Twenty-Five Years of HPLC Column Development—A Commercial Perspective

November 1, 2015
Ronald E. Majors
Ronald E. Majors

Ron Majors, editor of "Column Watch" and "Sample Prep Perspectives," has been with LCGC North America for over 26 years. Currently a senior scientist with Agilent Technologies, Wilmington, Delaware, Ron is known industry-wide as one of the premier chromatography experts in the field. He is also a member of LCGC's editorial advisory board.

Special Issues

Special Issues, Special Issues-11-01-2015, Volume 33, Issue 11
Page Number: 20–27

An unbiased summary of the state of the art up to mid-1994, which actually marked the first 27 years of HPLC.

 

The Significance of This Article-Then and Now

Before writing this article, I already had a history of writing review articles on column technology. At this point, I was on my way to becoming a regular reviewer of column technology developments. This article, published in LCGC in 1994, illustrates the practical emphasis of most of my writings, a focus that has continued to the present. I have focused on practical technologies that users can apply today to solve real-world problems, rather than on esoteric research or technologies that may not become commercially available for many years, if ever.

I chose this article for inclusion here because of its historical significance and its unbiased summary of the state of the art up to mid-1994, which actually marked the first 27 years of high performance liquid chromatography (HPLC) since HPLC really had its beginning in 1966. This review of the early decades of HPLC includes discussion of the first commercial HPLC packings and chemically bonded phases that led to the explosion in popularity of reversed-phase chromatography in the 1970s that continues today. It also discusses the cartridge column concept, the rise of the guard column and the integrated guard-analytical cartridge column, and the growth of specialty columns.

The subsequent 20 years of column development are now covered in the installment of “Column Watch” that appears in the November 2015 regular issue of LCGC, thus bringing us up to date on HPLC columns.

ABSTRACT

This article is based on a paper by Ron Majors presented at the Chromatography Symposium in honor of J.J. Kirkland at the 32nd Annual Eastern Analytical Symposium, which was held in Somerset, New Jersey, November 15-18, 1993.


During the last three decades, high performance liquid chromatography (HPLC) column development has paralleled, and sometimes exceeded, HPLC instrumentation development. The main reasons for HPLC’s rapid development in the late 1960s were the advent of pellicular packings, a low-flow rate reciprocating pump, and the flowthrough UV detector. The pellicular packings provided efficiency that was an order of magnitude higher and speed that was similar to the large, porous packings previously used. 

In the early 1970s, the microparticulate packings (<10-µm dp) that dominate today were sized and packed using high-pressure slurry techniques. They provided even greater speed, efficiency, and sample capacity advantages. Also, stable, chemically bonded phases replaced liquid-liquid chromatography and spurred the development of reversed-phase chromatography, the technique that dominates column usage today (1). 

The 1980s saw the introduction of biochromatography materials, which provided better analytical and preparative problem-solving tools for life scientists. In addition, solid-phase extraction (SPE) cartridges enabled more convenient sample cleanup.

In the 1990s, further refinements in column technology produced chiral columns that provided large separation factors, more-stable and -durable reversed-phase columns, and specialty columns for solving specific environmental, chemical, and other specialty applications problems.

Some early developments in packing materials occurred in the academic community, but when the commercial sector developed reasonably priced, packed columns, general chromatographers began to apply HPLC to real-world problems. This trend continued and an entire field of HPLC column technology evolved, including various bonded phases, microporous particles, biocompatible columns, column hardware designs, and specialty columns for carbohydrate and organic acid analysis and ion chromatography. Most of these developments occurred in the commercial sector; now many companies, not just the instrument manufacturers, are developing, manufacturing, and marketing HPLC columns.

This installment of “Column Watch” reviews some of the major contributions made by the commercial sector during the last 25 years. Rather than covering all aspects of HPLC column development, I’ll focus on the developments and improvements in particle and bonded-phase technology. In particular, I’ll look at reversed-phase chromatography; column hardware design for analytical, preparative, and process chromatography; and some of the major contributions in other HPLC modes. This installment will also explore the future directions of commercial column development.

