More affectionately known as HPLC 2005, the 29th International Symposium on High Performance Liquid Phase Separations and Related Techniques is the premier scientific event for bringing together the myriad techniques related to separations in liquid and supercritical fluid media. Organized by Professor Douglas Westerlund of Uppsala University, Sweden, who also chaired the HPLC 1989 Symposium, HPLC 2005 assembled over 1200 scientists from a total of 48 countries. This number included vendor representatives from 78 exhibitors for the three-day instrument, software, and consumables exhibition. and conferees, one-day registrants, exhibit-only registrants and press.  

The four-day-plus event had a total of 784 symposium papers and 124 oral presentations, many presented during simultaneous sessions, 650 posters in sessions with 26 themes and 10 vendor seminars. Thus, attendees had their hands full deciding how to allocate their time. With an ample social event schedule and seven short courses held during the previous weekend, most attendees who stayed for the week were ready for some relaxation. The short course topics reflected areas of current interest and included peak shape and retention in liquid chromatography (LC), mass spectrometry (MS) detection, preparative LC, FDA method development and validation, enantiomeric separations, monolith columns, and bioanalytical sample preparation. 

This year's event featured a special program for young scientists who had completed their highest degree within the past 10 years. Of the 24 lectures presented, surprisingly, 38% of them were female scientists. At the opening ceremony, a number of Halasz Foundation scholarships were awarded to aspiring graduate students. It is satisfying to know that the future of liquid chromatography is in good hands. The late Prof. Halasz of Saarbrueken University in Germany was an early pioneer in gas chromatography (GC) and high performance liquid chromatography (HPLC) technology.  

 Table I:  HPLC 2005 papers presented by technology or technique			

Obviously, HPLC was the predominant technology used by presenters in the technical sessions at the symposium. From a perusal of the poster and oral presentation abstracts, Table I provides a rough breakdown of the coverage of liquid phase technology and techniques in the separation sciences. Table II is a similar breakdown for application areas. This year, I also tabulated the detection principles (Table III) that were used. Not every abstract indicated the detector used, so only those abstracts that provided this information were counted. The category assignments were based on the main emphasis of a particular scientific paper as well as separation and detection techniques used.  

 Table II:  Presented by application area			

In this installment of "Column Watch," I will present some of the scientific highlights of HPLC 2005. Because it was virtually impossible for one person to adequately cover all oral and poster papers, my coverage will somewhat reflect a personal bias, although some of the presenters provided summaries.  

 Table III:  Types of detection techniques used at HPLC 2005			

The mainstay of HPLC 2005 was the poster sessions, in which more detailed applications and methodology studies were reported, often in very specific areas, and face-to-face discussions with the authors were conducted. Fortunately, many of the poster authors were kind enough to provide small reproductions of their poster papers that could be taken for later perusal. Some authors collected business cards and addresses for sending poster reprints by mail or e-mail. This year, posters were up for all four days, allowing participants to adequately cover these sessions.  

This year, I once again had the privilege of leading the Poster Committee that chooses the top three posters at the symposium. With over 650 posters to cover, poster review was a challenging task. Fortunately, I had the able assistance of 32 members of the Poster Committee, who worked very hard to narrow down the huge collection of posters and helped select the three winners by the end of the symposium's fourth day. I want to individually recognize them by providing their names and affiliations in Table IV. Overall, the committee felt that the quality of posters was outstanding this year and showed a continued improvement over previous symposia in this series.  

The Best Poster Awards, sponsored by Agilent Technologies (Waldbronn, Germany) were announced at the closing session. The winners received Amazon.com gift checks. This year's number one winning poster was titled "Separation of Probe Substrates of Cytochrome P450 Using Temperature Responsive Chromatographic System," written by H. Kanazawa, Y. Okada, E. Ayano, Y. Matsushima of the Kyoritsu University of Pharmacy, Tokyo, Japan, and T. Okano, Tokyo Women's Medical University, Tokyo, Japan. Their novel paper demonstrated the development and application of a temperature-responsive polymer grafted to a microparticulate silica substrate. The "green" chromatographic system used only water as a mobile phase. By changing the temperature only, the column's characteristics could be changed from a hydrophilic to a hydrophobic surface and allowed chromatographic resolution of a number of probe drugs under isocratic conditions. 

The second place poster was "Molecular Dynamic Simulations of Chromatographic Surfaces" by K.A. Lippa, L.Sander, and R.D. Mountain of the National Institute of Standards and Technology (NIST), Gaithersburg, Maryland, and was a last-minute poster that was not even in the final program. Their poster covered their work on molecular dynamic simulation models that describe the structural and dynamics of shape selectivity processes in reversed-phase LC when employing polymeric and monomeric phases with alkyl chain lengths, surface coverages, bonding chemistry, and temperature conditions that are typical of actual materials prepared in the laboratory. These resultant bulk structural features were related to the observed shape selectivity properties and were consistent with experimental observations. Among the findings were: alkyl chain segments proximate to the surface are disordered and chain segments at the distal end are more ordered; alkyl chain ordering is enhanced at lower temperatures; and models representing shape-selective phases have a significant region (~11 Å) of alkyl chain order and an increase in cavity order whose depths increased for polymeric C30 models, and with lower density monomeric C30 models, highly ordered chain "walls" are observed. The group is further modeling solute retention of solvated phases in acetonitrile–water systems as well as the retention relationships of constrained shaped solutes. 

The third place poster, titled "Anisotropic Siloxane-Based Monolith Prepared in Confined Spaces," was written by Y. Suzumura, K. Kanamori, K. Nakanishi, and K. Hirao of the Graduate School of Engineering, Kyoto University, Kyoto, Japan, and J. Yamamichi of Canon, Inc., Kanagawa, Japan. This same group was the first place winner at HPLC 2004. These authors extended their earlier work in this paper. Using sol-gel chemistry, the workers successfully prepared monolithic columns in situ with an oriented pillar structure in a variety of 2-D confined spaces. Due to wetting of the siloxane phase onto the interface of the mold and the effect of gravity, in this confined space, the gel skeletons in the vicinity of the interface are elongated perpendicular to the interface. Using scanning electron microscopy and laser scanning confocal microscopy, geometrical informatioin on gel morphologies was derived. The long range implications are that these technologies should allow more efficient separations in chip-based separation systems. 

Photographs of all three winners receiving their prizes are shown in Figure 1.  

Figure 1:  (Top) Ron Majors presenting first prize to Hideko Kanazawa of the Kyoritsu University of Pharmacy, Tokyo, Japan; (middle) Ron Majors presenting second prize to Lane Sander, NIST, Gaithersburg, Maryland; and (bottom) Ron Majors presenting third prize to Kazuki Nakanishi of Kyoto University, Kyoto, Japan.			

Topics of opening sessions usually are chosen to reflect current research areas in which separation science has played a key role. In this year's opening session on Sunday afternoon, Peter Schoenmakers of the University of Amsterdam, The Netherlands, a member of the Permanent Scientific Committee, discussed the state-of-the-art in LC-LC, comprehensive two-dimensional separations. However, as next month's column will be devoted to a review of this technology, I will review his plenary lecture at that time. 

Two other plenary lectures were by notable life scientists Ulf Landegren of the University of Uppsala and John Yates of the Scripps Research Institute, La Jolla, California. Landegren's lecture discussed the analysis of proteins at the single-molecule level using molecular tools that represent detected DNA, RNA, or protein molecules as short, circular rings of DNA. His approach allows highly specific analysis of large sets of molecules in solution or for in situ analysis. Using a technique called proximity ligation, antibodies or other suitable reagents that bind a target protein are equipped with these DNA strands that can be joined by ligation when pairs of reagents bind the same target protein molecule. The process effectively reverse translates target proteins into linear or circular signature DNA molecules that can be amplified and identified for highly sensitive, precise protein detection in homogenous or solid-phase assays. In the future, the process might be used for single-molecule detection inside of a cell. Interestingly, Landegren's techniques can be applied to any molecule that binds — not just proteins. Yates reviewed some of his early shotgun proteomics work in which a protein mix undergoes proteolysis to form complex peptide mixtures that are then analyzed by a combination of micro LC combined with tandem MS. The resulting massive amounts of data are then interrogated to hopefully come up with some useful information about the original proteins. The data analysis still represents the biggest challenge. The original multidimensional protein identification technology (MUDPIT) approach was described, in which cation-exchange and reversed-phase columns are connected in series to a 5-μm picospray nozzle into the MS ionization source. He further elaborated on work involving more targeted identification of specific proteins such as phosphorylated proteins. The identification of low stoichiometric amounts of phosphorylated proteins represents a challenging problem, so an additional reversed-phase enrichment column added downstream permits focusing of the analytes. Aside from the common use of trypsin, the use of multienzyme digestion using more specific enzymes also allows the determination of multiphosphorylation sites. The development of new MS detection tools with high mass accuracy, high sensitivity, and higher resolution will further aid the entire field of proteomics. 

As seen in Table I and similar to the past five years of HPLC Symposia coverage (1–5), the development and study of columns and stationary phases still dominates the new technologies. If one combines all papers pertaining to column technology, including monoliths, about a quarter of the presentations at HPLC 2005 covered this topic. Despite all the advances made in column technology to date, investigations on further developments in academia as well as the commercial side still are taking place. 

At HPLC 2005, monolithic phases dominated column technology and applications papers with 40% of the presentations devoted to this technology. Overall, more emphasis was devoted to the preparation of monoliths in capillaries and in microchannel chip-based systems than to columns of conventional dimensions. 

Monolith columns exhibit high permeability (due to bed porosity), absence of frits to confine the packing material, good separation efficiency, and ease of fabrication. Although this technology has been around for several years, as a routine tool it has yet to see widespread acceptance, but things are changing. Frantisek Svec of the University of California, Berkeley, California, gave a keynote lecture that presented an excellent overview of the development, state-of-the-art, and future directions of both polymeric and silica monolith technology. He discussed some of the newer or emerging synthesis techniques such as atom transfer radical polymerization (ATRP) and ring opening metathesis polymerization (ROMP). Ionic polymerization and polycondensation are other techniques that might also prove useful in the production of new types of monoliths. New inorganic materials such as hafnia and zirconia have been investigated to provide monoliths that can stand up in an extreme pH environment.  

