Without a doubt, life science technologies are providing valuable information and have become the core that discovery relies upon. Life science instruments are used predominantly by four industrial segments, which include academia, biotechnology, government, and pharmaceutical research labs.  Due to the amount of money being spent, these industrial segments are under pressure to deliver exciting new discoveries.			

While life science or discovery research was separated by genomic and proteomic applications, the dividing line between the fields is beginning to fade. Instead, researchers are integrating the two fields and adding another level of difficulty by taking on the challenges of systems biology. Researchers are broadening their focus to gain a comprehensive understanding of biological systems and are communicating with colleagues across different technology platforms. This integrated approach is correlating genetic data with phenotypes of whole organisms, essentially creating a map between the points at the molecular biology and cellular level, and thus providing a true understanding of life and disease.

Worldwide Market for Protein and Analysis in Life Science Research

Systems biology is pushing the boundaries of throughput and performance for bioinstrumentation technologies, especially for chromatography and mass spectrometry, as well as cell analysis and high content screening.  Information is being generated at a phenomenal rate and methods to analyze the data are in high demand. The overall demand for protein and cell analysis for life science research applications  accounted for nearly $4.5 billion in 2004, with 12â15% growth over the last few years.

The foregoing data was extracted from Strategic Directions International Inc. Market Analysis and Perspectives (MAP) report titled, “Life Science Instrumentation: Supporting Systems Biology in Integrating Genomics, Proteomics and Drug Discovery.” For more information, contact Glenn Cudiamat, Vice President of Research Services, Strategic Directions International, Inc., 6242 Westchester Parkway, Suite 100, Los Angeles, CA 90045; tel. (310) 641-4982, fax (310) 641-8851.

Articles

Departments

Without a doubt, life science technologies are providing valuable information and have become the core that discovery relies upon. Life science instruments are used predominantly by four industrial segments, which include academia, biotechnology, government, and pharmaceutical research labs. Due to the amount of money being spent, these industrial segments are under pressure to deliver exciting new discoveries.

Market Profile: Life Science Research

Bruker Daltonics and German agency announce collaboration

Bruker BioSciences Corporation (Billerica, Massachusetts) and the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) announced a collaboration for the identification and classification of microorganisms from peptide and protein profiles measured by MALDI-TOF mass spectrometry. The DSMZ uses Bruker Daltonics' CLINPROT BioTyper system to generate protein profiles and to perform the required data analysis. 

The DSMZ is the German National Resource Centre for Biological Material, and currently stores and maintains approximately 14,000 cultures, representing 6900 species from different types of microorganisms, including archaea, bacteria, yeasts, and fungi. In the collaboration, a database will be generated that will include species and strains for many different application areas such as microbiological classification, clinical and diagnostic infectious disease research, as well as environmental analysis, food safety, and water quality.   

Applied Biosystems completes acquisition of division of Ambion

Applied Biosystems Group (Foster City, California) has completed its acquisition of the Research Products Division of Ambion (Austin, Texas).  			

Applied Biosystems will continue to deliver the products, services, and support for both Applied Biosystems and Ambion.  

Applied Biosystems' product portfolio combined with Ambion's broad range of RNA products allows the company to offer complete solutions for application workflows.   

PerkinElmer Acquires Agilix Proteomics Technique

PerkinElmer (Shelton, Connecticut) recently announced their acquisition of Agilix Corporation's (Woodbridge, Connecticut) proprietary protein labeling technique that uses isobaric mass tags to simultaneously quantify expressed proteins from multiple samples.  

This technique brings quantitative proteomics to PerkinElmer customers in drug discovery and proteomics research. It provides a method for enhancing the utility of mass spectrometry for proteomic analysis.   

Waters Corporation (Milford, Massachusetts) announced that it has purchased the food safety technology business and associated net assets of VICAM (Watertown, Massachusetts) for undisclosed terms. 

According to the Annual Report of the European Commissions' Rapid Alert System for Food and Feed, the largest number of Information Notifications by identified risk in 2004 was for mycotoxin contamination in food imported to, or grown within, the European Union. VICAM's proprietary products employ monoclonal antibodies that are highly specific for certain mycotoxins in a sample. 

Peaks of Interest

Pittcon 2006 — the 57th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy — will return to the Orange County Convention Center in Orlando, Florida on March 12–17, 2006. This year's event will host more than 1000 instrument manufacturers and laboratory suppliers in more than 2300 booths. In addition to attending the exposition, the conferees will have the opportunity to listen to more than 2200 oral and poster presentations, which will include invited and award symposia, check out 50 seminar rooms, or attend one of 100 short courses. 			

Pittcon remains the most important yearly international analytical exhibition where companies introduce their latest instruments, instrument accessories, columns, sample preparation products, and other consumable products. Because many past attendees have purchased one or more new products within three months after attending the show, most exhibitors attempt to maximize their booth traffic to meet as many potential customers as possible. 			

The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2006. In some cases, products that were introduced during 2005 but after Pittcon 2005 (1,2) have been included for reasons of completeness. The information is based upon manufacturers' responses to a questionnaire mailed in December 2005. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered an exhaustive listing of all new products that will be introduced in Orlando. However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, columns and other products recommended by their manufacturers primarily for biomolecule separations or sample preparation are denoted in the tables with the designation BIO. Some of these products can be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples. 

In last month's coverage, I described new columns for reversed-phase, normal- and bonded-phase, ion-exchange and ion, size-exclusion, and specialty chromatography (3). In this month's coverage, I look at gas chromatography (GC) columns, supercritical fluid chromatography (SFC) products, sample preparation products, and hardware, and accessories and kits for chromatography and sample preparation. 

Although GC is considered to be a relatively mature separation technique, new GC columns continue to be introduced each year at Pittcon (Table I), 20 in all for this year. All columns were the wall-coated open tubular (WCOT) format with the exception of one — the Agilent (Santa Clara, California) GS-OXYPLOT, a specialty PLOT column for oxygenate analysis in the petrochemical industry according to the American Society for Testing Materials (ASTM) methods (4). This column has been optimized for this analysis and can be "dropped-in" with current oxygenate analysis with improved peak shape and better reproducibility compared with current columns. 

Table I: Gas chromatography columns at Pittcon 2006 			

In recent years, there has been a trend in the introduction of ultra low-bleed WCOT stationary phases that allowed better detection limits, less baseline drift during temperature programmed separations, and improved compatibility with gas chromatography–mass spectrometry (GC–MS) analyses. This year, almost every GC column had lower than normal bleed specifications. Figure 1 provides experimental data on two of these phases that demonstrates inertness (Figure 1a) or low bleed (Figure 1b). For Figure 1a, the low tailing of pyridine is a good indication of the absence of basic active sites. For Figure 1b, the bleed profiles for a regular 624 column and a new low-bleed column are compared. A column that has lower phase bleed will allow better detection and improved quantitation over a higher bleed column. 

All of the general-purpose GC capillary columns displayed at Pittcon 2006 were crosslinked phases. Crosslinked, chemically bonded, or immobilized phases are more stable, have lower bleed, and usually operate at higher temperatures than coated phases. Another advantage of the bonded phases is their ability to be solvent-rinsed. If a column becomes contaminated by high boilers, inorganic compounds, and other nonvolatile impurities that might cause drifting baselines, bleed, and other undesirable occurrences, it can be disconnected, washed (outlet to inlet direction) with various solvents, and placed back into the gas chromatograph for further use. Capillary columns with internal diameters ranging from 0.10 mm (small bore) to 0.53 mm (large bore or megabore) were the most popular sizes.  

High-polarity capillary GC columns always have been tricky to construct because the high-polarity phases are difficult to stick to the fused-silica inner wall. SGE (Austin, Texas) introduced an extremely polar column coated with 90% biscyanopropylpolysiphenylene phase that offers a completely orthogonal approach to the popular nonpolar polysiloxane phases for two-dimensional (2-D) GC. The phase shows strong π–π selectivity for polyunsaturated fatty acid methyl esters and various types of isomers. 

Table II: Sample preparation products at Pittcon 2006			

Applications-specific columns are always popular introductions at the Exposition. In addition to the oxygenate column, columns for volatile enantiomers, 

 fatty acids, pesticides, semivolatile, and volatile organic compounds, and PCBs were among those classes of compounds mentioned. Most of the application-specific columns are pretested and are shipped with test chromatograms. 