 

HPLC Column Developments

Providing an exposé about the history of chromatography and liquid chromatography (LC) is not the purpose of this column. Rather, I want to provide background about how the commercialization of HPLC was a driving force in its rapid acceptance and growth.

Particles and Columns

In the early 1960s, HPLC as we know it today hardly existed. Although researchers had predicted that LC theoretically could be more efficient and faster, they had done little practical work to realize these predictions. At that time, most liquid chromatographers used open columns packed with well larger than 100-µm dp
particles, usually sorted into particle-size ranges by sieves. Chromatographers used gravity as the pressure source, collected analytes at the column exit, and measured the analytes using off-line techniques such as UV-vis spectroscopy.

The first work, which seeded the commercialization of LC, focused on gel chromatography-the forerunner of modem size-exclusion chromatography. The research of Porath and Flodin (2,3) in cross-linked dextrans and of Moore (4) in polystyrenes pointed the way to commercial development of Sephadex (Pharmacia) and Styragel (Waters Associates), respectively.

In particular, James Waters recognized the importance of the Dow technology, licensed its material, and built a commercial instrument around the material that used a refractive index detector; thus began Waters Associates. His product offerings included a gel-permeation unit for polymer characterization and an organic acid analyzer. Still, the particles in these columns were rather big, and the separations were slow. The reciprocating pumps used at that time had large pulsations that had to be damped out, and the refractive index detector offered limited sensitivity.

Based on the theoretical predictions of Purnell (5) and Golay (6) and experimental gas chromatography (GC) results in Horváth’s Ph.D. thesis (7,8), using a support with an impenetrable, hard core coated with a thin outer layer of a porous solid should provide high-speed solute mass transfer in the stationary phase. Horváth and co-workers (9) first demonstrated the so-called porous-layer head (or pellicular) support for ion-exchange chromatography of nucleosides. As depicted in Table I, this medium became the first commercial HPLC packing (Pellosil, Northgate Laboratories) that provided convincing results.

 

 

The medium consisted of spherical, solid glass beads that were coated with a poly-(styrene-divinylbenzene) resin containing ionogenic groups such as tetraalkylammonium and sulfonic acid groups. Northgate Laboratories, founded by Horváth and Lipsky, eventually sold their pellicular packing technology to Reeve Angel Ltd., which later became Whatman Inc. Scientists at the now-defunct Picker Nuclear developed the low-volume flowthrough UV detector that Horváth and Lipsky used in their work; this detector technology was sold to Laboratory Data Control (LDC), part of the Milton Roy Corp., which, in turn, recently became part of Thermo Electron Corp.

At about the same time, Kirkland (10) worked on a similar approach, but he developed media for use in partition chromatography. Designed as a proprietary product for DuPont, the medium comprised submicrometer silica particles bonded onto glass beads with average particle diameters of 40 µm. The product, Zipax (DuPont Corporation), was called a controlled surface porosity support and was used in liquid-liquid partition chromatography.

In Kirkland’s approach, the Zipax surface was coated with a polar liquid phase such as (β,β′ oxydipropionitrile (ODPN) or Carbowax (Union Carbide), and an immiscible, nonpolar mobile phase that was presaturated with stationary phase was passed through the column. This method was similar to the method used at that time for coated GC stationary phases.

This technique was standard procedure for performing HPLC in the late 1960s. DuPont, which manufactured instruments at that time but dropped out several years ago, also developed a low-cell volume UV detector, the model 410, which also helped to accelerate HPLC’s acceptance.

Waters Associates, through the work of Bombaugh and Little, introduced Corasil, which was a similar product that had a thin film of porous silica gel. Corasil could be used for liquid-solid (adsorption) and liquid-liquid chromatography.