Monoliths are starting to see a variety of applications. Monoliths in the capillary format with advanced detection techniques such as matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-MS and nuclear magnetic resonance (NMR) have been applied to solving problems in proteomics, metabolomics, and other biorelated disciplines. Monoliths have been used as solid-phase extraction (SPE) and immunoaffinity phases, as sensors, and for ion-selective electrodes. Monolithic columns as the second dimension provide an answer to the speed mismatch problems with 2-D systems. Monoliths also provide a solution to some of the problems encountered in capillary electrochromatography (CEC) such as the lack of need for frit formation. Bilayer chemistry can provide protection for basic peptides and proteins by preventing them from touching the underlying ionized surface.  

In the future, Svec sees monoliths employed for even smaller devices such as micro- and nanofluidic chip-based systems. They can be fabricated easily inside narrow channels, and such approaches already have been shown to provide useful applications. Monoliths might prove useful as a support to immobilize enzymes such as trypsin that should allow rapid proteolysis and on-line systems for proteomics research. Monoliths also can be prepared in the orifice of MS electrospray probes and functionalized using photografting procedures. Such procedures enable miniscule amounts of orthogonal stationary phases to be placed inside of nanochannels, allowing for separation and concentration to occur in a single operation. 

In a tutorial, Christian Huber of the University of Saarland, Saarbrücken Germany gave a more basic overview that was useful for nonspecialists. His tutorial was divided into three sections. The first section contained the facts about monoliths. He covered the preparation and functionalization of silica and polymeric monoliths and showed examples of high-resolution separations on polystyrene–divinylbenzene (PS-DVB) monoliths and showed via scanning electron micrographs how the morphology of the monolith surface can have great impact on chromatographic performance. He then covered the challenges and dispelled the myth that monoliths cannot be manufactured reproducibly by showing comparative data on silica and PS-DVB monoliths and microparticulate columns. The challenges remaining for monoliths are improving their peak symmetries, retention factors, and loadabilities through higher surface coverage with C18 bonding. Finally, he demonstrated the achievements so far with monolith technology and showed a variety of applications ranging from preservatives to phosphorylated and glycosylated peptides to ion chromatography to nucleic acid analysis to preparative separations on monolithic tubes to MS interfacing to multidimensional separations of peptides in proteomics.  

Because they possess high permeability, monolith columns can be run at relatively high flow rates and still show good chromatographic efficiency. Pavel Nesterenko and co-workers from Moscow State University, Moscow, Russia, and the Dublin City University, Dublin, Ireland used flow programming to elute polyaromatic hydrocarbons (PAHs) and phthalates from reversed-phase monolith columns without the use of gradient elution. In addition, monolith columns under extremely high flow rate conditions give a solute zone profile that resembles that observed in turbulent flow chromatography. These workers were able to demonstrate that 

 curves at these high flow rates could not be described by the conventional Van Deemter equation. 

New monolith stationary phases were the subject of several papers at HPLC 2005. Cabral and co-workers from Concordia University, Montreal, Quebec, Canada developed a tunable stationary phase for CEC based upon a photopolymerized methacrylate-based stationary phase with grafted chemical moieties. This phase with various alkyl side chains permitted the separation of biomolecules. Using similar strategies, the authors were able to develop reactive monoliths that permitted the immobilization of trypsin and other enzymes by chemical reaction. These immobilized enzyme phases could be used for on-line protein digestion. 

Brett Paull and co-workers from Dublin City University, Ireland, and Lomonosov Moscow State University, Moscow, Russia presented work on the application of new zwitterionic silica-based monolithic columns for the rapid separation of inorganic and organic anions. The new short columns (2.5 cm length) were based upon an immobilized phase of a long chain carboxybetaine type surfactant, which exhibited both unusual selectivity toward inorganic anions compared with standard anion exchangers, and a strong pH dependent capacity effect. The new column was shown to be highly stable, with 400 repeated injections of a mixture of inorganic anions showing < 2% variation in retention times, and could be applied directly to rapid isocratic anion separations (< 2 min) without the use of excessively high flow rates. However, the real novelty of this work was the application of a so-called "double gradient" approach to the separation of diverse mixtures of anionic species. The combination of an eluent pH gradient with an eluent flow gradient, meant analysis times with the column for complex mixtures of common inorganic, organic and polarizable anions could be reduced from > 50 min to under 4 min, while still maintaining both efficiency and resolution. Current work is underway to exploit the new double-gradient approach for the rapid analysis of even more challenging mixtures of anions.  

Violaine Augustin and co-workers from the ESPCI in Paris, France presented an interesting application of monoliths. They studied the in situ synthesis of polymethacrylate monoliths in capillaries for use in CEC. Efficiency and monolith uniformity were influenced strongly by the various pretreatment procedures. The energy of photopolymerization was important in the formation of a successful monolithic phase. 

Flux that was too strong caused polymer degradation and a decrease in performance. Once they had developed a successful in situ procedure, they transposed the methodology to a microdevice format, resulting in a monolithic bed showing 200,000 plates/m efficiency. In addition, they were able to use the monolith to perform a preconcentration step that allowed a sensitivity increase of 140 for a series of PAHs. 

Joseph Pesek and coworker Maria Matyska (California State University, San Jose, California) described a stationary phase in which hydride groups (Si-H) have replaced most of the silanols (Si-OH). One interesting property of the hydride material is that, in aqueous solution, the retention of acidic and basic compounds increases as the amount of organic solvent increases similar to hydrophilic interaction chromatography (HILIC). Termed aqueous normal phase chromatography, at neutral to slightly basic pH values, acids display aqueous normal phase behavior but at low pH, only reversed-phase LC behavior was noted. For basic compounds, they appear to have both aqueous normal phase and reversed-phase LC behavior depending upon the composition of the mobile phase. As the percentage of acetonitrile increased, Pesek noted that retention goes through a minimum so there is a dual mechanism occurring. Elution order reversals can occur that can be used to the chromatographer's advantage. 

To enhance the high pH stability of silica-based phases, César Silva, Carol Collins, and co-workers from the State University of Campinas, Sao Paulo, Brazil have explored the modification of silica supports using titanium alkoxide, followed by silanization with C

 and endcapping. During an accelerated aging test using aqueous phosphate at pH 10 and 50 °C, column failure was observed at 3700 column volumes, while for a similarly prepared C18 phase without titanization, column failure was observed at 2800 column volumes. In spite of better stability, the presence of titanium oxide increases surface acidity and the separation of bases requires the use of buffered mobile phases. To minimize this effect, a C

 urea alkoxysilane was used for silanization of titanized silica. Better peak shapes for bases were observed in nonbuffered mobile phases. However, the stability of the C18 urea titanized phase was somewhat lower than for the C18 titanized phase without embedded urea. The influence of the titanium oxide concentration also was investigated by performing two successive titanization reactions. The double titanized surface did not change the overall performance nor significantly improve stability at high pH, possibly due to the continued presence of residual silanols, which are the points responsible for silica dissolution in high pH. To obtain more stable phases, alternative procedures still must be investigated. In poster presentations, a simple, clean (less solvent), and efficient procedure to prepare reversed phases with different functionalities (C1, C8, C14, and phenyl) was explored using deposition and immobilization of polyalkylmethylsiloxanes onto silica, as an alternative to the conventional silanization reaction that requires the use of reactive chloro- or alkoxysilanes under reflux in organic solvents.  

Kenneth Markus and co-workers from Clemson University, Clemson, South Carolina presented a talk on capillary-channeled polymer fibers as a packing material for HPLC. These fibers display high wicking action and a higher surface area than traditional fibers. They are about 25–60 μm in diameter with eight capillary channels running on their periphery. In this study, polypropylene, polyester, and nylon fibers were studied. In their approach, a fiber bundle is pulled through a conventional column (about 20,000 fibers in a 4.6-mm column). These packed columns show 70% less pressure than commercial packed columns. They appear to work best with large molecules like proteins and can be operated at a higher flow rate than typical protein columns. The use of a single fiber for the separation of dyes was demonstrated with the wicking action being the only driving force. Sample preparation and process scale separations could be other potential areas of applications. Column efficiencies have a way to go before these columns are comparable to typical packed reversed-phase columns. 

Two oral sessions and one poster session were devoted to theoretical presentations. Many of the theoretical presentations had practical consequences. The first day, Georges Guiochon of the University of Tennessee, Knoxville, Tennessee presented a keynote titled "Retention Mechanisms in RPLC Are Still More Complex Than We Thought," in which he discussed the behavior of the retention of polar basic and acidic analytes on reversed-phase packings in methanol and acetonitrile. For a typical C18 reversed-phase packing, there are two types of sites: high energy and low energy. The high-energy sites, yet unidentified, mainly control retention since they are where polar analyte molecules go first. Unfortunately, their density is low, hence the saturation capacity is small compared to the surface area of the packing. In the 0–60% methanol range, the energy and density of the high-energy sites are sensitive to the organic solvent content, while the properties of the low-energy sites are not. The organic modifier influences these sites, forming an adsorbed layer of solvent at the interface between the packing surface and the bulk mobile phase. Methanol, which mixes fully with water, gives a monolayer, swelling the bonded alkyl chains. Acetonitrile is merely a hydrogen bond acceptor, not both a donor and an acceptor as methanol. Its molecules tend to associate in aqueous solution, forming a thick (~14 Å) adsorbed layer separating the bonded alkyl-chain layer from the acetonitrile–water mobile phase. The presence of buffer also influences adsorption isotherms. Because most reversed-phase LC packings are heterogeneous with both high- and low-energy sites, retention mechanisms usually are mixed, which makes it difficult to compare different packings. 