Table II: Sample preparation products at Pittcon 2006 continued			

As indicated in Table II, Pittcon 2006 was another productive year, with 23 new sample preparation products or families of products displayed. Not counted were sample preparation accessories (Table III) that will be covered later. Among the more popular introductions were products that deal with increasingly smaller amounts of available sample. Cartridges, disks, pipette-tips, well plates, and spin tubes were among the formats introduced.

Table III: Chromatography and Sample Preparation Accessories at Pittcon 2006 			

In the past several years, the 96-well plate design for SPE and filtration has proliferated, driven by the needs of high-throughput sample preparation automation requirements. Compared with last year (2), this year numerous sample preparation plate products were again in evidence. Most of them were solid-phase extraction (SPE) plates or variations of SPE-plates. For example, Glygen (Columbia, Maryland) introduced their lab-on-a-plate well plates where the SPE medium was affixed to the inner surface of the well and rather than a flow-through design, one performs a liquid–solid extraction in a conventional well. Nalge-NUNC's (Rochester, New York) ArrayCote product was of similar design but the wells within a glass plate were functionized with reactive ligands that could be derivatized to form in-situ affinity phases. 

Table III: Chromatography and Sample Preparation Accessories at Pittcon 2006 continued			

Argonaut Technologies, now part of Biotage (Charlottesville, Virginia), displayed their flow-through supported liquid extraction plates. Here, the wells are filled with high-purity diatomaceous earth that serves as a support for an extractable aqueous sample, such as blood plasma. Under proper conditions, organic solvent is passed through the wells and a liquid–liquid extraction take place. The organic solvent containing the extracted analytes is collected in a well plate for further treatment.  

Other variations of SPE plates were those of Varian (Palo Alto, California), whose 96-well plate had removable flow-through wells that enable users to customize the plate configuration. Such flexibility is useful in method development where several SPE phases can be present in a single plate so that phase scouting can be performed.  

Several companies introduced SPE pipette tip–based products that can be used with 

 automated liquid-handling systems. The SPE stationary phase was packed, bonded, or affixed to the tip by various means. For example, in the Thermo Electron (Waltham, Massachusetts) tips, packing was affixed on the inner surface so that flow through the opening was not impeded. In Varian's OMIX, a monolithic packing is used to cut down flow resistance. The opening of the pipette tip is quite small and if the packing is restricted or the small packed bed is plugged with sample particulates, flow can be erratic and nonreproducible results can occur. Other formats for SPE introduced were disk and spin tubes. An example of one of the SPE pipette-tip based products from SeQuant (Umea, Sweden) is depicted in Figure 2. One version, the Zic-tip mini (Figure 2a), has a packed bed containing a zwitterionic hydrophilic interaction chromatography (HILIC) chromatographic resin. The other version, the Zic-tip micro (Figure 2b), has the same packing coated on the inner walls of the pipette tip. Both are used for the extraction of hydrophilic biomolecules, such as glycopeptides, from microliter volumes of biological solutions using typical extraction–elution steps. 

One area of special focus in sample preparation was the high-abundance protein-removal products from Agilent Technologies, BIA Separations (Ljubljana, Slovenia) and Sigma Aldrich (Bellefonte, Pennsylvania). These kits and columns are used in proteomics research where investigators are interested in the presence or absence of low-abundance proteins that can be indicative of disease states. The high-abundance proteins, which constitute 90+% of the proteins in human serum, can mask the lower abundance proteins in 2-D gel or HPLC separations. By removing them, there is a better chance to see the smaller ones. By the use of antibodies to the high-abundance proteins like albumin, IgG and so on, they can be removed or greatly reduced. 

Specialized SPE application chemistries also were introduced. Polymeric packings can be used under a wide variety of experimental conditions. Polymeric SPE products were displayed by Hamilton (Reno, Nevada), Polymer Labs (Amherst, Massachusetts), BIA Separations, OroChem Technologies (Lombard, Illinois), and Sepax Technologies (Newark, Delaware). Molecular-imprinted phases are quite popular in Europe, less so in the United States. These phases are very selective due to the presence of molecular imprints of a specific template on the surface of a polymer. Molecular-imprinted phases are prepared by adding a target template molecule to the monomer reaction mixture and forming a polymer around the template. The template is extracted away, leaving behind its imprint on the surface. When a mixture of compounds containing the target molecule is introduced to the molecular-imprinted phase, usually packed into a column or cartridge, there is a stronger affinity for the target molecule by the imprinted site. This is viewed by some as a "lock and key" mechanism. So the process very much resembles affinity attraction. Usually molecular-imprinted phases are customed synthesized but MIP Technologies (Lund, Sweden) has uncovered at least one high-volume application and has developed a molecular-imprinted phase exclusively for chloramphenicol, an antibiotic drug often used in cattle and poultry but banned in many countries. For this molecular-imprinted phase, detection limits well below the minimum performance limit are achieved. Glycopeptide sample preparation by HILIC SPE and a mixed-mode THC phase were some other specialized chemistries shown at Pittcon. 

More traditional markets also are addressed. Environmental and food samples often contain low concentrations of contaminants that have to be analyzed and enrichment is often required. SPE is a technique often used for this purpose. New products addressing U.S. EPA methods were introduced by Xenosep Technologies (Boonton, New Jersey) (oil and grease, semivolatiles, chlorinated pesticides, PCBs, and phthalate ester) and Supelco (multiresidue pesticides). The Xenosep Technologies new triple layer SPE filter has been designed for use with EPA Method 1664A. This method is designated for oil and grease analysis and recently the EPA has allowed the use of SPE if equivalency the more accepted hexane extraction method is proven. One of the problems in using SPE devices such as cartridges or disks is that occurrence of plugging due to the presence of particulates, particularly in untreated effluents and in-process wastes. The Xenosep SPE filter (Figure 3) has a coarse layer (traps particles 5–25 μm) followed by an intermediate prefilter layer (traps particles 1–5 μm) followed by the SPE adsorbent layer, which retains the oil and grease compounds. This triple layer concept enables fitration much faster filtration (in a matter of seconds) and provides improved SPE recovery and precision performance. 

Chromatography and Sample Preparation Accessories

Table III lists a record-breaking 31 new hardware products and accessories for HPLC, GC, capillary electrophoresis (CE), capillary electrochromatography (CEC), and sample preparation. This year, I have extended the columns and sample preparation accessories to include kits that are designated to solve specific applications problems. All of the products introduced were practical devices designed to make the chromatographer's life easier. I will cover each area individually. 

An automated system for preparation of chemical solutions including HPLC mobile phases including pH adjustment of buffers should make life easier in the laboratory. New vials, caps, and septa including a integrated vial insert system was introduced. Reference standards are always appreciated. A convenient small laboratory cooler that can be used for a variety of cryogenic experiments and sub-ambient storage of samples was displayed. 

Column switching is having a comeback driven by the needs to tackle complex samples such as tryptic protein digests. Systems, trapping columns, and orthogonal column sets were introduced. With the miniaturization in HPLC with shorter and smaller diameter columns as well as high-pressure requirements for longer columns and smaller particles, new lower dead volume and higher pressure rating hardware designs are becoming available so that performance is not compromised. A "see-through" union for low-volume connections for fused silica tubing shows promise. Figure 4 shows the PicoClear Union from New Objective (Woburn, Massachusetts). Unlike fused-silica unions used in nanoLC and CE–CEC that use frusto-conical compression, the absence of mechanical twisting with the PicoClear Union eliminates fused silica grinding and compression and reduces the subsequent likelihood of contamination. Through visual inspection, PicoClear Technology eliminates any question that a connection is leak-free and uncontaminated. Because high-temperature LC is becoming of increasing interest, a standalone mobile phase preheater or post column cooler should aid column performance and detector baseline stability. A new nanoliter flow sensor should help to ensure stable reagent addition or pump flow direct or split-flow output is constant.

A large number of convenience products were shown at Pittcon. Gas generators can replace inconvenient and dangerous gas cylinders. New hydrogen and zero air generators were introduced. Gas cylinders in the field are a hassle so that a hydrogen storage system that can operate for long periods of time might be a boon for field GC testing. Improved septa and inert inlet liners are always appreciated. Guard columns, either integrated or external, help to slow down column deterioration. Leak-free unions that do away with press-fit should help those interested in connecting capillary columns, retention or guard columns together. Figure 5 compares a typical GC union stainless steel hardware design with the Ultimate Union Design (Agilent Technologies, Santa Clara, California). In the latter union, the seal is made at the contact of the ferrule (light blue) and the metal fitting (green). The amount of dead (or unswept) volume is limited to a very small space near the tip of the column while the conventional union allows the sample to migrate into long unswept cracks and crevices causing peak tailing. A variable flow splitter for feeding GC column outlet flow to multiple detectors should prove valuable.