Compared with the large, porous particles used earlier in LC, porous-layer beads provided much faster separations (in the 20-30 min range, rather than several hours), more efficient separations (heights equivalent to a theoretical plate [HETP] in the 1-2 min range), and better limits of detection. However, because the surface areas were greatly reduced, sample capacity was very small, typically in the 0.1 mg/g range. Users could dry-pack the dense, solid-core pellicular packings easily using mechanical vibration. However, liquid-liquid partition chromatography had severe limitations: using gradients and polar mobile phases was not feasible, and presaturating the mobile phase was extremely inconvenient.

Using smaller, porous particles overcame the limitations of the porous-layer beads, but the unavailability of suitable narrow particle-size distributions in commercial quantities and the lack of packing methods for them precluded their use. For experimental usage, small amounts of thin-layer types of silica-gel particles could be sized into fairly narrow ranges by sedimentation and elutriation techniques. However, the larger quantities of reasonably priced, small-particle, irregularly shaped silica gels needed for commercialization could be obtained only by using the newly developed air centrifugal classifiers.

Dry packing particles <20-µm in diameter was extremely difficult, but work by Huber (11) showed that analysts occasionally could obtain a high-efficiency column with careful hand packing and tamping using a small PTFE-tipped rod.

 

 

Majors (12) successfully and reproducibly applied balanced density slurry techniques to a small-particle silica gel supplied by E. Merck; this method enabled commercial production of microparticulate packed columns. Used for liquid-solid and liquid-liquid partition chromatography, the initial commercial product, called MicroPak (Varian Associates), was available in 5- and 10-µm dsizes packed in 2.1-mm i.d. columns and provided plate heights in the 0.1-mm range. The separation speed and efficiency shown in the separation of Figure 1 was a marked improvement over other columns available in the early 1970s.

 

With the high surface area, the microparticulates displayed sample capacity typical of the older LC columns but with even better efficiency than the pellicular columns (values in the 0.05-0.1 mm range).

Kirkland (13) developed Zorbax, a spherical silica-gel material that also was a suitable base material for further developments. The packing had a 7-µm average particle diameter and provided excellent efficiency. In recent years, DuPont sold its analytical column business, and Rockland Technologies now manufactures the Zorbax products and their successors. Over the years, spherical silica gels replaced the irregular materials, and today most chromatographers prefer them (1). The spherical packings seem to pack more consistently and reproducibly. Special synthesis methods produce the spherical silicas, and they are generally more expensive.

Much of the early work in HPLC dealt with applications for traditional organic molecules--particularly polymers, additives, pesticides, and petrochemicals-and for small ionic or ionizable compounds such as nucleic acid constituents, pharmaceuticals, and inorganic ions. With the exception of the size-exclusion materials, most of the frequently used packing materials had pore sizes in the 60- and 100-Å range.

 

 

Beginning in the late 1970s, interest arose in biotechnology and in the separation and purification of larger biomolecules, and researchers required new types of biocompatible packing materials. Initially, larger-pore silica gels, 4000 Å in diameter with 300 Å being the most popular size, were prepared because proteins as large as several hundred thousand daltons could penetrate these larger pores.

Companies began to introduce wide-pore polymeric-based materials. High-speed separations of biomolecules were of interest in the mid-1980s, and analysts fell back on pellicular packings. However, the so-called nonporous resins had much smaller particle diameters, in the 1.5-3.5 µm range, than the earlier porous-layer beads. These materials provided exceedingly fast separations but were difficult to pack and produced high back pressure, as might be expected based on their small particle size. Less useful for general preparative work because of their small capacity, these materials could be used for micropreparative separations of a few milligrams.

In the 1990s, PerSeptive Biosystems introduced perfusion supports, and Pharmacia and Sepracor debuted similar products. These packings provide two classes of pores: large through pores, which permit the biomolecules to pass through quickly, and small pores, which line the through pores and create a higher surface area and short diffusion paths. These media are designed primarily for the high-speed purification of biomolecules and to reduce the time and cost required for biopharmaceutical development and manufacturing.