David McCalley of the University of the West of England, Bristol, U.K., followed up with a talk on overloading of basic compounds in reversed-phase LC as a function of pH in the range of 2.7–10. He studied not only silica-based packings but also inorganic–organic hybrids and pure polymeric packings. At low pH for 4.6-mm i.d. columns using phosphate buffers, he observed overloading problems for ionized solutes, even at the 0.1-μg level. Overloading was especially severe for weak acid buffers such as formic acid, a volatile buffer system popular in LC–MS. Figure 2 provides some overloading data for a series of neutral and ionizable compounds on a hybrid reversed-phase column. As the pH of formic acid was raised from 2.7 to 3.7, overloading was reduced, possibly due to higher ionic strength at the higher pH, or to increased ion pair effects. Using phosphate buffers on pH stable columns, high pH (up to 10) decreases solute ionization and thus reduces overloading, in the absence of interaction with ionized silanols. Many of the results were in agreement with theories from the earlier presentation of Guiochon.  

Figure 2:  Comparison of overload behavior of three neutral compounds (caffeine, phenol, 3-phenylpropranolol) and three ionized compounds on Xterra MS (Waters, Milford, Massachusetts). Mobile phase ACN / 0.02M formic acid pH 2.7 (6).			

Torgny Fornstedt and co-workers of Uppsala University, Sweden, presented a lecture entitled "Why Are the Injected Solutes Not Always Found in the Peaks?" An equilibrated system consisting of an eluent and a solute establishes a concentration plateau. If a small excess of solute is injected into this system, a single peak will appear in the chromatogram. This peak is known as a system peak or perturbation peak and consists of displaced plateau molecules whereas the injected molecule is coeluted later with a deficiency of plateau molecules and is therefore not normally detected. This present study investigated the multicomponent case of this phenomenon. For this study, both the theory and methodology were extended for the 

-component case. The researchers cleverly designed a new experimental approach using enantiomers of the two solutes to be studied and a binary reversed-phase LC system. Fractions were collected from the eluate from the nonchiral system and then reinjected into a chiral column. In this way, the molar ratios of the nonchiral eluate could be measured and retention times for the two tracer peaks be determined. It was shown that the retention times of the binary tracer peaks allowed the straightforward calculation of the real isotherm data points without any isotherm model assumptions. The 2-D isotherm surfaces could easily be plotted directly from the retention times. The results of this study should be important for large scale process design as well as for characterizations of new LC columns. The difficulty in this experiment is to selectively detect only the injected molecules among a population of identical ones already present in the system. The next steps will involve the use of different solute isotopes and MS detection to perform these more difficult experiments. 

Attila Felinger, now of the University of Veszprém, Veszprém, Hungary, presented a lecture on combining linear and nonlinear chromatography to investigate the adsorption behavior in HPLC. The goal of his presentation was to show (through several examples) that not only in nonlinear but also in linear chromatography, heterogeneity of the adsorption–desorption kinetics could be characterized. Although the information furnished is more limited than the one obtained with the measurement of nonlinear equilibrium isotherms, peak shape analysis can reveal several details of the chromatographic process without the need for lengthy isotherm determinations. With the mostly ignored stochastic model of chromatography, he determined the number of adsorption–desorption events and the kinetic rate constants through peak-shape analysis. With this approach, the presence of adsorption sites on which desorption is slow was recognized. Among other things, he studied the retention behavior of phenol in reversed-phase LC and found, in agreement with Guiochon, that phenol is retained on two different types of sites with interaction energies that differ roughly by 10 kJ/mol. Furthermore, he characterized how the number of adsorption events and the kinetic rate constants depend upon temperature, mobile phase composition, and solute concentration. With a simple peak-shape analysis, he determined the contribution of the individual sites to the retention of the analyte. 

Piotr Gzil and co-workers from the Vrije Universiteit Brussel, Brussels, Belgium challenged the audience to think outside the box and represented plate height and flow resistance data in a kinetic plot instead of in the classical Van Deemter plot format. Circumventing the need for a characteristic mass transfer length that would be common to all possible support shapes, the kinetic plot method provides a universal, morphology-independent basis to compare the separation efficiency and speed of different support types directly. To further promote the kinetic plot method, it was shown that it requires no rigorous mathematics, only the multiplication of a few data columns in a spread-sheet program like MS Excel. The audience was referred to a freely downloadable MS Excel template file (http://

) providing an automatic readout of all possible curve characteristics and kinetic performance data. Applying the method to an extensive set of silica monolith and packed-bed literature data showed at a glance that the open-porous structure of the monolithic type of supports is more advantageous for separations requiring large plate numbers, while the more compact structure of a packed bed column is better suited for easy, high-speed separations. The obtained plot also revealed the existence of a currently "forbidden region," which can only be invaded by preparing homogeneous open-porous monoliths with smaller domain sizes than currently achievable or by working under ultrahigh pressure LC conditions in densely packed systems, provided the frictional heating problem can be controlled. 

M.C. Garcia-Alverez-Coque with coworker J.R. Torres-Lapasio of the University of Valencia, Valencia, Spain presented a tutorial lecture on the search for the best conditions in chromatography: A Magical Mystery Tour. Some new tools for interpretive optimization supported by different models were presented. The use of peak purity as a resolution measurement that expresses the area fraction free of interferences was outlined. The most important peak purity concept was that of the limiting purity, which represents the maximum separation capability for a given compound in the chosen sample and chromatographic system. Limiting purities were shown to allow establishing the resolution threshold of the chromatographic system, or the maximal practical complexity in a gradient program. 

Optimization levels were classified as total, partial, and specific strategies. Total strategies try to resolve all compounds in a sample within a single run. Partial strategies resolve only certain target compounds. All compounds in the mixture can be resolved using more than one run with wisely chosen chromatographic conditions in the so-called complementary optimizations. Conventional criteria have the pitfall of offering noninformative resolution maps in low-resolution situations. A straightforward criterion accounting for the success in the separation, instead of the fail level (that is, the limiting peak count) was shown to discriminate the resolution power in these situations. Difficult coelutions cannot be resolved only chromatographically and require deconvolution. For these situations, a resolution measurement oriented to deconvolution, namely, the multivariate selectivity, was presented. 

As can be seen in Table II, protein separations and proteomics again dominated liquid-phase applications. The analysis of drugs, drug metabolites, and endogenous compounds in biological samples had a strong showing in the applications arena. This is an area in which sample preparation using protein precipitation and SPE in the 96-well format seems to be catching on. 

In proteomics, 2-D gel electrophoresis is still in widespread use but 1-D and 2-D LC coupled to MS is growing rapidly for the investigation of these complex mixtures of proteins and peptides. Hence, a large number of contributions at HPLC 2005 were devoted to such studies (see Detection section). This year saw the emergence of a number of investigations for biomarkers for various disease states. With techniques now available for the removal of high-abundance proteins, trace levels are more readily detectable.  

Biomarkers are often small polar molecules present in the mammalian body at micromolar-to-millimolar levels. Sara Bergstrom and co-workers from Uppsala University, Uppsala, Sweden, and Karolinska Institute, Stockholm, Sweden investigated the use of microdialysis for fast monitoring of biomarkers in tissue samples. Small biomarkers like glucose, glycerol, and pyruvate readily pass through a membrane much faster than larger biomolecules. In their experiments, microdialysis probes were implanted into rat livers and chemical stimulations were performed by the addition of potassium chloride or paracetamol to the perfusion solution. Samples were taken before and after stimulation. After microdialysis, C18 columns are used for preconcentration and desalting. Finally, the samples were directed online to a mass spectrometer for detection and identification. After analysis, the average mass spectra of the elution plug were compared by principle component analysis to look for sample trends. Using this approach, they were able to investigate potential biomarkers in the 200–1100 Da molecular weight range.  

Articles

Column Watch

HPLC 2005 was held in Stockholm, Sweden, June 26-30. In this month's installment of "Column Watch," Ron Majors covers this year's hot topics including proteomics, column technology - especially monoliths - microfluidics, and multidimensional and high-throughput separations.

Highlights of HPLC 2005

						Millipore acquires NovAseptic AB					

Millipore Corporation (Billerica, Massachusetts) announced that it recently has acquired over 90% of NovAseptic AB (Gothenburg, Sweden) shares. Millipore is in the process of acquiring the remaining shares from the minority shareholders. 

				Martin Madaus, Millipore's chairman, CEO, and president, noted, "NovAseptic brings innovative and differentiated products that our biotech and pharmaceutical customers value highly for manufacturing biologics and for monitoring biomanufacturing processes."			

NovAseptic provides a range of solutions for aseptic-processing applications in biotech and pharmaceutical manufacturing operations.  

						Applied Biosystems supports research into DNA signatures					

The Translational Genomics Research Institute (Tgen, Pheonix, Arizona) and Northern Arizona University (NAU, Flagstaff, Arizona) announced recently the award of a multimillion dollar grant from the U.S. Department of Health and Human Services to improve the understanding of two leading causes of death for most age groups worldwide, sepsis and community-acquired pneumonia (CAP).  

Applied Biosystems (Foster City, California) is supporting the project to discover pathogen-specific DNA signatures. These signatures will be used to design and validate Applied Biosystems TaqMan real-time PCR assays for the accurate identification of these life-threatening infections.  

						EMD opens chromatography center					

EMD Chemicals, Inc. (Gibbstown, New Jersey) recently unveiled its new Chromatography Technical Center in Gibbstown, New Jersey. The center will be equipped with a wide range of chromatography columns and staffed by expert personnel. 

The new center is expected to be operational by the end of 2005. Standard training sessions will be offered regularly, and custom-designed programs will be available to respond to individual customer scheduling and content requirements. In addition, EMD will continue to provide training at customer locations. 

						GTC Biotherapeutics receives notice of allowance on purification patent					

GTC Biotherapeutics, Inc. (Framingham, Massachusetts) announced recently that the U.S. Patent and Trademark Office has issued a notice of allowance for the claims in its patent application 10/635,117 covering tangential flow filtration technology. These claims provide the company with a unique position in the use of this technology to recover recombinant therapeutic proteins that are expressed in the milk of transgenic animals.  

The patent that issues from this application will expire in 2023. 

						Inverness Capital Partners invests in NCSRT to expand operations					

Inverness Capital Partners (Philadelphia, Pennsylvania), a $125 million private equity fund, has invested in NCSRT, Inc. (Apex, North Carolina), a designer, developer, and manufacturer of filtration solutions.  