 The use of vacuum systems for SPE sometimes creates problems with uneven flow and the possibility of drying out cartridges. A positive pressure manifold with individual flow regulators should help out. A low-volume SPE glassware system which is cost effective might prove helpful to sample prep chemists. The automation of solvent evaporation should prove to be a time saving and more reproducible operation and a couple of units for controlled evaporation were introduced.  

I would like to thank the manufacturers and distributors that kindly furnished the requested information in advance of Pittcon 2006, thus, allowing a timely report on new product introductions. For those manufacturers who would like to be considered for inclusion into Pittcon 2007 coverage, please send the name of the primary company contact, the mailing address, fax number, and e-mail address to Patrick Kempf, Managing Editor, LCGC North America, c/o Advanstar Communications, 485 Rte. 1 South, Bldg. F, Iselin, NJ 08830, Attn.: Pittcon 2007 "Column Watch." 

 "Column Watch" Editor Ronald E. Majors is business development manager, Consumables and Accessories Business Unit, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column to "Sample Prep Perspectives," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

(1) R.E. Majors, LCGC 23(3), 248–265 (2005). 

(2) R.E. Majors, LCGC 23(4), 358–369 (2005). 

(3) R.E. Majors, LCGC 24(3), pages TK (2006). 

(4) I.A.L. Rhodes and A.W. Verstuft, Environmental Testing and Analysis, March/April pp. 24 2001.  

Column Watch

This month's installment of "Column Watch" is the conclusion of a two part series in which Ron Majors examines the trends in columns and sample preparation at Pittcon 2006.

New Chromatography Columns and Accessories at the 2006 Pittcon Conference: Part II

In previous columns, we have discussed the development of the first gel-permeation chromatograph (1) and the work by Csaba Horváth at Yale University (New Haven, Connecticut) in the laboratory of Professor S.R. Lipsky, leading to the first modern, high-pressure liquid chromatograph (2). However, although these instruments usually are called the first liquid chromatography (LC) instruments, they actually were preceded by another complex instrument that was based upon the principles of LC on ion-exchange resins: the automated amino acid analyzer developed in 1958 at The Rockefeller Institute for Medical Research (New York) by S. Moore, W.H. Stein, and D.H. Spackman (3,4).			

The amino acid analyzer opened an entirely new field for research, permitting the elucidation of the composition of proteins. We might even say that the rapid expansion of biochemistry would have been impossible without it. However, because the majority of chromatographers are not in the biochemical field, this development is relatively unknown to the general public. In this installment, we try to fill this gap in the history of chromatography. 

To understand the importance of this development, a brief summary of proteins and amino acids, and the development of separation based upon ion-exchange, is useful. 

Amino acids are the building blocks of proteins: very complex, mostly α-helical, three-dimensional polymers consisting of hundreds of amino acids connected by peptide bonds. Proteins are the key gene-expressed compounds in life processes, and this is reflected by their name: "protein" comes from the Greek word 

, meaning "of primary importance." This term was first used in 1839 by the great Swedish chemist JÖrs Jakob Berzelius (1770–1848). The study of the amino acids has a long history, leading to the slow recognition of the individual amino acids. The 1902 Nobel laureate Emil Fischer (1852–1919) showed in the early 1900s how amino acids are bound to each other forming polypeptides, the building blocks of proteins (5).  

In the first part of the twentieth century, research on proteins and amino acids was carried out along two lines: as part of nutrition studies and investigating their chemistry and composition. Decades of painstaking work by many researchers established that the proteins of the humans and animals contain 20 amino acids, from which 10 cannot be synthesized by the human body, but must be taken nutritionally. The proper amount and balance of the essential amino acids in our food intake is very important. However, these 20 natural amino acids are only part of the amino acids occurring in nature: we know of the existence of hundreds, or even thousands of additional ones. 

Naturally, researchers in biochemistry, nutrition, and other life sciences fields wanted to identify the individual amino acids present in proteins and determine their quantitative levels. For this, the proteins must first be hydrolyzed to break the peptide bonds. This usually is done by boiling the peptide with an acid for an extended time; it is a delicate operation, as some amino acids can be destroyed during hydrolysis, and various safeguards are used to prevent and compensate for it.  

Various methods have been developed for the determination of the individual amino acids. Emil Fischer was the first to show that the esters of the amino acids can be distilled, and he used the fractional distillation of the esters prepared from protein hydrolysates to obtain a more precise understanding of their original composition. Other methods introduced in the first part of the twentieth century involved selective precipitation as insoluble salts, colorimetric analysis, and microbiological assays, but these were complicated and time-consuming operations (6). A.J.P. Martin and R.L.M. Synge first described partition chromatography in 1941 for column chromatography (7) and then its paper chromatography version — introduced in 1944 by Martin's group (8) — represented a major breakthrough, permitting separation and simultaneous determination of amino acids in a straightforward manner. Martin and Synge received the 1952 Chemistry Nobel Prize for the invention of partition chromatography. 

Using partition chromatography on columns packed with starch, in 1944 Synge started to establish the sequence of amino acids in peptides, leading to the structure elucidation of gramicidin S, a cyclic decapeptide (9–12). In the subsequent 10 years, Frederick Sanger at Cambridge University (Cambridge, United Kingdom) further advanced Synge's methodology, finally succeeding in the establishment of the sequence of the 51 amino acids forming the molecule of insulin (13). His achievements were recognized with the 1958 Nobel Prize in Chemistry. 

Ion exchange in soils was first described in 1850, but for decades, only sodium aluminosilicates ("zeolites") were available as exchange material. Thus, the possible utilization of the phenomenon was severely limited. In the late 1930s at Columbia University (New York), Harold Urey tried to separate lithium and potassium isotopes on very long (30–100 ft) columns filled with zeolite and was able to achieve partial separation (14). A new area in ion-exchange began in 1935 when B.A. Adams and E.L. Holmes (National Chemical Laboratories, Teddington, United Kingdom) prepared synthetic organic ion-exchange resins (15). These were first used in Sweden by Olof Samuelson in 1939 (16,17). Such synthetic polymeric ion-exchange resins also were produced in the United States by Rohm & Haas (Bridesburg, Pennsylvania) and Dow Chemical Co. (Midland, Michigan) under the respective trade names of Amberlite IR-100 and Dowex 50. 

Such synthetic resins were used extensively during World War II — even on a preparative scale — as part of the Manhattan Project (the development of the atomic bomb) for the separation of fission products and rare earths. This work was carried out at Iowa State University (Ames, Iowa) and Clinton National Laboratories (Oak Ridge, Tennessee) and was highly classified: its publication was forbidden for years. Finally, at the Fall 1947 National Meeting of the American Chemical Society (September 17, 1947), a special symposium was held where members of the two teams presented detailed reports on their work. Parallel to the symposium, 

 published a report on the activities carried out at Ames and Oak Ridge (18). The text of the 13 papers that were presented at the symposium was published in November 1947 as a special issue of the 

. In 1949, at the Chromatography Conference of the Faraday Society in London, the two team leaders, F.H. Spedding of Ames and E.R. Tompkins of Oak Ridge, presented detailed reports on the whole project. This was how the scientific community learned about the possibilities of the use of modern ion exchangers. (A detailed summary of the work carried out as part of the Manhattan Project was published in 1999 in an installment of 

's "Milestones in Chromatography" column [19].) 

Amino Acid Research at the Rockefeller Institute

The research leading to the development of the amino acid analyzer was carried out at The Rockefeller Institute for Medical Research. This institute was founded in 1901 by John D. Rockefeller (1839–1937) and built on farmland along the East River. The first laboratory opened in 1904 and its hospital for the study of human diseases was established in 1910. Soon after, the institute developed into one of the principal research organizations in biomedical sciences. The institute started to grant Ph.D. degrees in 1954, taking the status of a graduate university, and finally, in 1965, it changed its name to Rockefeller University. Because our story is related to the period before 1960, we shall use the name of Rockefeller Institute in our narrative. 

In March 1933, Hitler and his Nazi party came to power in Germany. A number of Jewish scientists soon had to leave the country, many of them immigrating to the United States. A prominent scientist involved in this intellectual migration was Max Bergmann. 