Chemically Bonded Phases 

The limitations of liquid-liquid partition chromatography caused researchers to look for alternatives that could extend HPLC’s usefulness. Bonding chemical phases to the surface of the porous-layer beads was the first approach that provided some advantages. The first commercial phases, Waters’ DuraPaks, were the “brush” type of phases in which the Si-OH of the silica was chemically reacted to form the Si-O-C bond (14). Bonded-phase HPLC first used various chemically bonded phases such as ODPN and Carbowax. Unfortunately, the Si-O-C bond was unstable to hydrolysis and could be cleaved with water and other proton-donating liquids. Thus, the use of silane chemistry proved more stable and organochlorosilanes or alkoxysilanes then could be purchased commercially. The reaction of silica gel silanols and the organosilane formed the Si-O-Si-C siloxane bond. These chemically bonded phases could withstand a greater range of mobile-phase conditions and, in particular, could be used with aqueous media. 

DuPont’s Permaphases were the first commercial packings using this silane chemistry, and the company introduced a series of different functionalities in the early 1970s. Other companies quickly developed their own phases on both pellicular and microparticulate materials. The siloxane phases could be used within the pH 2-8.5 range; at pH values >9, the siloxane bond of the underlying silica structure could be attacked. With high coverage, analysts can use longer chain lengths and protective steric groups with slightly higher pH; many companies have developed reversed-phase packings with these characteristics. Polymeric packings were developed for the highest degree of stability.

In general, the cross-linked polymeric packings, including polystyrene and polymethacrylates, can withstand strongly basic mobile phases. They display somewhat lower efficiency than the silica gel-based packings, although the efficiency has improved in recent years. Japanese companies have been particularly active in the synthesis and commercialization of polymeric materials (15), but a few American and European companies have introduced improved polymeric packing materials. These manufacturers include Hamilton, Dionex, Bio-Rad Laboratories, Interaction Chemicals, Polymer Laboratories, Pharmacia, and Tessek.

 

 

The availability of bonded siloxane phases brought a particularly favorable fallout: the development of reversed-phase chromatography. Most chromatographers in the early 1970s used polar stationary phases (for example, Carbowax and silica gel) and a nonpolar mobile phase (for example, isooctane and hexane). This new mode of chromatography (never very popular in liquid-liquid partition chromatography) used the opposite combination-a nonpolar bonded stationary phase (octadecylsilane) and a polar mobile phase (water and water-miscible organic solvents such as methanol and acetonitrile)-hence, the name reversed phase. The regularly practiced form of chromatography then became normal phase, a name still used today but only popularized after reversed-phase chromatography came on the scene.

Because many organic substances show low solubility in water and high solubility in the water-miscible organic solvents, reversed-phase chromatography’s popularity exploded in the late 1970s, and today it represents the most widely used HPLC mode (1). Manufacturers have introduced at least 250 reversed-phase packing materials since then, and subcategories of the reversed-phase mode such as ion-pairing, ion-suppressed, and hydrophobic interaction chromatography have evolved. Additionally, companies have debuted many hydrophobic reversed-phase bonded phases ranging from C2 to C30 alkyl, phenyl, and diphenyl phases. Highly loaded or high-coverage, endcapped, polymeric, carbon, and mixed-mode phases extended separation possibilities to include a wide variety of organic and inorganic compounds, which can sometimes be separated in the same chromatographic run.

Another major advance in HPLC was the development of the base-deactivated reversed-phase materials. Early reversed phases had problems during analysis of very basic compounds such as polyamines. These analytes would tail or be strongly adsorbed when they were used with regular mobile phases such as water, methanol, and acetonitrile. The presence of unreacted silanols (≡Si-OH) located deep within the silica gel micropores caused the tailing. These silanes would be sterically hindered from reacting with the bulky octadecylsilane reagents used to prepare the bonded phases. Often users could prevent tailing by adding competing bases, such as tetramethyl ammonium chloride, to the mobile phase. However, adding these bases was inconvenient, and often the bases were impure, which caused background interferences and sometimes reduced column life.