Henry Kopf III, NCSRT president and CEO, says that the investment will allow NCSRT to expand operations and continue to provide customers with industry-leading technical support.

Peaks of Interest

Millipore Corporation (Billerica, Massachusetts) announced that it recently has acquired over 90% of NovAseptic AB (Gothenburg, Sweden) shares. Millipore is in the process of acquiring the remaining shares from the minority shareholders.

Go into nearly any gas chromatography (GC) lab and you will see shelves or cabinets stocked with columns; new, used, favorite, and even broken ones will be on display. Yet in the midst of an abundance of columns, many chromatographers take them for granted. In the years since 1979, when fused silica became generally available to the chromatographic community as a column tubing material, the art of making capillary columns has advanced tremendously. Today's fused-silica columns, while outwardly indistinguishable from all but the earliest predecessors, deliver both general and application-specific separations with much higher mechanical stability, much lower column bleed levels, greatly enhanced inertness, and a high degree of column-to-column reproducibility. 

In this installment of "GC Connections," we'll review some of the developments in fused-silica column technology and then take a brief tour through the process of column manufacturing. 

In June of 1979, I was about to finish graduate school when word came of the presentation by Dandenau and Zerenner (1) at the Third Hindelang Symposium, held in April that year, in which they discussed their development of flexible capillary columns made of fused silica. Dandenau and Zerenner recounted their experiences in an 

 article in 1990 (2). As far back as 1960, the chemical advantages of silica over softer borosilicate or sodium glasses as a column material had been recognized (3), but the difficulties in working at the much higher temperatures required to soften silica for drawing out and then coiling rigid silica capillaries had inhibited much progress up to 1979 (4,5). Dandenau and Zerenner's breakthrough was the successful application of optical fiber technology to the production of flexible fused-silica capillary tubing. With access to Hewlett-Packard's optical fiber facilities, where the production of step-refractive-index, silica-clad optical fibers utilized hollow fused-silica tubing preforms, they made the leap from flexible fibers to flexible capillaries. 

I had personally drawn and coated innumerable rigid glass capillary columns in the course of completing my research, and so I was very interested in the properties of flexible fused silica; especially considering that I might have saved all those midnight hours nursing a cantankerous glass capillary drawing machine. Early in the summer of that year, a 30-m length of the new capillary tubing found its way into the lab, and as the senior GC student, I was elected to coat the tubing with a stationary phase and give it a try. I did so, using one of the chiral organosiloxane polymers from my research. I carefully cut the ends of the column with a razor blade (the "proper" cutting techniques for fused silica were completely unknown to me) and installed it in the GC inlet and detector. I set up the carrier gas pressure and split flow, lit the flame ionization detector, closed the oven door, and heated the column to about 100 °C. Upon injecting a test mixture, I was thrilled to see the expected peaks emerge, and I repeated the test several times. Then I turned off the oven heater and opened the oven door, which turned out to be a fatal mistake for the column: it broke into about 50 pieces! Everywhere that the column had been in contact with its metal cage created a stress point that broke as the column cooled off. 

Fortunately, I was already set to graduate and did not need any more experimental results. Less than a year later, now employed at an instrument company, I was thrust into the midst of the rush on the part of column producers to adapt their processes to the new material. I was tasked with determining the feasibility of making fused-silica columns in-house and with comparing that route to the purchase of columns from an outside supplier. At the time, there were no fused-silica column suppliers with any track record, and so the decision was not trivial, especially considering that nearly all suppliers of glass columns were engaged in essentially the same pursuit.  

Modern fused-silica columns resemble their predecessors from the 1980s in several superficial ways, while they depart from them in many significant respects. Were I to hold an early column and a recent one next to each other, I would see a long, thin silica tube coiled to about 6–8 in. diameter and restrained on a metal cage. The most obvious visible differences would be the color and external appearance of the column's outer protective coating and the details of the cage itself. Not apparent to the eye would be the chemical differences in the silica material, its inner surface, and the stationary phase coating. 			

Fused silica makes a superior material for GC columns primarily due to its high level of chemical purity and not to the flexibility of the thin tubes drawn from it. The material properties of capillary fused-silica tubes are a fortuitous side-effect that has relieved those who started practicing GC after 1980 of the joys of straightening and connecting coiled rigid glass capillary tubing. This was a manual procedure that required some skill. It involved heating one end of a column with a blowtorch just enough to soften the glass so that it would straighten out under the influence of gravity; at least that's how I did it. The stationary phase was destroyed, of course, so the ends had to be deactivated each time a new length was straightened. We routinely made columns an extra meter or two long in anticipation of some breakage while trying to connect them. The flexibility of fused-silica eliminated this concern, and made it easier to remove some of the column on purpose (for example, if it became contaminated with sample reside).  

There are three basic types of silica glass that have been used for capillary columns: sodium, borosilicate, and fused silica. Lead glass, a fourth type of silica glass, has not been used at all to my knowledge. All of these glasses consist primarily of SiO

, but there are significant differences. Sodium and borosilicate glasses are the easiest to form and work with. Sodium (also called soda-lime) glasses have the lowest working points at around 1200 °C. Borosilicate glass, which is used for laboratory glassware, is workable at around 1600 °C. Their relatively low working temperatures are obtained by adding significant quantities of sodium and calcium, or boron, respectively, and they contain about 70–80% silica. Other impurities are present at the percent or lower levels. For capillary columns, the impurities cause peak distortion, tailing, and sometimes partial or complete loss through active reversible or irreversible adsorption of many of the polar, polarizable, and aromatic compounds commonly separated by GC. These adsorptive effects are more significant at trace levels of the analytes. A tremendous amount of effort had been expended toward deactivating glass capillary columns before 1979 that resulted in innumerable methods to remove, replace, or mask the active sites. I had employed some of these in my graduate research on borosilicate glass with varying degrees of success. 

As had been recognized early by Desty (3,4), pure fused silica does not cause the undesired effects of impurities in softer glass. However, as researchers began to work with the new material, they quickly discovered that fused silica was definitely not perfect, and many of the surface preparation techniques used for earlier glass columns did not necessarily work with fused silica. To confuse matters, some early fused silica was produced from highly purified quartz sand while other material was synthetically produced through the hydrolysis of silicon tetrachloride. The former is known as fused quartz and the latter as synthetic fused silica. Fused quartz tends to contain low but significant impurity levels, and it was soon abandoned as a capillary GC column material. Synthetic fused silica contains less than less than 1 ppm of metallic impurities, while good fused quartz can include around 20–30 ppm metallic impurities. Both fused quartz and synthetic fused silica have very low coefficients of thermal expansion, so they tolerate temperature extremes well. 

The glass drawing process (see Figure 1) feeds a relatively large preform tube into a furnace that heats the glass to its flowing point. The inside of the tubing and the furnace cavity are continuously purged with inert gas to avoid oxidation and prevent the influx of ambient atmospheric contaminants. The glass is drawn out more rapidly than the incoming preform feed rate, which produces a smaller and longer tube with reduced cross-sectional dimensions. The draw rate ratios used for fused-silica material typically yield several thousand meters of finished tubing for each meter of preform that is fed into the furnace. A laser optical gauge constantly measures the outgoing tubing diameter and provides feedback to the rate controllers: The tubing dimensions are controlled very tightly as a result. Immediately after passing the laser gauge, the tubing enters multiple stages of external coating application and curing, and is tested for tensile strength. Finally, it is wound up on large spools, sealed, and stored prior to being coated internally with stationary phase. 

Figure 1:  Fused-silica capillary tubing drawing tower schematic. The preform feed mechanism (1) feeds a fused-silica preform (2) into a 2000 ÃÂ°C furnace (3) at a slow rate. The tubing softens in the furnace and is drawn out at a higher speed. A laser gauge (4) measures the tubing size and adjusts the rates to maintain a constant diameter. The drawn tubing is externally coated with a protective coating (5), and the coating is cured (6). A draw capstan (7) controls the rate at which the tubing is drawn. After testing for tensile strength (8), the tubing is wound on a spool (9) for storage. This diagram is not to scale. Figure used with permission of Polymicro Technologies, Phoenix, Arizona.			

The outer surface of the tubing is very sensitive to physical stress. The slightest imperfection in the silica will propagate into a major crack and cause breakage under any significant strain. The silica preforms are carefully fire-polished before the tubing is drawn to ensure a defect-free result. Bending fused-silica tubing produces high stress levels that the tubing tolerates due to its very high tensile strength. 

Immunity to external abrasion is achieved by coating the outside of the column immediately after it is drawn. Several different types of protective coating have been employed for fused-silica columns, including silicone, polyimide, and aluminum. Of these, the latter two are still in use. The main limitation of silicone coatings is their relatively low temperature limit, which conventional GC analyses can exceed easily. The vast majority of fused-silica columns today are coated on the outside with multiple layers of the familiar brown to yellow polyimides. Polyimide coatings, which originated in the aerospace industry, can tolerate temperatures of up to about 400 °C for brief periods, and of course aluminum has a much higher limit. The multiple column breaks that I and many others encountered early on have not been seen in new columns for more than two decades, due to improvements in external coating materials and processes. 

The process of drawing fused-silica capillary tubing subjects the material to temperatures close to 2000 °C, effectively removing all surface water and leaving Si-O-Si bond bridges and some Si-OH functionality on the inner and outer surfaces. This initially pristine surface would rapidly take up atmospheric water upon cooling and during subsequent storage and handling if the tubing were not purged with inert gas. The amount of water and how it is attached to the inner surface plays a crucial role in deactivation and stationary phase chemistry. The level of bulk hydroxyl groups in the material has important effects on its optical properties that are not of much concern to gas chromatographers but may be of interest in liquid chromatography systems that use the column end, stripped of the external coating, as an optical window to the mobile phase. 