(1884–1944) was a student and associate of Emil Fischer and had been involved in his amino acid research. He had a distinguished career in Germany, was a founder and director of the Kaiser Wilhelm Institute for Leather Research (Dresden), which he developed into a world-renowned, leading center for protein chemistry research in the 1920s. He left Germany in 1933, joining the Rockefeller Institute, where he soon established a laboratory and became a central figure in protein research in the United States. Bergmann surrounded himself with the most talented young, postdoctoral scientists, launching them to become important members of the international group of protein chemists. Two who are the key figures in our story are William H. Stein and Stanford Moore (Figure 1). 

 (1911–1980) studied at Columbia University, graduating in 1937 with a thesis on the analysis of the amino acids of the protein elastin. He then went directly to Bergmann as a postdoctoral associate.  

 (1913–1982) studied at Vanderbilt University (Nashville, Tennessee) and the University of Wisconsin (Madison), graduating with a thesis on the characterization of carbohydrates as benzimidazole derivatives. It is interesting to note that during his undergraduate years, Moore also took engineering courses (in fact, his first thought was to major in engineering) and as a graduate student in the laboratory of Professor Karl P. Link, at the University of Wisconsin (Madison, Wisconsin), he received an extensive training in microchemistry, including Pregl's microanalytical methods. After graduating in 1939, he joined Bergmann at the recommendation of Professor Link. 

There were two main lines of investigation in Bergmann's laboratory: the field of proteolytic enzymes and the structural chemistry of proteins. Both Moore and Stein had been involved in the second field and their task was to further improve gravimetric methods of amino acid fractionation through the formation of sparingly soluble salts (20–22). America's entry into the war interrupted their work: Bergmann's group started to work for the Office of Scientific Research & Development (OSRD), investigating the physiological effects of mustard gas and related compounds, while Moore left the laboratory and served with OSRD in Washington and at the headquarters of the U.S. Armed Forces in the Pacific Area. At the end of the war, Moore returned to the Rockefeller Institute. Bergmann died suddenly in 1944 and his former group was dissolved, but the director of the institute allocated part of Bergmann's laboratories to Moore and Stein to develop their own research program. This is how their close cooperation began, and it lasted for 40 years, eventually leading to the quantitative analysis of amino acids and its automation, and culminating in the elucidation of the 124 amino acid sequence of ribonuclease, honored by the 1972 Chemistry Nobel Prize. As noted by Moore, "We approached problems with somewhat different perspectives and then focused our thoughts on the common aim. If I did not think of something, he was likely to, and vice versa, and this process of frequent interchange of ideas accelerated our progress in research" (23). 

When Moore and Stein returned to peacetime research, the wartime issues of the British

 had just became available in the United States and there, they read about the work of Synge describing the separation of free amino acids by partition chromatography on columns containing starch as the stationary phase (10,11). They immediately adapted the technique to their investigations, further improving it. They collected column effluent in small fractions and by establishing the amount of amino acids in each fraction, constructed chromatograms showing separated peaks. Quantitation of each fraction was carried out by adapting the color reaction of the amino acids with ninhydrin. In the beginning, the small fractions were collected manually, but this was much too tedious due to the large number of fractions. Therefore, they developed a very sophisticated, fully automated fraction collector. 

In November 1946, The New York Academy of Sciences held a two-day conference on chromatography and there, Moore and Stein presented a report on the early results of their work. The publication of the proceedings of the meeting was delayed by more than one year and, thus, in the printed text of their presentation they also could include a detailed description of their newly developed fraction collector (24). This was followed by four publications reporting on their further results and the determination of the amino acid composition of β-lactoglobulin and bovine serum albumin, using partition chromatography on starch columns (25–28). 

The starch columns worked well, but they were very slow. Therefore, Moore and Stein were looking for other possibilities. Meanwhile, the detailed publications on ion-exchange chromatography became known (described previously) that led to the pioneering work of Waldo E. Cohn at Oak Ridge (Tennessee), on the separation of nucleic acid constituents by ion-exchange chromatography (29,30). Reports from England also involved ion-exchange chromatography for the separation of amino acids in protein hydrolysates, although in the displacement and not in the elution mode (31). These reports initiated intensive activities by Moore and Stein, who were joined by C.H.W. Hirs (born in 1923), who graduated from Columbia University in 1949. Dr. Hirs remained at the Rockefeller Institute for 10 years and participated in much of the amino acid research. He was the first young postdoctoral associate in Moore and Stein's group. In the next 20 years, 20 additional young scientists spent a few years with them, cooperating in their research and learning the intricacies of protein investigations. 

Dr. Hirs provided a very vivid narrative of the intensive research carried out by Moore and Stein's group (32). Their first report on the use of ion-exchange resins for amino acid separation was published in 1951 (33), followed by six more papers in the early 1950s. Studying these, one can follow the careful, painstaking, and systematic investigations approaching every aspect of ion-exchange chromatography and the possibility not only of quantitative amino acid analysis, but also of separation on semipreparative scale. 

The 1972 Chemistry Nobel Prize was awarded to Moore and Stein (together with C.B. Anfinsen) for their "contribution to the understanding of the connection between chemical structure and catalytic activity of the active centre of the enzyme ribonuclease." These investigations started in 1954 and aimed at the determination of the complete amino acid sequence of the molecule. In the course of these studies, a very large number of protein hydrolysate analyses had to be performed and the manual procedure, one complete analysis taking a few days, was just not satisfactory: analysis time had to be reduced and some kind of automation was needed. Moore always had been interested in engineering work — let us not forget that in college, he also took engineering courses. Thus, they decided to investigate the possibility of developing an automated amino acid analyzer. In this work, they had a new associate: D.H. Spackman (born 1924), who received the Ph.D. degree from the University of Utah (Provo, Utah) in 1954, and moved from there to the Rockefeller Institute. 

The development proceeded along two lines: further improvement of the ion-exchange separation process and the construction of an instrument. With respect to separation, they selected a two-column system: a long (150-cm) column for the separation of the acidic and neutral amino acids, and a separate, short (15-cm) column for the basic amino acids. Because the basic amino acids remained on the first column in the standard run, the column had to be regenerated after each use. To ensure the possibility of continuous operation, two long columns were included in the system: while a sample was analyzed on one, the other was regenerated. In this way, when one analysis cycle was finished on one column, the second was ready for the next sample. Meanwhile, improved sulfonated polystyrene resins became available; they further fractionated the commercial product to obtain a narrower particle size cut. This method, based upon hydraulic flotation, was developed by P.B. Hamilton at the A.I. DuPont Institute of the Nemours Foundation (Wilmington, Delaware) (34). This improved packing permitted the use of higher flow rates without any loss of resolution. 

The analyzer contained the three columns in a thermostat. Reciprocal pumps maintained a constant flow of the various buffer solutions used as the eluent and for column regeneration. Other major components were photometric detectors used at 570 and 440 nm, connected to a multipen potentiometric recorder, and a vessel between the columns and the detectors in which column effluent was continuously mixed with a ninhydrin solution. In this way, the amino acids reacted with ninhydrin, forming colored compounds. The system also included other devices, such as deaerators, manifold, valves, and gauges. Figure 2 shows a simplified schematic of the system. The sample was added manually by pipette to the top of the column, but from then on operation was unattended. One full analysis of a protein hydrolysate took one day and a more complex physiological fluid sample took about two days. 

A preliminary description of the system was given at the April 1956 meeting of the Federation of American Societies for Experimental Biology (FASEB), in Atlantic City, New Jersey (35). The constructed breadboard system was used in a number of investigations at the institute. Finally, on February 28, 1958, the Rockefeller Institute team submitted two papers to 

, describing in detail the preparation of the column packing, the operation of the column, and providing a very detailed description of the apparatus and its components, even giving an engineering drawing of the photometer consisting of three units. These two papers, entitled "Chromatography of Amino Acids on Sulfonated Polystyrene Resins" (3) and "Automatic Recording Apparatus for Use in the Chromatography of Amino Acids" (4) were published in the July issue of the journal. They represent the start of automated amino acid analysis. 

Although the two papers provided a detailed description of the instrument constructed in the workshops of the Institute, probably only a few large laboratories would have been able to construct it. The Rockefeller Institute team realized this, and they turned to the Spinco Division of Beckman, with which they already had a good relationship, to transfer the design to production. 

Specialized Instruments Company ("Spinco") was founded in 1946 by M.C. Hanafin and E.G. Pickels to commercialize the analytical ultracentrifuge developed at Rockefeller Institute under the direction of Dr. Pickels, constructed originally to aid the isolation of pure polio virus (36). On December 30, 1954, Beckman Instruments acquired the company that from then on, operated as the Spinco Division of Beckman. 