Exhaustive endcapping was another way to eliminate silanols. In this process, analysts used a small silane reagent, such as trimethyl chlorosilane, that could penetrate the micropores after the reaction with the primary silane. The base-deactivated phases introduced in the late 1980s provided much better performance than the early phases. These base-deactivated phases were not deactivated with base, as the name implies, but were specially synthesized or treated silicas that contained a more-homogeneous distribution of silanols, which permitted higher surface coverages; in these phases, the unreacted silanols were either blocked or nearly totally reacted.

Most of the bonded-phase developments described above came from the commercial sector as internally developed products that were generated from customer requests or as technology transferred from universities to industry.

 

Column Hardware Designs 

Initially, analytical HPLC columns consisted of stainless steel tubing pieces, usually 50 or 100 cm × 2.0 mm for porous-layer beads and 25 or 30 cm × 4.0 or 4.6 mm for microparticulates. Parker Hannifin and Swagelok manufactured the most popular compression fittings.

After columns were exhausted, users threw them away or, to save money, cleaned and repacked them. A whole industry of column refiners sprang up, especially in Europe. Because endfittings often represented a major part of a column’s cost, analysts wanted to eliminate endfittings or find ways to reuse them, and the cartridge column concept was born.

Brownlee Laboratories, which designed the first cartridge column, was bought by Applied Biosystems in the late 1980s. The Brownlee column had a sleeve (cartridge holder) in which the column would fit. When the columns were “dead,” users merely removed them from the holder and inserted a new one. The endfittings were an integral part of the holder and, therefore, required no replacements.

E. Merck designed an alternative type of cartridge that had no sleeve but included reusable endfittings. In this design, the column had grooves in which the reusable endfittings were retained by a nut placed at the column’s end. Column replacement costs were lower for cartridge columns than conventional columns with compression fittings. Some cartridge columns could be finger tightened, eliminating the need for wrenches.

Over the years, the use of in-line filters and guard columns increased. An in-line filter placed between the pump and the injector prevented debris such as pump-seal wear material and solvent particulates from getting lodged in the injector or at the head of the analytical column. Some in-line filters, built with especially low dead volumes, could be placed between the injector and the analytical column.

Guard columns were short versions of analytical columns filled with the same packing material. Placed between the injector and the analytical column, the guard column collected debris-such as pump-seal fragments and sample particulates-and strongly retained sample components, thus protecting the analytical column. Analysts needed a short piece of tubing to connect the guard and analytical columns. Because some extracolumn effects were unavoidable, these connections usually caused some loss of efficiency. Guard columns were considerably less expensive than the analytical columns that they protected. Therefore, users could afford to replace them on an as-needed basis. The use of guard columns has continued to increase over the years (1).

 

 

The concept of an integrated guard-analytical cartridge column combination came about in the mid 1980s. Designed to avoid almost all dead volume, the guard column in these systems often butted against the analytical column, thus maintaining the full efficiency of the analytical column-guard column combination. Many of the systems could be finger tightened.

In preparative chromatography, many unique hardware designs came in the late 1980s. Early preparative work was performed using open-column chromatography, in which gravity was the driving force. Large columns ensured large sample capacity, which was of utmost concern. Sometimes, the whole synthesis mixture was pipetted onto the top of a column and moved through the column with a series of solvents--a crude step gradient.

As HPLC developed, so did preparative column design. Because porous-layer beads had a very low sample capacity, analyses required very large columns. But because porous-layer HPLC packings were expensive, it was economically painful to use these packings preparatively. The advent of the microparticulates allowed chromatographers to use more-realistic dimensions for preparative chromatography. For a few milligrams of sample, conventional analytical columns performed well. However, as the need for large-scale, preparative high performance separations developed in the early 1980s, manufacturers introduced columns with inner diameters as large as 1 in. For the most part, these columns were large-diameter varieties of the packed analytical stainless steel columns without any major changes in the column design.