The raw inner surface of freshly drawn fused-silica capillary tubing contains very little water. The surface Si-O-Si bridges are strained, however, and will readily absorb water from the atmosphere or from solvents and solutions used in the deactivation and coating processes. Additional water can be adsorbed on the surface as well. Controlling the amount and type of -OH functionality on the inner surface — whether as reversibly adsorbed water molecules or as terminal silanol (Si-OH) groups — is vital to successfully engineering the desired stationary phase coating, not only in terms of its retention characteristics, but also for producing desired levels of residual chemical activity, bleed levels, and coating stability. The reagents used for surface treatment and coating must themselves be highly purified because the fused-silica surface will easily adsorb contaminants, including trace metals, from them as well. 

The task of coating the inside of a fused-silica capillary with stationary phase is a multistep process consisting of pretreatment and deactivation, coating and final polymerization, conditioning, and testing. Each class of stationary phase coating — nonpolar siloxanes, moderately polar siloxanes, highly polar siloxanes, carbowaxes, and others — requires different treatments that also can depend on the film thickness, column length and inner diameter, upper temperature limit, and application scope.  

The purpose of these initial steps is to create a consistent surface that is ready to be coated with the stationary phase, while also ensuring that the surface is suitably deactivated. In the finished column, chemical activity is measured relative to one or more specific compounds, including acidic, basic, and aromatic types. Capillary column test mixtures contain compounds such as octanol, dimethylphenol, dimethylaniline, and naphthalene that exhibit peak tailing, peak area loss, or retention shifts on columns that lack suitable deactivation, have exposed silica surfaces, or are contaminated. 

It is difficult to balance residual chemical activity to achieve high performance for all compounds: some treatments produce better results with acidic compounds, while others are better for basic compounds. In general, however, fused-silica material with significant levels of surface silanol groups tends to be acidic and requires different treatments when basic compounds are the target analytes; many capillary columns are specifically tailored for basic or acidic compounds. In any case, appropriate expectations for test mixture results should be determined according to the specific application.  

Excess surface activity, as well as the presence of impurities on the surface, also can lead to higher column bleed levels. Column bleed refers to the increased rate of release of material from the stationary phase and inner surfaces as column temperatures rise. Bleed is observed as a rising baseline profile that obscures small peaks and adds noise to the detector signal, and eventually as reduced retention times and increased solute activity due to bulk loss of the stationary phase. One source of column bleed is the breakdown of the stationary phase polymer under the influence of active chemical groups on the surface. Bleed also occurs in the presence of introduced impurities from sample injections as well as from the action of free oxygen in the carrier gas. Producing low bleed columns requires careful attention to reagent chemistry and purity at all stages of column production. Silica surface activity is masked to some extent by thicker stationary film coatings, but at the same time, thicker coatings tend to exhibit lower efficiencies and higher bleed levels themselves.  

The initial stationary film must form a smooth stable layer upon coating inside the column. Its stability depends upon the surface tension of the treated inner silica surface, as well as some other variables that are described later, and so any pretreatments must leave the silica surface in an appropriate state of readiness to accept the coating.  In the case of some bonded stationary phases, the coating material chemically bonds with surface silanol or siloxane groups or with other intermediary reactive groups introduced as part of the initial preparatory treatments. Here, the term "bonded phase" means that there are chemical bonds between the bulk of the stationary phase polymer mass and the surface of the column. In theory, a column with the stationary phase chemically bonded to the tubing will exhibit better mechanical and thermal stability. However, in the face of external destabilizing influences such as free oxygen or sample impurities, surface bonding can give limited improvement. Thus, the importance of operating and storing the column clean and oxygen free cannot be overemphasized. 

A stationary phase film or a layer of pre-polymer reactant solution is deposited onto the column inner walls in one of two ways, either by dynamic or by static coating. Figure 2 illustrates these two techniques. In dynamic coating, a plug of solution passes through the column at a (hopefully) controlled rate, under positive gas pressure. The trailing meniscus leaves behind a layer of solution the thickness of which depends upon the speed at which the plug moves as well as the surface tension and wettability of the solution on the column inner surface. After the excess solution has left the column, continued gas flow evaporates the solvent and leaves the dissolved material behind. The dynamic coating method is difficult to control because the plug grows shorter as it sheds liquid and therefore, the pressure required for constant plug velocity decreases during the process. In my hands, the plug velocity would go up exponentially as it got near the end of the column, which always ruined the last few meters of the column. I simply cut that last section off after the column was finished, but I was never very satisfied with this method. 

Figure 2:  Dynamic and static coating techniques. (a) Dynamic coating: A plug of coating solution (1) is forced through the tubing under pressure (2). The meniscus (3) moves through the tubing and leaves behind a layer of coating solution. (b) Static coating: The tubing is filled with coating solution (1), and one end is plugged (4). A vacuum is drawn on the open end, and the meniscus (3) moves through the tubing as the solvent evaporates, leaving behind a layer of coating solution. 			

In static coating, the entire capillary tube is filled with a solution of the stationary phase or its precursors. One end is carefully capped without a gas bubble, and then a vacuum is drawn on the opposite open end. I remember the anticipation at the moment of first applying vacuum to a new column and waiting to see if a bubble would form in the solution. If you were quick enough to spot it then the column could be recovered, otherwise it had to be drained and rinsed out before starting over. 

Once started, the solvent evaporates under the influence of the vacuum and leaves the dissolved material behind: as the solvent evaporates, the meniscus moves back along the column towards the closed end. The rate of static coating also varies during the process, and the speed of evaporation slows down as the meniscus progresses along the length of the tubing. However, in this case, the concentration of the solution is what determines the amount left behind and not the evaporation rate. The main trick with static coating is to control the tubing temperature well; the liquid in the tubing acts like a very sensitive thermometer and small temperature variations can cause the meniscus to fluctuate and deposit an uneven layer. In my limited experience, the static technique gave the best results, and I often was able to make columns that performed at close to theoretical efficiency. 

All of the columns I made were coated with a solution of the stationary phase in its final state: once the solvent evaporated, the column was done and ready for conditioning. I worked with fairly low molecular weight polymers that I had synthesized, and I was not concerned with temperature limits. To obtain columns that are useful at higher temperatures, the polymer molecular weights become much higher and the polymer structures more highly cross-linked. Such large polymers have high viscosities and are more like gums than liquids. Even when in solution, if they can be dissolved, they are difficult if not impossible to force to fill an entire capillary tube for static coating at concentrations that deposit enough material for the desired phase ratio. 

Instead, the tube is filled with a solution of oligomers or other precursors of the final material plus polymerization initiators. The polymerization reaction is performed in situ, either before or after the solvent is evaporated, leaving the newly formed stationary phase material behind. The polymerization chemistry is chosen so that only volatile by-products are produced, which can be purged out of the column during initial conditioning.  

After coating and initial drying, the new column is conditioned by gradually programming its temperature to the upper limit with highly purified carrier gas flowing. The temperature is then held there long enough to vaporize and remove residues that otherwise would be seen as column bleed later on. After an initially high impulse of exiting residues, primarily the leftovers from coating and polymerization, the background level decreases rapidly until it reaches a nearly steady value. This will be the initial bleed level of the new column. The bleed level will tend to decrease gradually over time in the absence of external influences like trace oxygen or chemical contamination, but it cannot be expected to go to zero. The amount of bleed emitted from modern low-bleed columns is remarkably small when the subnanogram detection capabilities of many GC detectors are taken into consideration. This is both a benefit to gas chromatographers and a challenge to column manufacturers who must strive to meet end-users' expectations. 

Finally, after conditioning is complete, the column is tested for chromatographic performance with a test mixture that is appropriate to the intended application. There are many different test mixtures available from chromatography suppliers. In addition to the fundamental polarity test compounds mentioned earlier in this article, hydrocarbons, fatty acids and their methyl esters, polyaromatic hydrocarbons (PAH), and alcohols, plus common herbicides and pesticides are all available in various general purpose test mixtures. Many analytical protocols call for the use of a standardized mixture of the actual target compounds to test columns and this is certainly a good idea. However, the occasional use of a general column test mixture often can reveal developing problems before they surface in the day-to-day methodology. Before putting a new column into routine service, I like to test it with the same or a similar test mixture as was used by the column manufacturer. These mixtures usually cover a broader range of polarities and can constitute a more stringent test. Later on, periodic tests with the same column test mixture can be compared with the original test that was performed at installation, which may reveal a developing problem. 

Although very similar in appearance to the earliest fused-silica columns of the 1980s, modern fused-silica columns and the tubing they are made of have undergone decades of development and improvement. The process of making capillary GC columns has evolved from something that a graduate student could perform starting right from drawing out the tubing to a technically sophisticated procedure that involves precision machinery, sophisticated polymer chemistry, and highly trained technical specialists. As far as I know, all of the old soft-glass drawing machines have been retired or put into museums. 

"GC Connections" editor John V. Hinshaw is senior staff engineer at Serveron Corp., Hillsboro, Oregon, and a member of 

s editorial advisory board. Direct correspondence about this column to "GC Connections," 

 Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

 For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at http://

(3) D.H. Desty, J.N. Haresnape, and B.H.F. Whyman, British patent 899,909 (applied 9 April 1959, issued 27 June 1962). 

GC Connections

In this installment of "GC Connections," the author reviews some of the developments infused-silica solumn technology and then takes a brief tour through the process of column manufacturing.

The Making of a Column

The purchase of expensive capital instrumentation is critical to the success of any analytical laboratory. In the process of selecting instrumentation, careful deliberation of the scientific, economic, and organizational factors, in the context of a laboratory's specific charter, is required to obtain the maximum benefit for the life of the equipment. Consideration of the important factors, in the context of our evaluations of HPLC–MS-MS systems primarily intended for quantitative bioanalytical applications, is explained. The use of a simple Excel-based "Golf Score Card" tool has facilitated our assessments and instrument acquisition decisions by collating and weighting the relative importance of the many criteria that impact the final purchase decision. This approach can be adapted readily to the evaluation of any analytical instrumentation. Additionally, the overall process is examined, practically and strategically, from the perspective of those who are both end-users and stewards of vital mass spectrometry-based technologies within our industrial health care organization. 

Scientists using analytical instrumentation are faced with the need to evaluate and select technologies they will use to perform their jobs. In most cases, there are a number of instrument choices that could be purchased to perform the required task. However, within a given application and context, there will almost always be one selection that will have advantages over the alternatives. There is essentially no formalized training and little information in the literature (1–4) to guide scientists in the acquisition of critical tools. 