Spinco built the first prototype of the amino acid analyzer — the so-called model MS (for Moore and Stein) — in the spring of 1958 (before the two papers were actually published), but originally, it had many of the usual problems associated with the transfer of a complicated design from research to production. At that time, Darrel Spackman left Rockefeller Institute and joined Spinco. He remained with the company for over three years and was involved in the improvements of the instrument; in 1962 he moved to the University of Washington (Seattle, Washington) and from then on, had academic affiliations. With Dr. Spackman's help, the instrument was soon "debugged" and from then on, the production of the model 120 Amino Acid Analyzer (its final designation) proceeded smoothly (Figure 3). Spinco also maintained a continuing contact with Dr. Moore, who regularly visited them (36). 

In the subsequent years, the technique and the ion-exchange resins were improved further, permitting the use of a single column (37) and significant reduction of the analysis time. Spinco introduced the Model 120B in 1963 and the model 120C in 1966. Figure 4 shows a typical chromatogram from this period: a complex physiological sample now could be analyzed in 11 h instead of the two days that were required originally in 1958. For a simpler protein hydrolysate, the analysis time could be reduced to 4 h or even shorter. (In Figures 4 and 5, we do not detail the analytical conditions.) 

Spinco introduced additional models of the amino acid analyzer. Soon other instrument companies also entered the field, most notably Hitachi (Japan) whose amino acid analyzer was based primarily upon the investigations of H. Hatano (1924–1998), a professor at Kyoto University (Kyoto, Japan). 

To illustrate the present status of amino acid analysis by ion-exchange chromatography, Figure 5 shows a typical chromatogram of a complex physiological fluid sample, with a total analysis time of 2 h. This chromatogram was obtained at the Chemical Laboratories of Missouri State Experiment Station at the University of Missouri (Columbia, Missouri). The laboratories have 10 Hitachi model L8800 fully automated amino acid analyzers in operation and handle about 1300 samples each month. Depending upon the sample complexity, each run takes 10 min to about 2.5 h. These data illustrate how advanced automated amino acid analysis has become. 

Today, ion-exchange chromatography is not the only method used for the determination of amino acids: it is complemented by gas chromatography (GC), LC, and capillary electrophoresis (CE). To conclude our story, a few words about these developments are given here. 

The use of GC for amino acid analysis became possible by the development of methods to prepare stable volatile derivatives, most notably the 

-butyl esters. At the University of Missouri, Gehrke and associates, in a decade of painstaking investigations, established the optimum methods for sample treatment and derivative formation, and for the optimum GC conditions permitting routine analysis (38,39). This work culminated in their investigation of lunar samples from Apollo 11 through 17 missions, for the possible presence of amino acids (40,41). 

In LC, the key step in sample treatment involved the preparation of less polar derivatives permitting the use of reversed-phase chromatography and enhancing the possibilities of UV, fluorescence, and mass spectrometric (MS) detection. More recently, methods for the LC analysis of underivatized amino acids, with MS detection, also have been developed. Today, automated LC systems for amino acid analysis are available from a number of instrument companies. 

Table I:  Identification of amino acids in Figure 5			

The use of CE for amino acid analysis is relatively new and is still growing. CE is very tolerant to biological fluids and less or even no sample treatment is necessary. UV absorbance, fluorescence, and electrochemical detection as well as combination with MS is used. 

	The literature of amino acid analysis is vast, and we refer here only to a couple of useful recent review articles (42,43), as well as to two general monographs dealing with the state of art of the various analytical techniques (44,45).			

With their pioneering work in the 1950s, Moore and Stein opened up an entirely new field for biochemists and it rightly can be considered as one of the great milestones in the century-old evolution of chromatography. 

The activities of Moore and Stein leading to the amino acid analyzer have been documented in numerous literature sources, such as their autobiography (46), their Nobel lecture and the associated material (47), as well as the recollections of C.H.W. Hirs (32) and M.J. Gordon (36), and our report is based upon these sources. We would like to express our particular appreciation to Ms. Pat Ashton of the Heritage Exhibit at Beckman-Coulter, Fullerton, California, for information on Spinco and their activities related to the amino acid analyzer, and for the photos used in Figures 3 and 4. Dr. Thomas P. Mawhinney, Missouri State Experiment Station, kindly provided us the chromatogram shown in Figure 5. We also should note the help of Dr. Robert L. Wixom, Columbia, Missouri, concerning information on early amino acid research, and of Ms. Rebecca Graves, education services librarian at the Health Sciences Library, University of Missouri at Columbia, in literature search. 

References to publications of Moore and Stein and their associates are given here to document the progress in their work leading to the amino acid analyzer. It was not our intention to present an exhaustive bibliography of their publications or to provide a review of the work of other scientists who contributed to the investigations of amino acids and proteins and to the development of analytical methods for amino acid analysis. 

MILESTONES IN CHROMATOGRAPHY

This installment of "Milestones in Chromatography" discusses the events leading to the development of the amino acid analyzer near the end of the 1950s at the Rockefeller Institute of Medical Research by Moore, Stein, and Spackman.

The Development of the Amino Acid Analyzer

MS — The Practical Art, this column's running rubric, reflects a knotty truth about using mass spectrometry (MS) in general analytical practice. Despite the well-considered physics and engineering involved in mass spectrometer technology — that is, in creating ions and passing them through a series of lenses — it nonetheless seems that the quality of one's results is as much the product of art as it is of science. 

Years ago, an acquaintance of mine was pursuing graduate studies involving the physics of the vibrating string. I do not know what came of his efforts, but I do remember observing with interest that the mathematical precision of the science used to predict the string's behavior and measure outcomes often went unappreciated by the end-users — the musicians. 			

The term "intonation," when applied to a musical instrument, denotes how well the instrument plays in tune with itself. For example, as long as you immobilize a string at a specific point along its length, it should theoretically produce the same note each time. Yet on a finely crafted guitar, striking a harmonic at the 12th fret by dampening its vibration with your finger versus fretting the string  (binding it behind the fret) to form the note can produce inconsistent results. In some positions along the length of the strings, one chord can sound perfect and another out of tune. These phenomena arise from the guitar's system of intonation, what musicians call its "temperament." 			

An acoustic guitar's frets maintain a fixed tuning. The guitar's 12th fret is used to gauge whether the instrument's "equal temperament" (a system that divides the chromatic scale into 12 equal half-steps) produces the desired intonation. So for any guitar, what frustrates some refined ears is that the intervals between notes are not always perfect, or "pure," making it hard to accept idiosyncrasies in an instrument designed for precision. So, each guitar maker tries to create its version of optimal performance knowing well the physics that underlie the vibrating string, but knowing also that their instruments' particular brand of idiosyncrasy will play well in the hands of some musicians but not all. 

Like musical instruments, mass spectrometers operate with the same constancy of physics. Source and ion optics interface an atmospheric ionization inlet with a mass spectrometer, delivering sensitivity and economy of function.      Producing ions from analytes must obey the same physical laws, such as the (still-debated) formation of electrospray ions, gas dynamics, and desolvation to produce ions in the gas phase and conserve them via the source and ion interfaces. This is true regardless of whether those interfaces are attached to an ion trap, quadrupole, or time-of-flight instrument. Yet results often differ when parallel experiments are attempted using different instrument designs. Or worse, the same experiment is attempted on different days using the same instrument. So you might ask: How can this be when electrospray ions created from the same analyte are identical?  

Apart from differences between instruments themselves, artifacts occur such as sample degradation and other vagaries of the sample handling process. Even more significant in terms of guaranteeing unequal results are the idiosyncrasies of practitioners. People can differ in their approach to calibration and tuning their instruments, and to some degree an assessment of a mass spectrometer's "tone" can be subjective. Producers of mass spectrometers design their instruments to work well within specific parameters. The constraints vary among manufacturers — otherwise all four major instrument manufacturers would offer the same design. Assessing the common points as well as the differences, real and alleged, can appear to be quite an art. 

Most in-service liquid chromatography–mass spectrometry (LC–MS) courses fail to address specific operational problems, and retaining consultants can be financially prohibitive. So one source that practitioners often turn to for advice is online discussion postings. These are perhaps the best way to appreciate that the rest of the MS world shares problems similar to yours. Yet, it is often a bit vexing to find the answers to those problems are not so clear-cut. With that in mind, I want to focus on sources to which you can go to find help, on helpful hints, and some anecdotes on spectral interpretation in upcoming columns. 