Chromatographers later discovered that sample introduction and distribution had to be different and that the columns would often settle or dissolve with the continual passage of mobile phase at high flow rates. In addition, conventional HPLC pumps lacked the necessary flow-rate capacity for the larger columns. Therefore, new HPLC companies (or divisions of existing companies) emerged to deal primarily with preparative HPLC.

To handle preparative separations, manufacturers modified columns and developed new designs. Waters Associates developed radial compressed columns. These soft-sided columns fit in a special holder that applied pressure to the sides of the column to prevent flow along the walls during preparative elution. This design grew from observations that the wall-packing interface in conventional columns offered a low-resistance flow path that enabled the mobile phase to move faster, thereby distorting the peaks.

To prevent resolution loss as the column settled, manufacturers developed axial compression columns. In these columns, pressure was applied in an axial manner rather than a radial manner. One design included a movable piston that provided an adjustable bed height. The piston would apply continuous pressure to the column head, and the piston would move with the top of the packing as the level of packing changed due to settling or dissolution, thereby eliminating void formation. An alternative design includes a movable inlet plug that users can adjust manually. Other designs allow chromatographers to take up slack in a preparative column and ensure a tight bed.

 

 

One unique design from SepTech is the macrobore annular-expansion column, which has a stainless steel spindle inserted lengthwise into the center of the column (16). The plunger-frit-distributor assembly is attached at the head of the conical spindle and sealed against the column wall. The assembly moves in tandem with the spindle, and the entire plunger assembly presses against the stationary phase, creating axial and radial expansionary forces within the column and resulting in a tightly packed bed that can be adjusted if the bed settles.

Other hardware developments included the introduction of new column tubing materials. Stainless steel type 316 has been the mainstay for many years, but PTFE- and glass-lined stainless steel, titanium, Hastelloy, and other stainless steels have been used as substitutes. Companies introduced high-pressure glass columns for both analytical and preparative work. Biochromatographers sometimes prefer the inertness of glass and the ability to see the condition of the packed bed. Polyetheretherketone (PEEK), which resists most of the popular HPLC solvents, has been used frequently for connecting tubing. Several companies have made entire columns from this material. Acrylics and other polymeric materials found niche applications; these non-metallic columns are popular for ion chromatography, in which metallic ions can leach from tubing and interfere with trace analysis, and for biochromatography, in which metals might interfere with analysis of metal-scavenging proteins.

Microbore columns are versions of analytical HPLC columns with smaller inner diameters (0.5-2.0 mm). For the last 15 years or so, chromatographers have flirted with smaller inner diameter columns, but the columns never became a mainstream success, as predicted in the early 1980s. The main reasons to use microbore columns are increased solvent savings, greater sensitivity for limited sample situations, and the ability to use solvent-limited detectors such as certain gas-phase detectors and mass spectrometer inlets.

In the early 1980s, these reasons didn’t offer enough incentives, and most of the HPLC instruments were not optimized for these smaller diameters. As column diameters become smaller, flow rates decrease because the inverse square of the column radius ratio and system dead volume play bigger roles in maintaining resolution. Most HPLC systems were designed for use with 4.6-mm i.d. columns, so many are taxed when used with columns in the 1-2 mm i.d. range.

In recent years, solvent consumption has become more important as the cost of HPLC solvents increases, and disposal costs now are equal to or greater than solvent purchase cost. Worker safety and reduced overall use of chemicals in laboratories brought microbore LC back to the attention of users. In a recent survey, 42% of the chromatographers thought that they would be considering microbore columns in the future (17). As this need increases, columns and instrument companies will respond with new products.