High performance liquid chromatogrpahy (HPLC)–mass spectrometry (MS)-MS using triple quadrupole mass spectrometry is well-established as the technology of choice for measuring ultratrace concentrations of small molecules (drugs, metabolites, and biomarkers) in biological samples. Due to the vast array of bioanalytical needs spanning all stages of drug discovery, development, and market support (5), such quantitative measurements have grown to represent the single largest segment of support provided by our health care MS laboratories. In providing such support over many years, we have accumulated considerable experience not only in HPLC–MS-MS-based applications but also in continually assessing and optimally integrating new MS-based tools to improve capabilities and capacity to meet project needs. In this article, we describe our general philosophy and process for mass spectrometer assessment and selection. These same principles have also been applied to the acquisition of other types of instruments (such as liquid chromatographs and autosamplers). 

A primary objective for instrument acquisitions has been to increase the capacity for addressing the growing needs across our health care businesses. At the same time, we continually seek to improve the technical capabilities of our bioanalytical arsenal. To deliver against bioanalytical quantitation objectives alone, we currently have 14 triple quadrupole HPLC–MS-MS systems across three laboratories, spanning pharmaceutical discovery, development, and over-the-counter R&D and manufacturing. A decision to acquire a specific instrument should not be taken lightly, especially when the price tag is up to $400,000 for a total system (with chromatograph and autosampler). Through each of the many deliberations we have had, for these and other types of mass spectrometers (about 60 instrument acquisitions over two decades), we have worked toward continual improvement in our process of assessing and selecting instrumentation. In addition to making the best choice for the business need at hand, attention is focused toward efficient use of our (and vendors') time and assures that evaluations will result in predefined measures that can contribute to an objective selection.  

For experienced and established laboratories such as ours, the instrument evaluation can transition through several stages. Through vendor contacts and discussions with our counterparts at other companies, we first determine if various vendor offerings have evolved (improved) sufficiently since our prior instrument evaluation exercise, to warrant a multivendor instrument evaluation. If not, we move directly into value negotiations with the preferred vendor. If an evaluation is needed, contending instruments are selected, and a Golf Score Card tool is constructed. First-tier technical assessment often is conducted remotely (without travel) by submitting prescribed experiments to the vendor that will provide a clear assessment of their relative ability to meet our needs. If there is a clear winner that has hardware and software platforms that we are also very familiar with, we proceed without the cost and time of a demo trip. If the first-tier exercise uncovers a less-familiar leader (or no leader), we would only then opt to do a hands-on evaluation, which would include a more in-depth (comprehensive) set of experiments. For less established laboratories, an in-person evaluation of instrumentation would be recommended as the first course of action. 

Multiple vendors are now able to market quality HPLC–MS-MS instruments (and other types of analytical tools), each offering somewhat unique attributes in order to differentiate their product (or find a special niche) relative to the others. Obviously, this benefits endusers in several ways. The resulting competition tends to drive list prices down over time and strengthens customer leverage in value or pricing negotiations at the point of purchase. This situation also has fueled vendor R&D efforts, further accelerating the evolution rate in instrument enhancements, providing access to increasingly effective HPLC–MS-MS tools for bioanalytical applications. That said, this situation does create a more difficult task in sorting through the pros and cons in the instrument selection process. We would argue that even though a number of different instruments could accomplish a given analytical task, if one considers the cost of the capital investment and the length of time an organization will utilize (and depreciate) that selection, even a small overall advantage that one system demonstrates relative to others will provide a significant benefit over the life of the analytical tool (typically 7–10 years for mass spectrometers and chromatographs). 

Recognizing this dilemma, we evolved toward a systematic process for objectively measuring the relative value and appropriateness of candidate instrumentation for the intended use. The process involves four steps: 					

Develop a list of primary factors that might influence the relative effectiveness of an instrument in its intended application area. This includes not only the usual analytical attributes (sensitivity, linear range, and so forth) but also factors such as software versatility, quality and affordability of vendor service, receptiveness of vendor to meet our specific needs, and especially practical implementation considerations such as our familiarity with hardware and software platforms.

Stratify these factors into one of three categories, prioritized in terms of relevance (importance) within the context of the intended application area (most important, important, less important).

Collect data that allows a rank-ordering of the contending instruments in terms of each factor. Depending upon the factor, data might be collected as part of the experimental evaluations or through a survey of the experiences of others (for example, quality of vendor service).

Tabulate the results and weight each value, according to the importance category within which the factor resides. 

We then total these weighted values for each instrument. Low score wins. Thus, we have termed this the "Golf Score Card" tool. This is admittedly not an exact science, however, we have found it to be a practical tool not only to help us sort through myriad  considerations, but also to distill findings more clearly into a form that can be digested readily by our managers, researchers, and technical peers throughout our company. We explain here in detail our process and rationale. We show results from an extensive evaluation but do not name specific manufacturers because we intend to describe our process and not a relative evaluation that is in a sense a snapshot at a point in time. Instruments are continually evolving and a specific evaluation will be less relevant the following year. 

To use the Golf Score Card process, regardless of technology being acquired, careful consideration of the key criteria is required. For our first example, triple quadrupole MS-MS systems for quantitation, 10 technical or vendor factors were identified as being important in the instrument selection. 

The smaller the amount of target compound that can be detected and quantified from a complex biological matrix, the more problems can be solved quickly and with less overall effort. For example, more sensitivity often translates to less sample preparation or allows additional dilution (reduction of matrix). Certainly, other factors contribute to the bottom-line sensitivity (for example, selectivity, ion source ruggedness), but in our view, this represents the composite of the single most important HPLC–MS-MS performance attribute for quantitation.			

 When a primary objective is to maintain (through replacement) or increase capacity within a given application area, significant efficiency advantages are realized by maintaining common (identical) hardware and software platforms. A few positives gleaned in the areas of training multiple users are method troubleshooting and transfer (for example, from discovery to development); instrument repair (part swapping) and spares stocking; and ease of maintaining regulatory compliance. Moreover, we have a "knock out the champ" philosophy. All other things being equal, we would go with the technology we already know and can utilize seamlessly and quickly. This is one example of why context is such an important qualifier in this process. In the case of a lab purchasing its first HPLC–MS-MS system, this factor would have no relevance at all, but to us it is a very important consideration. 

 Ideally, this is user-friendly and intuitive. Power and versatility of software must allow streamlining of data acquisition, reduction, reporting, and archiving. Also, the ability to set-up and control parameters of peripheral tools such as chromatographs and autosamplers is critical to exploiting increasingly automated sample and data management capabilities.

 The ability to analyze samples over a wide range is key to supporting pharmacokinetic studies in which target compound levels can vary by over 1000-fold across study samples. 

 The volatilization–ionization process must cause minimal analyte degradation and be applicable across a broad range of compound classes. Ion sources that can handle a wide range of liquid flows without compromising performance provide versatility for high-throughput applications and are readily adaptable to a broad range of chromatographic inlet technologies are obviously advantageous. Other factors include probe or mode switching (for example, from electrospray [ESI] to atmospheric pressure chemical ionization [APCI]). 

 Minimal maintenance and downtime are ideal for optimal instrument performance and throughput. This can be ascertained from experience and discussions with other instrument users (internal and external). 

This often is determined from the recent track record of field engineer responsiveness and experience. There usually will be local variability in terms of quality and responsiveness, as service usually is directly related to the ability of a few individuals, but vendor organization and cost are also factors. 

This is a factor that definitely plays into a purchase decision, although to what extent will vary by organization. However, one way to look at this is to consider it in terms of value. Lower cost for equivalent performance is better, but cost ideally is not the primary driver. Paying $100,000 less for an instrument that cannot meet the business needs optimally is not a cost savings. It is a waste of money. 

This reflects the vendor's track record of working with us to address technical needs specific to our applications (for example, provide custom programming to control a preferred accessory) or in defining next-generation software and hardware improvements. 

This consideration was added because of the potential of this advantage (6). Improved mass resolution capabilities (better than nominal) provide a potential means of increasing specificity for quantitative applications. This capability could be advantageous when detection limits are chemical-noise limited and the target compound contains one or more hetero atoms (such as P, Cl, and Br) so that the analyte has a negative mass defect relative to the predominating biological matrix components. 

The preceding are the factors we value for consideration of mass spectrometers intended for quantitation of potential pharmaceutical and related compounds. For some types of instrument acquisitions, we also deliberate on potential instrument redeployment and life-cycle management considerations. Other organizations might consider additional factors including GLP compliance, interfacing to LIMS, and manufacturer stability–viability. Evaluation of different types of instrumentation necessarily will involve other considerations. For example, for an evaluation of autosamplers for ultratrace quantitation, injection-to-injection carryover would be an important consideration. For HPLC systems performing that job in conjunction with MS-MS, extracolumn volume, retention time reproducibility and data system interfacing would be considered. An important point to reiterate is that, for each type of instrumentation being considered, it is absolutely essential to first clearly define the context or task for which that instrument is targeted to devise the optimal list of evaluation considerations and weight them appropriately. 

Information that allowed a rank-ordering of contending instruments, in terms of each factor, was collected in various ways. Four factors (sensitivity, ion source–interface, linearity–dynamic range, and mass resolution) were addressed by data obtained from vendor (and in-house) execution of our prescribed experiments (see next section). Five more subjective factors (vendor service, common platform, up-time–ruggedness, influence on vendor, and software–data system) were assessed through surveys and discussions with users (internal and external) having the most relevant experience. The cost factor reflected the standard corporate discount from each vendor (first-cut negotiation). To yield the "Golf Score Card," all rank orderings were entered, weighted, and then compiled using an Excel spreadsheet. 			