One well established source (since 1995) is the discussion group (SciTechniques), maintained by moderators David Bostwick (Georgia Institute of Technology) and Sarah Shealy (Georgia State University) at http://

. John Bartmess (University of Tennessee) offers a (more or less) weekly reprise of the discussions at https://

. Perusing the discussion thread can be useful and sometimes even amusing. Perhaps, best of all, it is free. 

I edited some samples of the exchanges, hoping to improve their coherence and also to ensure against unintended emphasis on either products or individuals. The exchanges lie buried as discussion threads, and digging them out can prove daunting. But it is simple enough to subscribe to the mailings, and have them show up periodically in your inbox. The helpful hints range from the obvious to what appear the beginnings of an advanced-degree thesis. 

Modifier, Linearity, and Response Problems

This plea for help is by a chromatographer with a modifier problem: 

I have several positively charged (amine) compounds, which, for chromatographic reasons, must be analyzed using a basic (triethylamine) buffer mobile phase. Because the mobile phase is only 0.1% triethylamine and about 50% acetonitrile, I thought I could simply acidify the effluent with a postcolumn infusion of formic acid (10:1 effluent–formic acid) via a tee. Unfortunately, I cannot see anything, even at high sample concentrations. Where am I going wrong? 

Have you ever gotten an LC–MS signal from your substances? If you have, was triethylamine then included in the mobile phase?  

(Comment: Triethylamine is highly ionic and can compete unfavorably with your analyte, especially if its concentration is much higher than the analyte's. You might find some additives like triethylamine work better in negative rather than positive mode. Triethylamine can also present a persistent [m/z 102] background ion. Surprisingly, many do not first try to omit modifiers from their chromatography when transferring to the gas-phase environment of LC–MS from the condensed phase world of UV-only. Sometimes uncharted changes in equilibrium can and do take place in the transition. Sometimes it can be impractical — for instance, when it would require showing equivalence with an existing method based upon an accepted new drug application — but it can nevertheless provide a simplified comparison.)

Are you sure you are introducing the intended percentage of formic acid? 

Do the interface and postcolumn device work?  

(Comment: Anytime a novel — that is, a less-than-satisfactory — response is encountered, operators must verify that the system is working correctly. An easy way to do this is to include a small amount of "known good" analyte to the postcolumn infusion. At the very least, every practitioner should have a sample that gives a well-documented response at hand.)

This spectrometrist has a linearity problem: 

I have problems with the linearity of some compounds while using LC–MS in negative ion mode. The calibration curves are very nonlinear. The concentration is in the same range as that of other linear compounds. The compound is an acid, like the other compounds, but others do not exhibit this problem. I have tried several eluent compositions, but without success.  

(Comment: The distracted analyst comes to various seemingly reasonable solutions, such as considering what eluent additives he or she could try that would improve the method. Is acetic acid better than formic acid? Is methanol or acetonitrile better? Yet the solution could be much easier.)

This could be caused by adsorption (compound-dependent or analyte-dependent). Changing from stainless steel to PEEK might improve performance. 

I had similar problems when I worked with a [triple quadrupole]. We could not find the problem [replacing solvent type–acid, and so forth] and an engineer suggested we lower the temperature of the source. We were running at 180 °C and lowered it to 150 °C. That solved the nonlinearity problem. Sometimes solutions are very simple.  

(Comment: Amen. And sometimes they are not. Indeed, not all linearity problems can be resolved in the discussion group format.)

This chromatographer missed both linearity and response: 

I am hoping that someone can help me. I am developing a method to analyze alkylphenol ethoxylates by electrospray LC–MS, and I ran into a problem. The analytes in my method give exclusively (M+Na)

  ions, so I am monitoring those. During a sequence of three injections the chromatogram baseline remains stable for a period of time. Then it drops for a few minutes only to rise again. Any peaks during the baseline drop are much smaller than where the baseline does not drop. This plays havoc with my quantitation, especially when the internal standards are eluted during this time. 

The baseline suppression does not happen at the same time during each of the LC runs. It migrates all over the place, showing no discernable pattern. Because the depression of the peak height is much greater than the magnitude of the baseline suppression, I am assuming that what is happening is a disruption in the ionization process, leading to low and irregular ion formation and sampling efficiency. The first thing I thought was that this was perhaps a competition effect from coeluted interferences. So, I put 50 μM of sodium acetate in the mobile phase. All this did was junk up the spectrum with a bunch of clusters and reduce the overall sensitivity (10 μM seemed to be okay, though). In any case, the baseline problem persisted. My next thought was that it might be a problem with really nonpolar stuff sticking to the column and carrying over into the next run (my runs are short, about 15 min) because I was running isocratic. Nope, I inserted a 15-min flushing–equilibration step, and this still did not get rid of it. 

The problem seems to occur most often with samples. But when it happens with standards, it really ruins quantitation. It does not happen in negative mode; I get a nice baseline. Does anyone have an idea about what is going on here? Surely I am not the only person who has ever seen this problem. 

(Comment: In this case the submitter's reasoning was absolutely on target. The problem is widely recognized as the bane of quantitative MS in complex mixtures, and numerous publications are available for guidance. If I were to recommend one, it would be "Ion Suppression: A Major Concern in Mass Spectrometry," a tutorial written by Lori Lee Jessome and Dietrich Volmer, which appears in an upcoming issue of LCGC. It serves as not only a very comprehensive overview of what painstakingly has been collected in recent practice, but as a collection of references to pursue as needed.) 

We will examine this topic in detail soon, in another column. For the moment, though, consider that atmospheric ionization work often can create adducts that range from the common to the exotic, making spectral interpretation and quantitation difficult. 

A case of mystery ions — common sodium in a not-so-common mode: 

Could anybody give me some clues about the mechanism of sodium adduct formation under atmospheric pressure chemical ionization (APCI) so that I can control it? The Na

  adduct is commonly formed in solution in electrospray ionization (ESI). Is it possible the Na

  adduct can form in the gas phase by APCI? 

(Comment: The issue remained unresolved after a long but interesting and informative exchange. In the ensuing exchange, which I summarize here, the consensus was that sodium adducts form in solution requiring a considerably higher voltage rather than forming in the gas phase.)

Perhaps because of APCI's higher probe temperatures, certain analytes can be desorbed into the gas phase, resulting in a mixed electrospray and APCI spectrum. [A mixed spectrum is not uncommon and is in fact being exploited in various mixed or multimode ionization source designs]. If it is of interest to determine which mechanism predominates, a high-speed switching design like the ESCi multimode ionization source (which comes standard on all Premier products from Waters Corporation, Milford, Massachusetts) can be employed to characterize the response between the two mechanisms with good, reproducible fidelity. The Turbo V design (MDS Sciex, South San Francisco, California) offers similar common source abilities. If your purpose is simply to ionize the majority of molecules in a solution, a simultaneous design such as the Agilent (Santa Clara, California) multimode will suffice. All of these devices are of course limited by each vendor for use with their particular instrument line. 

You can minimize or avoid sodiation by using plasticware instead of glassware. Glass surfaces produce infinite amounts of sodium, which seems to affect some compounds readily. Also, you might see in the sample history that sodium buffers were used in the prep. 

Because the concentration of sodium in solution is not fixed, using sodium-adducts for quantitation can present problems. One reader suggests adding 1 mM sodium acetate to the eluent and then running in ESI mode rather than APCI mode. Even though APCI is preferred, studies indicate it is less susceptible to suppression. This suggestion is not so extreme these days: Z-spray and similar designs minimize contamination and clogging. 

You might find that expending a bit of effort to hold contamination to a minimum improves downstream interpretation and response. Two major sources of contamination in reagent prep are the containers used for preparing solvents and the transfer devices. Detergents and surfactants can increase propagation of ESI ions and therefore sensitivity. Unfortunately, the increased sensitivity is for the surfactant contaminants, not the analytes. So, as an old hand at electrochemistry once told me, do not use anything in your preparative practice that you would not want to see in your spectral background. Segregate LC–MS containers and measuring devices for LC–MS use only. Do not use detergents, and rinse containers repeatedly before setting them aside to dry. Good practice also dictates preventing acids and plastics from coming together. 

Readers' remarks, though they sometimes fail to offer technical help, always remind us that we do not face these problems alone — cold comfort, perhaps. So if your problem seems instrument-specific, you likely will find someone who can sympathize. Just remember that LC–MS practice can be quite a complex orchestration of sample preparation, instrument condition, and suitability for the intended use, not to mention your own level of understanding — all of which are evolving at different rates. Clearly, if there is one benefit to point out from among LC–MS courses, reference literature, and other self-help sources, it is the decision you make before purchasing your instrument. Weigh the accessibility and history of your service and sales contacts well in the equation. Unless you are blessed with a colleague who is an experienced operator — which aside from major pharma sites can be rare — these folks are your best allies. 