 

 

Packed capillary columns (0.25- and 0.32-mm i.d.) and open tubular LC columns (<100-µm i.d.) remain as research curiosities. A few companies, such as LC Packings and MicroTech Scientific, are starting to make headway in demonstrating the advantages of the smaller columns. Perhaps the future instrumentation and column technology requirements of capillary electrophoresis, which has detector and some injector needs similar to micro LC, will serve as the driving force for further HPLC column miniaturization.

The popularity of fast HPLC columns has gained momentum in recent years (17). These columns are merely short versions of columns with popular inner diameters. When these columns are packed with 3- and 5-µm packings and used with slightly higher flow rates (<6 mL/min), chromatographers can obtain very rapid separations in a few minutes. These rapid analyses seem to be popular in pharmaceutical quality control and in kinetic studies. However, extracolumn effects can negate the advantages of fast HPLC columns, much as they do in microbore LC, and the instrument design requires great care.

Other Advances

I have no intention of covering all aspects of commercial HPLC column developments in this column. Over the years, we have tracked column developments in a series of articles about products introduced at the Pittsburgh Conference (Pittcon), starting in 1984 (18) and continuing through this year (19,20).

The development of specialty columns is something we should review. In the early days of HPLC, chromatographers believed that most separations could be achieved with just a very few stationary phases by manipulating the mobile phase. No one thought that stationary phases would proliferate as they had in GC. However, as analysts applied HPLC to increasingly complex samples and compounds with subtle structural differences, they found that standard columns failed to separate the analytes of interest sufficiently even with systematic changes in mobile-phase composition. Thus, researchers developed specialty HPLC columns.

Table II extracts some of the specialty columns that companies have introduced since 1985. Many of these column types were synthesized especially for the compounds of interest, and most were tested and shipped with appropriate chromatograms. Introductions of specialty columns are likely to grow in the coming years.

 

 

In the main HPLC modes, some major milestones are worthy of mention. In size-exclusion chromatography, Waters Associates and some Japanese companies such as Toyo Soda (now Tosoh) pursued further developments that resulted in improvements in Dow’s original polystyrene materials. The synthesis of smaller particle beads (as small as 5-10 µm) made size-exclusion chromatography a faster technique for the separation and characterization of polymers. Also, aqueous-compatible size-exclusion chromatography phases based on polyether, polymethacrylates, and polyvinyl alcohols allowed analysts to use water-soluble polymers.

In ion-exchange and ion chromatography, Dionex took the lead and developed its proprietary process for making ion chromatography materials that possessed a highly cross-linked solid core of polymer coated with a pellicular latex material. Because of rapid diffusion rates, these columns enabled fast analysis of ionic samples and the use of organic solvents to help elute difficult compounds. Many companies also developed ion exchangers, usually based on wide-pore silicas and polymeric base materials, for the separation of proteins and other biomolecules.

Improvements in sample preparation also have helped the progress of HPLC. The development of SPE cartridges in the late 1970s gave chromatographers the opportunity to clean their samples before injecting them into the HPLC column. Because the SPE cartridges were small but less-efficient versions (40-µm particles) of regular HPLC columns, users quickly accepted them, and the technique’s popularity has grown steadily over the last decade.

Most SPE devices are syringe barrels packed with silica and bonded-phase packings. A new format appeared on the scene in the early 1990s-the SPE disk (21). The disk presents a large surface area that enables high flow rates and reduces plugging, which is common with the conventional small cross-sectional area SPE cartridges.

Commercial polymeric SPE materials had the major advantage of being washable and reusable, but few users would risk contaminating future samples by doing so. To reduce the extractables emanating from the plastics used in cartridges’ frits and barrels, companies introduced glass and PTFE bodies. Just as manufacturers developed specialty HPLC columns, specialty SPE cartridges debuted for applications such as the cleanup of drugs of abuse and catecholamines in body fluid, polynuclear aromatic hydrocarbons in water, and ketones and aldehydes in air.