The Golf Score Card, shown in Figure 1, was constructed from an evaluation conducted a few years ago. The totals of all ten of these weighted factors provided a measure of relative fit of each instrument toward meeting the lab's requirements. The rankings that were considered 

 were multiplied by three, so as to give these the most influence on the final score. Those considered 

 were multiplied by a factor of two. The golf score (weighted summation) for each instrument was calculated using a simple Excel formula (shown here for Instrument 1): 

=SUM(B3:B4)*3+SUM(B6:B10)*2+SUM(B12:B14)  

 Figure 1:  The Golf Score Card showing data from an evaluation of three triple quadrupole MS systems. The low "golf" score won and Instrument 1 was purchased. Weighting: most important category multiplied by 3 and important by 2, with data summed by the spreadsheet.			

As with the game of golf, the instrument with the low score wins. The score or weighted summation was influenced by each category, but to different extents based upon level of importance (associated weighting). In some cases, a straightforward ranking of first, second, and third place was not used. For example, instruments 1 and 3 were found to have equivalent sensitivity and instrument 2 was significantly less sensitive. Therefore, 1 and 3 received a 1.5 ranking (splitting the difference between first and second place) and instrument 2 a third place ranking. In a similar manner, as we already had several (previous generation) instruments from vendor 1 and no triple quadrupoles from 2 and 3, we assigned a first place ranking to instrument 1 and 2.5 rankings to the other two. Clearly, it would be simple to configure a Golf Score Card to reflect individual needs and emphasis. Categories and weightings can be adjusted easily depending upon instrumentation type, researcher experience, existing equipment and, importantly, the specific charter or task at hand. That we configured and weighted our Golf Score Card the way we did is a reflection of our context. This context included our experience, instrumentation on hand, lab charter, and so on. The cost differential was low (in this case) and up-time–ruggedness was based upon previous generation instruments, so these factors were de-emphasized by placing them in the 

 category. The primary point is that this simple Excel-based tool allows multifaceted data to be well-organized and encourages consideration of the relative importance of the various factors that lead to a decision. 

Additionally, we conducted a recent and more extensive evaluation of MS-MS quantitation systems that included ion trap mass spectrometers and additional evaluation categories (mass accuracy, full-scan sensitivity, high-flow source capability). A retrospective assessment of this evaluation data revealed a direct correlation between our golf scores and instrument pricing. The linear plot of the scores versus cost (from initial price negotiations) shown in Figure 2 supports this. The fit quality (

 = 0.9108) from our golf score data implies that vendors already know where their instruments rank against each other in terms of bioanalytical quantitation capability (a large application area for multistage MS systems). Furthermore, the resulting linear fit might be thought of as a "Value Line." Scores below the line tend to be good values, while those above the line are perhaps overpriced for what they might deliver in health care-oriented quantitative MS applications. Note that the golf scores from the more recent evaluation (Figure 2) are higher than those from the first example (Figure 1). This was simply a result of the math from additional categories (three more) and more instruments in the evaluation (that is, fourth through eighth place values also possible). 

Figure 2:  This golf score versus price correlation shows the relationship between cost and the relative bioanalytical quantitation capabilities of eight mass spectrometers.			

It is important to construct evaluation or "demo" experiments that directly assess technical performance in the application areas the instrument will be used for. In order to reduce variables, we are prescriptive in detailing experiments and in the formatting of results that the vendor must provide for our review. Collectively, this maximizes the chance that observed differences in instrument performance are not a reflection of location, demo chemists, or other factors we do not wish to test. A significant part of this is avoiding the "apples and oranges" dilemma that arises from ancillary variables that a vendor might impart as they execute the experiments, or the manner in which individual vendors might choose to display the data. For example, to eliminate chromatography variables in our assessment of mass spectrometers, we employ isocratic HPLC methods for analyte introduction and even provide the vendor with HPLC columns (all purchased from the same lot). We also mandate that vendors precisely adhere to our prescribed experimental parameters and procedures and provide a page-by-page listing of the key figures (spectra and chromatograms) that they must assemble and present to us at the conclusion of their experiments. Another way we minimize difficulties in comparing performance attributes is to select test compounds with which we have considerable historical experience. This not only gives us confidence that these experiments will work if executed as prescribed (very low uncertainty), it also permits us to generate the best possible in-house "control" data in our hands (on our existing instrumentation). 

Isocratic HPLC–MS-MS assays for dextromethorphan and fluprostenol were chosen as the basis for some of our recent instrument evaluation experiments (Figure 3), including the two examples discussed in Figures 1 and 2. Over the years, we have analyzed tens of thousands of biological samples containing one or the other of these compounds. Dextromethorphan, an ingredient in over-the-counter cough and cold products, represents a typical drug active. Its tertiary amine substructure is protonated easily via ESI and yields abundant and structurally specific fragment ions under typical MS-MS conditions, making it a compound that can typically be detected with high sensitivity, across a variety of MS-MS instrument types (7–10).We have found that dextromethorphan is an analyte that any system can determine, yet every system it has been tested on responds differently, allowing an interesting and reliable comparison. Fluprostenol represents a more difficult class of compounds to analyze by positive ion ESI-MS-MS detection (11,12). Fluprostenol ions are much more labile than dextromethorphan. [M + NH

 ions formed under all but the mildest of ESI conditions tend to degrade within many ESI source/interface types ([M + H]

 ions are not abundant). This makes fluprostenol an ideal compound to test ion source–interface quality and provide an indication of the breadth of compound class applicability of a given instrument. 

Figure 3:  The structures of two compounds used for MS-MS instrument evaluations, dextromethorphan and fluprostenol.			

Conducting an instrument evaluation at the vendor's site is an excellent opportunity to learn about their instrumentation and software. Demonstration lab chemists often are highly experienced and have a varied perspective on the type of work different customers intend to perform with their new instrumentation. Often, the prospective customer will have access to vendor experts and learn about the company and its support structure, so that overall these visits can be an important learning experience. After a number of iterations of the demonstration and acquisition experience, with the same vendors and instrument types, an on-site demo might not be required. We have managed the acquisition of 15 triple quadrupole HPLC–MS-MS systems since 1992. Although improved systems continue to be offered, this technology is fairly mature and we have a great deal of experience with it. In the absence of a revolutionary (as opposed to evolutionary) improvement or a significant offering by a vendor that we are less familiar with, our last several rounds of acquisitions have been conducted with assays sent to demo labs with the aforementioned carefully prescribed experimental conditions. In cases in which no significant evolutionary change has occurred and the acquisition might be tied to capacity needs, a paper argument will suffice (no demo at all). 

It is important to consider the many other sources of information that are readily available, in addition to a direct demonstration of instrumentation. These include vendor websites, literature, and, more recently, net meetings or "web casts" (conferences or presentations by vendors to groups over the internet). Other important sources of information include trade shows (PittCon, for example) and informal discussions with colleagues, counterparts from other companies, and contract lab personnel. 

The acquisition of expensive and important analytical instrumentation always is preceded by a significant decision. Typically, there will be several instrument choices that can perform the required analyses to different degrees. In addition, we would argue that the purchase decision impacts business success, careers, and future acquisition opportunities and therefore, even a small advantage, especially over time, is of concern to the successful scientist. Careful consideration, categorization, and weighting of the relevant factors can greatly increase the chance of an ideal outcome. Our experience is that the use of a tool such as the Golf Score Card helps focus, organize, and streamline this process. We cannot emphasize strongly enough the importance of carefully considering potential acquisitions within the context of the intended application. Instruments can have scientifically interesting attributes that do not directly impact the work an instrument is intended for and these factors should be minimized in an evaluation. Importantly, the demonstration and evaluation process is a key learning opportunity. Insights into instrument usage or related techniques can be applied in future efforts. 

The authors would like to thank Tom Eichhold, Mike Quijano, Mary Kay Dirr, Pete Stoffolano, Dave Foltz, Michelle Dunaway, John Tomlinson, Adam Lux, and Rose Marie Deibel for assistance during our recent instrument evaluations and acquisitions. 

Tim Baker has been with Procter & Gamble since 1990. After five years in the Health & Personal Care and Over-The-Counter Technology Divisions, he joined P&G Pharmaceuticals. He is currently principal scientist and group leader of the Bioanalytical MS Laboratory, and his research interests include finding new ways of supporting pharmaceutical discovery with mass spectrometry-based technologies. Tim received the Ph.D. in analytical chemistry from Northeastern University, Boston, Massachusetts, in 1990.

Steve Hoke joined Procter & Gamble Pharmaceuticals in 1994. After one year in Norwich, New York, he moved to Cincinnati, Ohio and joined the Oral and Personal Health Care (O&PHC) organization of Procter & Gamble.  He is currently a principal scientist and leader of the O&PHC mass spectrometry group.

Steve received the Ph.D. in analytical chemistry from Purdue University in 1994, where his graduate research focused on applications of mass spectrometry.

Roy Dobson joined Procter & Gamble's Health & Personal Care Technology Division in 1986.  He is currently a research fellow and technical manager of Health Care Mass Spectrometry.  Roy's research interests touch on most aspects of MS-related innovations, with greatest emphasis in the areas of ultratrace quantitation, drug metabolism, and high-throughput small molecule characterization. Roy received the Ph.D. in analytical chemistry from Iowa State University, Ames, Iowa, in 1986. 

*Procter & Gamble Pharmaceuticals, The Procter & Gamble Company

 †Health Care Research Center, Mason, Ohio.

Please direct correspondence to Tim Baker at 

Peer-Reviewed Article

The use of a simple Excel-based "Golf Score Card" tool can facilitate assessments and instrument acquisistion decisions by collating and weighting the relative importance of the many criteria that impact the final purchase decision. The authors describe this process.

Evaluating Analytical Instrumentation Using the Golf Score Card Tool

There are few things more frustrating in the laboratory than having a liquid chromatography (LC) method fail after working reliably for many hundreds or thousands of injections. For some workers, gradient elution LC methods are much more prone to problems than isocratic ones. There are some tricks of the trade that experienced chromatographers practice to help ensure reliable method operation for routine gradient assays. The LC system must perform in a reliable and reproducible manner, but it does not happen by magic. Some simple practices will help to make problems with gradient methods rare rather than frequent. This month's installment of "LC Troubleshooting" presents a baker's dozen of these tips. I hope that you find them useful in lowering your frustration quotient. 