 "MS — The Practical Art" Editor Michael P. Balogh is principal scientist, LC–MS technology development, at Waters Corp. (Milford, Massachusetts); an adjunct professor and visiting scientist at Roger Williams University (Bristol, Rhode Island); and a member of LCGC's editorial advisory board.

MS - The Practical Art

This month's column explores the idea that despite the well-considered physics and engineering involved in mass spectrometer technology, it nonetheless seems that the uality of one's results are as much the product of art as they are of science.

From the days of my involvement in the Boy Scouts, both as a youth and a leader, I have no trouble remembering the motto: "Be Prepared." This also is a good motto for those of us involved in using liquid chromatography (LC) methods to get our daily work accomplished. Just because LC is, by some accounts, the most widely used analytical technique in the world, does not mean that it is the most reliable technique. Every LC method will fail, sooner or later. One of the signs of a competent chromatographer is the ability to get that failed method back up and running quickly. Although wisdom and troubleshooting strategies are gained with years of experience, there are some fairly simple practices that can trump years of experience. This is the subject of this month's "LC Troubleshooting" installment. These strategies are all about being prepared — what you can do in advance that will shorten the down time when a method does fail. 			

The International Conference on Harmonization (ICH) provides a series of documents that provide guidance for workers in the pharmaceutical industry when developing, validating, and using LC methods for the analysis of pharmaceutical compounds and their impurities. Two key documents for the current discussion are "Q2A Text on Validation of Analytical Procedures" (1) and "Q2B Validation of Analytical Procedures: Methodology" (2). Both of these documents, as well as many other regulatory documents, can be found at the Food and Drug Administration's (FDA) website listed in the references. Although these documents are prepared specifically with agency-regulated pharmaceutical methods in mind, the information they contain applies equally well to most LC ethods for routine analysis.							

				The word that describes the task at hand is "robustness." Q2A defines robustness as follows (1):			

				"The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage."			

				This is complemented by a statement about robustness testing from Q2B (2):			

"The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters." 

Both quotations refer to "deliberate variations in method parameters." This implies that some formal testing should be done. We need to deliberately test what will happen when the LC method conditions are changed in a way that might happen by accident or other normal variation in the method. Let's consider which parameters might be good to test. 

Most methods will exhibit more sensitivity to changes in some experimental parameters as opposed to others. What should be tested? Q2B lists mobile phase pH, mobile phase composition, different columns, temperature, and flow rate. These all are parameters that are tested easily by making deliberate changes in the method. We will look at each of these parameters in a moment. Depending upon the method, one or more of these might not apply — and additional parameters can be important.  

The important practice here is that testing should occur before the method is put into routine use. This would be before validation, if you formally validate the method. Testing helps you identify the soft spots in the method, select the best conditions for operation, and develop troubleshooting strategies to correct problems when they do occur. If you know, for example, that the method is very sensitive to small changes in mobile phase pH, you will take extra care when adjusting the mobile phase pH, so as to avoid problems. If you know that the method will perform adequately at any temperature between 30 °C and 40 °C, you can choose to operate at 35 °C so that some tolerance to error is built into the method. If you know that the resolution for a critical peak pair gets worse with lower percentages of acetonitrile in the mobile phase and better when more acetonitrile is used, you will know how to adjust the mobile phase when you see the resolution decrease. All of this knowledge will improve the quality of the method, as well as your skills in running it. Finally, when testing the effect on the method of changing a variable, do not look just at retention time — the resolution of peaks of interest generally is more important than retention. 

If your sample contains ionizable compounds, the mobile phase pH will be a variable that will have a profound effect on retention times. For reversed-phase methods, it generally is advantageous to work at a mobile phase pH that converts the analyte(s) to the un-ionized form. Under these conditions, the analyte will be neutral and, thus, less polar, so it will be more strongly retained. Ionic compounds often are retained poorly under reversed-phase conditions. For an acid, more than 99% of the compound will be un-ionized at 2 pH units below the p

. The same holds for bases at 2 pH units above the p

. Often the situation is complicated by the presence of acids and bases in the same sample and by multiple p

s for various analytes, so that a single mobile phase pH will not convert all compounds to the un-ionized form. In addition, the column places some practical restrictions on pH selection. For typical silica-based reversed-phase columns, hydrolysis of the bonded phase takes place at pH < 2, and the silica dissolves at pH > 8. There are specialty columns that tolerate conditions outside the 2 < pH 

The net effect of all of these variables is that the mobile phase pH often is closer than 2 pH units to the p

 of at least some compounds in the sample. This means that the method will be susceptible to changes in pH. Once you have determined the best pH for the method, check the effects of a change in pH on the separation. I recommend checking at least ±0.5 pH units from the desired pH. When you find the pH range that is tolerated, generally it is best to run the method in the middle of the range so that some small variation in pH can be tolerated.  

A couple of other pH-related comments should not need to be made, but it is surprising how often they are ignored. First, use a buffer that is effective at the pH you have chosen. As a rule, a buffer is effective ±1.5 pH units from the pKa of the buffer. For LC methods using UV detection, most commonly phosphate is used for 2 < pH < 3.5 and 6 < pH < 8, whereas acetate is chosen for 3.5 < pH < 6. Also, remember to adjust the pH of the buffer before adding organic to the mobile phase — pH meters do not give the same readings when organic solvents are present as they do in water. Finally, the mobile phase pH and the p

 of sample compounds will change when the temperature is changed, so be sure to thermostat the column. 

During method development, usually you will identify the organic solvent that gives the best separation by testing acetonitrile, methanol, and sometimes tetrahydrofuran. Then you will adjust the mobile phase organic concentration to get the best isocratic separation or the starting and ending concentration plus the slope for the best gradient separation. Data you gather during this development process can be used to help you understand the sensitivity of the method to a change in the organic composition of the mobile phase. I often refer to "The Rule of Three," which states that a 10% change in organic solvent will result in approximately a three-fold change in retention factor 

, or for well-retained compounds, retention time. This converts to a 10–20% change in retention for a 2% change in organic. For your method, you should quantify this approximate rule, so you will know the impact of a small error in mobile phase preparation. I recommend checking ±2–5% organic for isocratic separations. For gradients, check -2–5% initial organic with +2–5% final organic, and vice versa. As with the other parameters, it generally is best to center your method conditions in the middle of a region where a small increase or decrease the in the parameter setting can be tolerated.  

Whereas the mobile phase composition can be varied in a continuous manner and is under the operator's control, column changes are discrete, and under the control of the column manufacturer. Eventually, the LC column will no longer provide a satisfactory separation and must be replaced. You would like to be sure that the next column you buy will give an equivalent separation — thus, the "use column X, or equivalent" statement in many methods. Just what is equivalent? First, you should check to make sure the column you have chosen will really do the job. A check of three columns is a good place to start. This should include two columns from the same batch and one from another batch. By batch, I mean the silica particles to which the bonded phase is attached. This test will give you an idea of the column-to-column variability you will encounter under normal conditions. Column reproducibility is much better with today's high-purity, type B silica columns than the type A columns that were more common 15 years ago. Hopefully, your understanding of the effect of the other variables (pH, percent organic, temperature, and so forth) will help you decide how to adjust the conditions to achieve a successful separation when minor changes in the column are encountered. 

Depending upon the method and your company policies, you might need to have a second source of columns, in case the primary supplier can no longer provide them. If you have been in the LC business for very long, you know that all C18 columns are not created equal. A separation on one manufacturer's column might look substantially different on one from another manufacturer. Recent publications on the comparison of column selectivity (for example, references 3 and 4) and column vendor's comparison guides can be used to help guide you in selection of a truly equivalent column as a backup for your primary column.  

I continue to be amazed at how many people operate their LC systems under "ambient" conditions — in many laboratories this can vary by 5 °C or more over the course of a year, and sometimes over a day's time! A good guideline is that retention for reversed-phase separations changes by approximately 2% for each 1 °C change in temperature. It is easy to see that in many laboratories, retention time changes of 30 s or more can occur during a day's run. This could cause a peak to drift out of a data system peak window or to be confused with another closely eluted compound. Furthermore, changes in selectivity as a result of temperature changes can compromise the resolution of a method, especially for ionic compounds. You should include temperature control as a standard part of every LC method. I recommend starting with a temperature slightly above room temperature, so that it is easy to control, such as 35 °C. When you have selected the final temperature, check the impact of the temperature on the separation by checking the method at ±5 °C from the normal setting. Select the final conditions in a region where a few degrees fluctuation will not cause the method to fail. 

