051015202530Rs (1,2)   4.84.6   250 mm, 5 µ m29.65 1234 Figure 2: Effect of shortening column length and decreasingparticle diameter on chromatographic separation. Columns:Zorbax SB-C18; mobile phase: 50% 20 mM NaH 2 PO 4 , pH 2.8:50% ACN; flow-rate 1 mL/min; temperature: RT; detection: UV230 nm. (Courtesy of Agilent Technologies, Wilmington,Delaware, USA.)  RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 4 Majors  with a porous packing of the same particle and pore size, theperfusion packings give better efficiency for large molecules. 9 In addition, compared with the older soft, organic porouspackings used for biomolecules such as fast-flow agarose orpolydextrans, which tend to collapse at higher linear velocities,the perfusion packings may be used at higher flow-rates. Atthese higher flow-rates they maintain their sample capacity making them useful for preparative separations andpurifications. Non-Porous and Superficially Porous Packings The use of non-porous packings represents another approachto improve the rates of mass transfer. There are two types of non-porous packings: non-porous silica (NPS) and non-porousresin (NPR). As depicted in Figure 1(c), the non-porouspackings are very reminiscent of the older pellicular or porous-layer beads (PLBs) used in the early days of HPLC, but thesematerials are of much smaller particle sizes, typically in the1.5–2.5 µm range. 10 The thin porous layer allows much fasterrates of mass transfer and separations of only a few minutes canbe achieved for both large and small molecules. Unfortunately,the thin layer of stationary phase also limits the capacity of thepacking making NPS and NPR unsuitable for preparativeseparations. In addition, because of their small particle size, thebackpressures from NPS columns are generally much greaterthan those experienced with microparticulate HPLC porouspackings of popular particle sizes (i.e., 5 and 3 µm). For moreinformation on the use and advantages of NPS packings,consult reference 10. Such particles are finding less use intoday’s chromatography laboratory.Superficially porous packings (Figure 1(d)) are similar toNPS particles described above but the particle diameter islarger (~ 5 µm), providing a much lower pressure drop. ThesePoroshell particles (Agilent Technologies, Wilmington,Delaware, USA) are recommended for larger biomolecules thatdiffuse slowly into porous packings. When flow-rates areincreased with porous packings, the biomolecule peaks broadenbecause of slow diffusion into and out of the pores. The thinlayer of stationary phase is derivatized with alkyl bondedmoieties such as C3, C8 and C18 providing rapid separationsof proteins by reversed-phase chromatography. Poroshell-typepackings combine the advantages of rapid mass transfer (i.e.,improved efficiency), a decent sample capacity and goodrecovery of biomolecules. Figure 3 shows the rapid gradientseparation of several protein standards in less than 1 min onsuch a column. Monoliths Monoliths are columns that are cast as continuoushomogeneous phases (just like concrete in a mould) ratherthan packed as individual particles. These types of columnshave been reviewed earlier in LCãGC  11 and in this present volume. 12 There are several types of monolithic column:ãagglomerates of polyacrylamide particles ãpolymethyacrylate block ãagglomeration of micron-size silica beadsãpolystyrene-divinylbenzene block ãsilica rods ãmembranes of various types (made by many manufacturers).Monolithic columns have great potential in offering a stable,easily replaced column for both analytical and preparativeseparations. Both silica-based and polymer-based monolithshave been extensively studied. The silica-based materials weredeveloped by Tanaka and coworkers in Japan, 13 introduced by Cabrera and coworkers 14 at HPLC ’98 in St. Louis, Missouri,USA, and commercialized by E. Merck (Darmstadt, Germany)as its SilRod column. These columns are solid rods of silicamonolith. Similar to the perfusion packings, they have bothflow-through pores with macroporosity (1–2 µm in width) anddiffusive pores (called mesopores). The silica rods can bemodified using the same derivatization chemistries that areused for regular HPLC packings (e.g., C18 bonded phase).The SilRods, now encapsulated in PEEK, have been introducedas Chromolith.There are two important characteristics for current silicamonolith columns: they have the efficiency equivalent to a 3–5µm silica particle and their pressure drop is approximately 30–40% lower than a 5 µm silica particle. Thus, columns can becoupled in a serial manner thereby generating higher platecounts for more difficult separations. The polymeric monolith columns have also made theirmark on separation science. These columns consist of acontinuous crosslinked, porous monolithic polymer usually polymethacrylates or methyacrylate copolymerizates. They can be fabricated into discs and tubes in convenient housingsfor easy connection to an HPLC system. Some examples of commercial products are the UNO from BioRad Laboratories(Richmond, California, USA), the CIM copolymers from BIA Separations (Ljubljana, Slovenia 11 ), and the Swiftpolystyrene-divinylbenzene monoliths from Isco (Lincoln,Nebraska, USA). To illustrate the use of a polymericmonolith, Figure 4 provides a 1 min separation of oligodeoxynucleotides (8- to 16-mers) on a CIM DEAE disc with a 3 mm thickness and a 16 mm diameter. This particularrun was a fast gradient elution using 6mL/min flow-rate thatpermits the use of a relatively thin disc of poly(glycidylmethacrylate-ethyleneglycol) dimethacrylatecopolymer. There are several polymeric monoliths Peaks: 1  angiotensin II, 2  neurotensin, 3  RNase,4  insulin, 5  lysozyme, 6  myoglobin, 7  carbonic anhydrase, 8  ovalbumin Time (s)015304560200250010050150     m     A     U 12345678 Figure 3: Fast high resolution separation of peptides and proteins with Poroshell column. Column: Poroshell 300SB–C18,2.1  75 mm; mobile phase: (A) 0.1% TFA (trifluoroacetic acid);(B) 0.07% TFA in AcN (aceonitrile); gradient: 5–100% B in 1.0 min; flow-rate: 3.0 mL/min; temperature: 70 ºC; pressure:260 bar; detection: UV, 215 nm. Majors commercially available with ion-exchange, hydrophobic-interaction, reversed-phase and affinity chromatography capability. Note that the functionalized membranes that havelong been used in the isolation of biomolecules are also aform of monolith columns. Inorganic–Organic Hybrids  Waters (Milford, Massachusetts, USA) has developed a uniqueapproach for making a hybrid packing, especially useful forhigh-pH applications, in which silica gel has been less useful.Traditionally, when high-pH conditions were required toachieve greater retention of basic compounds or for compoundstability reasons, polymeric packings, coated zirconia, aluminaparticles, or graphitized carbon materials were usually considered. For various reasons such as lower efficiency,swelling/shrinking problems, strong adsorption sites and otherundesirable features, these materials have never achieved thepopularity of silica gel as a base material. Waters has attemptedto combine the advantages of silica with those of organicpolymers.Most modern silica gels used in HPLC are produced by thepolymerization of tetrachloro- or tetraethoxy-silane monomerseventually resulting in a silica gel polymer with siloxane bonds(Si-O-Si) and various types of terminal silanols (-Si-OH) attheir surface. In its synthesis process, Waters starts with a silanemonomer that contains both a methyl group and three ethoxy groups thereby incorporating a methyl group into the silica-based final packing material. The column constructed from thismaterial (called XTerra) has proved to be more stable inalkaline conditions than its typical silica-based packings. 15 Thecompany has also used ethylene-linked triethoxysilane(RO) 3 SiCH 2 CH 2 Si(OR) 3 instead of MeSi(OR) 3 to form a sol-gel. At high-pH conditions, the particle from this lattermethod has a 30% longer lifetime than the particle from thesol-gel of MeSi(OR) 3.16  Another approach to make morealkaline stable silica bonded phases was used by Kirkland andcoworkers 17 in which a special bidentate bonded phaseanchored at two adjacent silanols combined with a high degreeof endcapping protected the underlying silica backbone fromattack by hydroxide ions. Sol-Gel Silica  As silica gel is the most widely used base material for bondedphases in HPLC, there is an interest in expanding the pHoperating range both on the acid and the basic sides of thepH scale. There are two types of silica particles used incommercial HPLC columns: sil-gel and sol-gel. Sil-gelparticles, usually made by gelling soluble silicates orcoalescing fumed silica, are characterized by higher porositiesand irregular pore shapes with variable wall thicknesses. Sol-gel particles, which are made by aggregating silica solparticles, have lower porosities and more-regular pores withthicker walls defined by the surrounding solid silica-solparticles. Sol-gels are generally more mechanically stable thansil-gels. Both silica gels types can withstand typical mobilephase buffers that are used on the acidic end of the scale. Atlow-pH values, the unprotected siloxane bonded phases arethe more vulnerable part of these bonded packings and may be subject to catalysed hydrolysis by the hydronium ion. 18 Pertinent to the discussion of intermediate-to-high pHstability, because of their thinner pore walls sil-gel particlesappear to dissolve more quickly than sol-gel particles. 19,20  Accordingly, researchers anticipate developing more long-term stable methods at intermediate-to-high pH usingcolumns made with sol-gel supports such as Hypersil(Thermo Hypersil Keystone, Bellefont, Pennsylvania, USA),Zorbax (Agilent Technologies) and Spherisorb (Waters)columns. Other Packing Materials Other non-silica–based packings have been introduced in thelast few years; among them have been polystyrene-divinylbenzene (PS-DVB) co-polymers, other organic polymers,zirconia, graphitized carbon and hydroxyapatite. As graphitizedcarbon was recently the subject of a review, 21 I will notelaborate on this packing material. The PS-DVB polymers have been around for a long time.Users often turned to them when they needed a high-pH,reversed-phase alternative to bonded silica gel. These polymericmaterials have a wide pH range, have high crosslinking, arepressure and temperature stable, and have no silanols tointeract with basic compounds. However, they suffer frompoorer efficiency than silica gel. Typically, a 5 µm polymericcolumn may exhibit about a third of the theoretical plates that Time (s)0102030405060708090010203040506090190140  1040    R  e   l  a   t   i  v  e  a   b  s  o  r   b  a  n  c  e   (   2   6   0  n  m   )  %  b  uf  f   er A 8 mer10 mer12 mer14 mer15 mer16 mersolvent peak Figure 4: Separation of Oligodeoxynucleotides on polymericmonolith column. Column: CIM DEAE (Anion Exchange) Disc,diameter: 16 mm  thickenss: 3 mm; bed volume: 0.34 mL;instrumentation: gradient HPLC System with extra low deadvolume mixing chamber; mobile phase: Buffer A: 20 mM tris-HC1, pH 7.4, Buffer (B): Buffer (A) + 1 M NaC1; gradient:shown on figure; flow-rate: 6 mL/min; detection: UV at 260 nm; temperature: ambient; injection size: 20 µL. (Courtesyof BIA Separations. 29 ) Number of Bases5'–3' SequenceShort Name8CCA TGT CT8 mer10CTC CAT GTC T10 mer12AGG TCC ATG TCT12 mer14CGA CGT CCA TGT CT14 mer15CCG AGG TCC ATG TCT15 mer16GCCG AGG TCC ATG TCT16 mer
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