Conclusions

As customers recognize new needs and HPLC grows, instrument and column companies will continue developing new products to meet the needs of the marketplace. However, the HPLC market’s growth has slowed and the prices of columns have eroded as competition has increased. Refiller companies have gained a foothold, especially in Europe. Companies that prospered during the high-growth times are forced to find new markets and other areas of expansion to fuel their growth.

Capillary electrophoresis (CE) represents an area in which some HPLC technology has been applied, but GC column manufacturers that are knowledgeable in fused-silica and polymer technology have also ventured into CE. The CE capillary marketplace is still relatively small compared with HPLC and GC, and most CE instrument manufacturers produce capillaries that fit only in their instruments.

 

 

Researchers have focused much attention on biochromatography and process chromatography in the last several years, sparked by continuing interest in techniques for separating and purifying biotechnology-derived materials and biopharmaceuticals. Growth will continue, especially in the perfusion and wide-pore supports that offer high speed and capacity. With the explosive growth in chiral separations stemming from the regulatory requirements for enantiomer purity in pharmaceutical and agricultural compounds, manufacturers have opportunities to develop a universal chiral phase that can work with a wide variety of compound classes.

SPE cartridges use technology similar to HPLC column production, but too many companies have ventured into this area because of low startup and production costs. Chromatographers need other sample preparation devices to decrease the amount of time they spend preparing samples for analysis. Usage of both SPE disks and functionalized membranes will continue to grow.

Aside from Japanese companies, few commercial enterprises have ventured into the polymer arena. Polymer material development seems to require some expertise to make reproducible materials and modify the polymer surfaces. Still, manufacturers can improve the efficiency of polymeric packings; after they do, the columns should achieve a higher market share.

Microcolumns still await more widely available, optimized instrumentation, but they offer great promise. All in all, HPLC faces no major competitive separations technology that will displace it in the near future, and the outlook for the HPLC columns business remains bright, especially for companies that are in touch with their customers’ needs.

References

  1. R.E. Majors, LCGC9(10), 686-693 (1991).
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  3. P. Flodin, Ph.D. thesis, University of Uppsala, Sweden (1962).
  4. J.C. Moore, J. Poly. Sci., Part A-2, 835 (1964).
  5. J.H. Purnell, Nature184, 20009 (1959).
  6. M.J.E. Golay in Gas Chromatography 1960, R.P.W. Scott, Ed. (Butterworths, London, 1960), p. 139.
  7. Cs. Horváth, Ph.D. thesis, Universitat Frankfurt Main, Germany (1963).
  8. I. Halasz and Cs. Horváth, Anal. Chem. 36, 1178 (1964).
  9. Cs. Horváth, B.A. Preiss, and S.R. Lipsky, Anal. Chem.39,1422 (1967).
  10. J.J. Kirkland, Anal. Chem.41, 218-220 (1969).
  11. J.F.K. Huber, J. Chromatogr. Sci.7, 85-90 (1969).
  12. R.E. Majors, Anal. Chem.44, 1722-1726 (1972).
  13. J.T. Kirkland, J. Chromatogr. Sci.10, 593-599 (1972).
  14. I. Halasz and I. Sebastian, paper presented at the Fifth International Symposium on Advances in Chromatography, Las Vegas, Nevada, January 1969.
  15. R.E. Majors, LCGC11(11), 778-788 (1993).
  16. R.E. Majors, LCGC7(4), 304-314 (1989).
  17. R.E. Majors, LCGC7(6),468-475 (1989).
  18. R.E. Majors, LCGC1(3), 146-147 (1983).
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  20. R.E. Majors, LCGC 12(4), 278-293 (1994).
  21. C. Markell, D.F. Hagen, and V.A. Bunnelle, LCGC9(5), 332-336 (1991).

How to Cite this Article

R.E. Majors, LCGC12(7), 508-518 (1994).

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