For most isocratic applications, mobile phase flow rate merely affects retention, so little impact on resolution will be seen. In gradient elution, on the other hand, flow rate can result in changes in selectivity. However, in either case, the change in flow rate will be much less than 10%, except in cases of severe leaks, check valve failures, or improper instrument settings. For modern 3–5 μm particle columns, these small changes in flow rate will not have any noticeable effect on peak width. Thus, normally encountered flow rate changes are unlikely to have any negative impact on the separation, but it is easy to check to determine the effect of a ±10% change in flow rate. 

Depending on company policy, government regulations, and personal preference, you might or might not have to include robustness testing as part of the validation. If you have shown in your method development process that changes in one or more variables do not negatively impact the method, that is probably ample evidence to obviate testing of the variable during validation. Some workers like to check each variable independently, whereas others use a multiparameter experimental design approach that will minimize the number of experiments required to evaluate the effect of the various parameters. In any event, it is a good idea to include some robustness testing in the method validation process. By showing the effect of changes in the various parameters, and including these data in your method document, you can make life easier on the end user of the method. For example, peaks A and B might move together as the column ages, but you have shown that A and B can be pulled apart by an increase in temperature, as little as 3 °C. Now, when you see the problem, you know how to adjust the method to keep it operating properly — and still be within the allowable method conditions. 

If you do your method development with a goal of gaining a better understanding of how peaks move relative to each other as conditions are changed, you will find that troubleshooting will be much easier. You will know what small change in one or more parameters will be required to bring the method back into specifications. A good system suitability test will allow you to evaluate the method performance before you run valuable samples. If you include allowable adjustments as part of the method document, you can make such changes when system suitability fails, and be back up and running quickly. If you do not build flexibility into the method, much more work will be required to test and document even small changes in the method. 

Some people look at the ICH recommendations for robustness testing as placing extra requirements on us that inhibit us from doing the important part of our work. A better perspective is that they encourage us to produce a better product — our method. If we do "good science," we should not have to worry so much about regulatory restrictions. 

 "LC Troubleshooting" Editor John W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of LCGC's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

(1) Guideline for Industry. Q2A Text on Validation of Analytical Procedures. http://

(2) Guidance for Industry. Q2B Validation of  Analytical Procedures: Methodology. http://

(3) J.W. Dolan, A. Maule, L. Wrisley, C.C. Chan, M. Angod, C. Lunte, R. Krisko, J. Winston, B. Homeier, D.V. McCalley, and L.R. Snyder, 

(4) L.R. Snyder, J.W. Dolan, and P.W. Carr, 

LC Troubleshooting

John Dolan ponders te question of whether or not you will be prepared when that method eventually fails.

It's Gonna Break

Chromatographic situations are created for varying peak resolution and relative peak size. In this case, the peak size of the smaller peak is always at least 5% of the larger peak, and resolution is varied from 4.0 to 1.0. All separations are then integrated, using both area and height, by four different baseline methods: drop, valley, exponential skim, and Gaussian skim. Integration errors are calculated using reference calibration injections. The results demonstrate that the drop and Gaussian skim methods produce the least error in all situations. The valley method consistently produces negative errors for both peaks, and the skim method generates a significant negative error for the shoulder peak. Peak height is also shown to be more accurate than peak area. As the relative peak size increases, resolution below 1.0 will generate unacceptable errors, and resolution greater than 1.5 is necessary to minimize integration errors. 

Integration of chromatographic peaks (determination of height, area, and retention time) is the first and most important step in the data analysis part of all chromatography-based analytical methods. This information is used for all subsequent calculations, including construction of calibration curves and calculation of unknown concentrations. Clearly, any error in measurement of peak size will produce a subsequent error in the reported result. 

Analysts have several choices for integrating peaks. Figure 1 illustrates the four most common options for drawing the baseline between two peaks: drop, valley, exponential skim, and Gaussian skim. The drop method (Figure 1a) involves addition of a vertical line from the valley between the peaks to the horizontal baseline, which is drawn between the start and stop points of the peak group. The valley method (Figure 1b) sets start and stop points at the valley between the peaks, thus, integrating each peak separately. Skim procedures separate the small peak from the larger parent with separate baselines. The parent peak is integrated from its starting point to the apparent end of the peak group. The small peak's baseline starts at the valley between the peaks, and ends when the signal nears the baseline. The area "under" the skimmed peak is added to the parent peak, not the skimmed peak. This approach has been described also as a tangent integration method, and the small peak variously labeled a skim, shoulder, or rider peak. 

Several variations of the skim procedure are possible. Early integration algorithms drew a straight line from the valley to the end of the peak. Figure 1c shows an exponential skim baseline. An exponential function is used to create curvature in the skim line, in an attempt to approximate the underlying baseline of the parent peak. While the exact procedures used to construct this line are a proprietary part of modern software algorithms, they all employ the same basic approach — use an exponential function to draw a curved baseline under the skimmed peak. More recently, another skim procedure has been developed, as shown in Figure 1d. Although referred to as a new exponential skim method, it will be described here as a "Gaussian" skim, because the intent is to more accurately reproduce the Gaussian shape of the parent peak.  

For any of these methods, analysts must also choose between measuring peak size by either area or height. It is clear that these integration options are likely to generate significantly different analytical results, and analysts must decide which approach provides better accuracy. 

Despite the importance of this issue, clear guidelines are difficult to find in the published literature. Snyder and Kirkland (1) discussed the relative quantitative errors for symmetrical peaks, suggesting that peak height with the drop method was more accurate for poorly resolved peaks, but area measurements were more precise. These recommendations seem to have been ignored largely by the chromatography community, as the drop method with area is used almost exclusively today. Foley (2) published correction factors for pairs of tailed peaks, allowing estimation of their true areas. More recently, Meyer published a series of papers (3–7) dealing with several integration issues. Peaks of varying symmetries, resolutions, and size ratios of as much as 10:1 were prepared by calculation, and integrated using the drop method and area (3), resulting in errors of 40% in some cases. In another study of 10:1 peak ratios, the height method was more accurate if the small peak was eluted first. However, when the small peak is eluted second, the drop method produced a large positive error and the skim method produced a large negative error (4). These general recommendations were extended to even smaller peaks (that is, 1000:1 ratios), although the better integration method for a small second peak varied with the relative peak widths (5). The effects on enantiomeric analysis and calibration curves were reported (6), and a mathematical procedure for quantification of shoulder peaks was developed (7). Again, the recommendation that peak height is more accurate than area has not been put into general practice. 

Although these studies provided some excellent general guidance, they only considered the drop method when the first peak was small, and only added straight skimming as an option for a second small peak. The valley and skim methods were not evaluated. When significant errors were noted, explanations for the errors were incomplete or missing, especially for the skim method. 

This study seeks to provide some guidelines for analysts by creating several peak resolution situations. The resolution between two peaks is varied from 4.0 to 1.0 through changes in operating conditions. The relative size of the two peaks also is changed from nearly equal size to approximately 5% of the other peak. Each peak pair is then integrated using each of the four baseline options, using both area and height, and integration errors are calculated through the use of reference calibration injections. This study is concerned with situations in which the size of one peak is at least 5% of the other peak. A separate study will report results when one peak is significantly smaller than the other (8). 

Finally, all resolution situations described here were created using a liquid chromatography (LC) system. However, the peak shape observed is typical for any well-behaved chromatographic system, so application to gas chromatography (GC) separations should be valid. Although only one data system was used for this study, the author believes that all modern chromatography data systems process data using similar procedures, and only minor differences would be produced by other software packages. The general conclusions still should be valid. 

 All chromatographic experiments were performed using a model 1100 HPLC, equipped with a binary pump and degasser, autosampler, column heating compartment, and diode array detector (Agilent Technologies, Wilmington, Delaware). The system was controlled by ChemStation software (Agilent Technologies, Palo Alto, California, Version B.01.03). A 100 mm × 4.6 mm Hypersil C18 column, packed with 5-μm particles, was used for this study. 

In part I of a two-part series, the author introduces several methods to reduce error in your chromatographic analyses.

LCGC North America-04-01-2006

Market Profile: Life Science Research

Peaks of Interest

New Chromatography Columns and Accessories at the 2006 Pittcon Conference: Part II

The Development of the Amino Acid Analyzer

Q&A

It's Gonna Break

Integration Errors in Chromatographic Analysis, Part I: Peaks of Approximately Equal Size