Reversed-phase gradient runs tend to concentrate nonpolar materials at the head of the column and release them as the gradient progresses. Under starting gradient conditions, the mobile phase is weak (very polar, with high water content). This means that nonpolar materials will be strongly retained. Nearly any organic impurity in the mobile phase will be held at the top of the column under these conditions. During equilibration between runs, perhaps 10 or more column volumes of weak mobile phase pass through the column. While the column is equilibrating, most of the nonpolar materials in the mobile phase will concentrate at the head of the column, only to be eluted when the gradient is run. These show up as ghost peaks in the chromatogram and can make quantification difficult and confuse interpretation of the chromatogram. For this reason, it is essential to use high performance LC (HPLC)-grade reagents for gradient work. Lower quality reagents might be suitable for isocratic applications, but with gradients, even the most minor impurities can cause problems. Discard aqueous reagents and buffers frequently (for example, weekly) to avoid contamination by microbial growth. Water impurities can be problematic, so buy HPLC-grade water or use a water purification system designed for production of HPLC-grade water.			

Just as reagent quality is important for a minimum of interfering peaks, a clean instrument also will help avoid unwanted peaks. Thoroughly flush the system with strong solvent at the end of the day or before shutting off the system. Because switching to pure organic solvent, especially acetonitrile, can cause buffers or salts to precipitate, I recommend switching to nonbuffered mobile phase first. Just replace the buffer bottle with water and run the gradient again. Run the gradient to 100% strong solvent (usually methanol or acetonitrile) and hold it at 100% for 10–15 min to thoroughly flush the column and equipment. Do not shut off a system that contains buffers or salts — either flush the system with nonbuffered mobile phase or reduce the flow rate to 0.1 mL/min. This will help avoid formation of buffer salt crystals and microbial growth in the equipment. Check valves will work more reliably and pump seals and columns will last longer with routine flushing. If you are using high-salt mobile phases (for example, > 50 mM), it is a good idea to use the seal-wash feature of your system to flush water behind the pump seals to remove any buffers or salts that leak past the seal. Clean up spills, leaks, and other potential sources of contamination. Be sure to wash or replace solvent reservoirs on a regular basis (for example, weekly). 

When asked how to make columns last forever, my usual answer is to never make an injection. That makes it hard to get our work done, so the next best thing is to inject clean samples. As a rule, the cleaner the samples are, the longer the column will last and the more reliable the method will be. There must be a balance, however, because it is possible to spend more time and money cleaning up the sample than you save in reliability and extended column life. You will have to figure out the economics of that yourself. However, for all methods, it is a good idea to avoid injecting any sample that contains particulate matter. Some workers like to filter samples with a syringe filter. I work primarily in the pharmaceutical industry and tend to question the value of filtering every sample. The syringe filters are expensive — a dollar or more each. Plus, you must validate the filtration process. Are any sample components selectively removed by the filter? Are any contaminants added to the sample by the filter? How much sample volume is lost during filtration? These and other questions make me want to avoid filtration. Instead, I like to centrifuge the samples in a benchtop centrifuge for a few minutes (for example, 10,000–15,000 rpm for 5 min). This settles out particulate matter that can clog column frits. Transfer the sample to a clean vial before injection. I am also a strong advocate of mounting a 0.5-μm in-line frit just downstream from the autosampler. This will trap any junk that would normally block the 2-μm frit at the top of the column and can be changed much more easily than the column frit. 			

Although some gradient LC systems will operate without degassing the mobile phase, every system will operate more reliably with degassed solvents. Trapped air bubbles and solvent outgassing are two of the most common problems with gradients, and these can be largely avoided by solvent degassing. Bubbles in the pump(s) cause pressure and flow fluctuations, which can distort the gradient shape and affect retention times. Helium sparging has been the gold standard for degassing for many years, but many of today's LC systems come with in-line vacuum degassers that are much easier to use and provide effective gas removal. It is a good idea to purge the pump(s) and solvent inlet lines daily by opening the purge valve(s) and operating at an elevated flow rate (for example, 5 mL/min) for a few minutes to remove any air bubbles that might have accumulated while the system was shut off. 

I recommend running a blank gradient as part of method setup to ensure that unexpected drift, noise, or peaks are not present. Simply set the autosampler to bypass the injection and run a normal gradient. (The systems in my laboratory can be programmed to make a zero-volume injection to accomplish this.) Look for peaks in the blank chromatogram. These come from reagent or water impurities. For methods run at maximum system sensitivity, it might not be possible to obtain completely smooth baselines, but the number of spurious peaks should be kept to a minimum and none should be eluted at the same retention time as sample components of interest. Although blank gradients might not be required on a daily basis for a routine method, the blank run is inexpensive insurance to help protect against loss of valuable sample data. 

Each analytical method should have a column dedicated to that method. I strongly advise against sharing columns between methods, because unimportant peaks in one method can cause interferences in a second method. Dedicated columns last longer, so fewer columns will need to be purchased in the long run if each method has its own column. While we are talking about columns, remember that a column is a consumable item — it should not be expected to last forever. In my experience, with a well designed method, a column should last for at least 500 injections, even under severe mobile phase conditions. This amounts to < 5% of the cost of analysis and usually is much less than the cost of other consumable items, such as solid-phase extraction cartridges. With adequate sample pretreatment and nonaggressive mobile phases, it is not unusual for a column to last for 2000 samples or more. 

Before each run, the column should be equilibrated to the same extent as the other injections in the run sequence. A good rule of thumb is to allow 10 column volumes of the initial mobile phase to pass through the column for equilibration between runs. For the most common column, 150 mm × 4.6 mm, the column volume is approximately 1.5 mL, so 15–20 mL should be sufficient. Remember that during equilibration and column washing, volume is more important than time, so the equilibration time might be shortened by increasing the flow rate during this phase of the method. Recent information (for example, reference 1) shows that complete equilibration might or might not be necessary, as long as the same amount of equilibration is used between every run. Operating under partial equilibration conditions can save time by shortening the wait between injections. 

It is a good idea to carefully examine the LC system each day before starting routine injection of samples. A good time to do this is during the initial equilibration of the column with a new batch of mobile phase. Just start at the mobile phase reservoirs and trace the solvent feed tubing through the mixer, connecting tubing, pump, and so forth on to the waste bottle after the detector. Are there any leaks? Is there white fuzz on any of the fittings? Do the reservoirs contain sufficient mobile phase? Is the autosampler wash solvent level sufficient? Does the waste solvent bottle have room for the anticipated waste stream? Is there anything else about the system that does not look, smell, or sound right? A few minutes each day spent examining the system pays great dividends when minor problems can be identified and corrected before they grow to the extent that they will compromise analytical results. 

Some methods will give better results if several "priming" injections are made before the first sample is injected. These injections of standards or mock samples can help to load slowly equilibrating active sites on the column so that more reproducible separations can be obtained. If you notice that it takes several runs before the retention time or peak area stabilizes, your method might be a candidate for priming injections. You can make a special priming solution that contains the analytes at high concentration or make several large volume injections at a lower concentration to load enough sample on the system to deactivate the column. Sometimes the system suitability injections serve as priming injections. Most methods do not require priming injections, but if your method does, this procedure reduces the time it takes to get reliable data from the system. 

Because some methods require the priming process (see above) and the first injection can be equilibrated differently than subsequent injections, I advise setting up a routine method so that the first injection is not used for quantitative purposes. The second and subsequent runs will be more reliable than the first injection. By making it a formal practice to ignore the first injection, you will not have the hassle of having to perform outlier tests or other procedures to justify throwing out the first injection when it does not meet method criteria. 

Many methods that run under the oversight of regulatory agencies (FDA, EPA, OECD, and others) will require a system suitability test before sample analysis. System suitability serves as a mini-validation to show that the equipment and analytical method are operating in a fashion that will produce reliable results. Requirements for system suitability tests vary, so the regulatory guidelines should be consulted to help select appropriate tests. Many workers use retention time, retention and area reproducibility, peak response, peak width, peak tailing, resolution, and column back pressure either alone or in combination as part of the system suitability test. The system suitability sample might be a diluted pure standard, a mock sample in extracted matrix, or some other sample selected to demonstrate system performance. Whether or not a system suitability test is required, I strongly suggest running such tests before routine analysis, even if it is just an injection of a standard to see if the retention and peak size are as expected. 

For quantitative analysis, the response of unknown samples is compared with the response for standards of known concentration. The range of standard concentrations, number of replicates, and sequence of injection will depend upon the specific application. External or internal standardization might be appropriate. In any event, running at least one standard before running unknown samples will provide assurance (system suitability) that the analytical method is working properly before potentially valuable samples are injected. 

Analytical methods operated under regulatory oversight can require inclusion of quality control (QC) samples. QC samples are mock samples made in the sample matrix at known concentrations. Their concentration is calculated against the standard calibrators as a check to show that the method is operating in a manner that will produce reliable results. Generally, it is wise to intersperse QC samples among unknown samples to demonstrate that reliable results can be obtained throughout an analytical run. The results of QC samples can be tracked over time with a control chart. Sometimes the control chart can be helpful in anticipating failure of the system or identifying subtle problems. 			

You might realize that these tips for reliable gradient method operation already are part of your routine, either in whole or in part. Or you may have other techniques you use to ensure that the system is ready for samples. Send me an e-mail to share your favorite practices. Whether required by regulations or not, you will find that your gradient methods will be much more trouble-free if you take a few minutes before injecting the first sample to make sure that the system is ready to go.  

"LC Troubleshooting" Editor John W. Dolan is Vice-President of BASi Northwest Laboratory of McMinnville, Oregon; a Principal Instructor for LC Resources, Walnut Creek, California; and a member of

's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," 

(1) A.P. Schellinger, D.R. Stoll, and P.W. Carr, 

LC Troubleshooting

Some simple practices will help to make problems with gradient methods rare rather than frequent. this month's installment of "LC Troubleshooting" presents a baker's dozen of these tips.

LCGC North America-09-01-2005

Highlights of HPLC 2005

Peaks of Interest

The Making of a Column

Evaluating Analytical Instrumentation Using the Golf Score Card Tool

A Baker's Dozen