Part II of our yearly report on new products introduced at Pittcon. This installment of "Column Watch"covers gas chromatography columns, supercritical fluid chromatography columns, and products for sample preparation, including small benchtop instruments.

Pittcon 2014 Conference and Expo (less formally known as the 65th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy) was held once again in the gigantic McCormick Place in Chicago, Illinois, on March 2–6, 2014. The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2014. In some cases, products that were introduced during 2013 but after Pittcon 2013 (1,2) may be included for reasons of completeness. The information is based on manufacturers' responses to a questionnaire mailed in early 2014. 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 were introduced in Chicago. 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. 

In last month's coverage (3), I described new introductions in the areas of HPLC columns for reversed-phase LC, hydrophilic-interaction chromatography (HILIC), ion-exchange and ion chromatography, and size-exclusion chromatography (SEC). Specialty columns for specific types of analyses were also covered last month. This month, I look at gas chromatography (GC) and supercritical fluid chromatography (SFC) columns, sample preparation products, and hardware, accessories and small tabletop instruments, mainly for sample preparation. For a synopsis of the overall trends seen at Pittcon, please refer to part I (3). 

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 may be used for general high performance liquid chromatography (HPLC) separations and for sample preparation as well, but their main emphasis is for biological samples.   

Gas Chromatography and Supercritical Fluid Chromatography

The year 2014 was the sparsest ever for new GC column introductions. Only two manufacturers supplied data on their new columns for Pittcon: Agilent Technologies and J&K Scientific.  

Agilent's offerings included a new line of porous open tubular (PLOT) capillary columns called PLOT PT. These unique columns virtually eliminate particle shredding through an integrated particle trapping technology on both ends of the column — giving the advantage of no need for cumbersome (and leaky) unions or downstream filters. The integrated particle trap is built directly into the column as one continuous length of fused silica. Overall, signal spiking and unnecessary downtime because of loose particles in the flow stream is less of a concern in the analysis of light gases and volatile organic compounds (VOCs), which are major applications of PLOT columns.  

Agilent's second introduction in the GC area was the J&W DB-Sulfur SCD column. With dimensions of 40, 60, or 70 m × 0.32 or 0.53 mm and a film thickness of 0.75–4.3 µm, the DB-Sulfur SCD column is designed to work with sulfur chemluminescence detection (SCD) to provide lower bleed performance than polydimethylsiloxane (PDMS) phases such as ASTM D 5623 and 5504. These phases typically bleed causing SCD ceramic tube fouling and thus more downtime and maintenance requirements. The new phase also provides improved peak shape compared to older phases. Custom configurations are available. To illustrate the capability of this new column, Figure 1 shows a separation of an actual cracked gasoline sample, along with sulfur compound standards that contain about 63.5% aromatic hydrocarbons and 25% nonaromatic hydrocarbons. Monitoring sulfur compounds in petroleum-related products is very important to protect expensive catalysts and to ensure product quality. Individual sulfur compounds in the cracked gasoline sample were identified by retention times and confirmed by gas chromatography–mass spectrometry (GC–MS). In the example of Figure 1, the very selective Sulfur Chemiluminescence Detector (SCD, Agilent Technologies) was used. In should be noted that trace levels of these volatile sulfur compounds are very difficult to detect and successful analysis requires a very inert flow path and a low-bleed stationary phase. 

 Figure 1: Chroma tograms of (a) a cracked gasoline sample and (b) sulfur compound standards. Column: 60 m  Ã 0.32 mm, 4.2-Âµm df Agilent J&W DB-Sulfur SCD; carrier gas: helium, constant flow at 2.8 mL/min; inlet temperature: 275 Â°C; split ratio: 10:1; oven temperature program: 35 Â°C for 3 min, 35â250 Â°C at 10 Â°C/min, then 250 Â°C for 10 min; injection volume: 1 ÂµL. (Courtesy of Agilent Technologies.)			

J&K Scientific is a Canadian company based in Nova Scotia that makes specialty columns. The company's newest introduction is the NSP-EUPAH GC capillary column. This column has a wall-coated proprietary stationary phase designed to separate EU and US EPA priority polynuclear aromatic hydrocarbon (PAH) analysis. The column can provide baseline separation of the EU 15 + 1 PAH on short columns of lengths of 8–15 m with analysis times of 25–40 min. Some unique characteristics are complete separation of triphenylene from chrysene (resolution >10) and benz(a)anthracene. The column can also provide a separation of critical isomer pairs of US EPA Method 610 Priority Pollutant PAH method. In all, separation of 32 EU and US EPA priority compounds with interfering compounds and isomers can be achieved.  

One dedicated column for SFC was introduced by ES Industries. Typical of columns specific to SFC, the GreenSep Triamine column resembles a regular HPLC column but with a special triamine bonded phase that is not normally used in LC. The silica-based bonded phase material (120 Å pore size) can be purchased in stainless steel packed columns in sizes ranging from microbore to preparative, as well as in guard column format and in bulk. Particle sizes range from 1.9 µm to 20 µm. It is recommended for the separation of polar compounds such as amines without the use of additives in the mobile phase. 

As indicated in Table I, Pittcon 2014 was another productive year with 17 new sample preparation products or families of products displayed. Not counted here are the sample preparation accessories (Table II) or sample preparation benchtop instruments (Table III) that are covered later. The most popular application areas included protein and phospholipid removal from biological fluids and pesticides and toxins in food. Formats included cartridges, 96- and 48-well plates, stainless steel columns, and pipette tips. 

Many of the products were for solid-phase extraction (SPE) and just about every mode (reversed phase, strong and weak anion and cation exchange, affinity, molecularly imprinted, adsorption and normal phase, partition, and mixed mode) was represented. Various media were introduced at Pittcon including polymer-based media, graphitized carbon black, carbon on Florisil, pepsin on silica hybrid, silica-based bonded phases, bare silica, zirconia, and diatomaceous earth. 

Table II: Chromatography and sample preparation accessories			

This year, sample preparation introductions were dominated by the 96-well plate format; obviously these products are destined for those interested in the application of automation. Very few chemists would have the patience to manually work with such tiny wells performing SPE, filtration, protein crashing, and other liquid-handling operations. If one doesn't have a robotic system, there are manual handheld multichannel pipettors that relieve some of the tedium. One interesting plate that was introduced by Tecan has an immobilized stationary phase inside each well. Thus, a form of SPE or liquid–liquid partition can be performed by using the well as a small extraction vessel. An aqueous solution is first introduced and, depending on the stationary phase, analytes can be sorbed onto the immobilized coating. Then, after a washing step, an organic solvent is introduced that desorbs the sorbed analytes, which then can be injected into a chromatographic or electrophoretic system. 

Table III: Sample preparation instrumentation			

Both single-tube and 96-well plate filtration devices are available for protein "crashing" experiments. Rather than using SPE and spending time developing methods, the trend in recent years has been to precipitate (crash) proteins out of biological fluids (such as plasma) by adding an organic solvent like acetonitrile. The precipitated proteins can be removed by centrifugation (somewhat time consuming) or can be filtered through a membrane filter and the supernatant can be analyzed by LC–MS-MS for the drugs and other compounds. Several new products were introduced that not only perform the protein precipitation but will also remove troublesome phospholipids by use of special media, sometimes based on zirconia or titania but more often referred to as proprietary, that will remove or substantially reduce the lipid content. Phospholipids are notorious for causing signal suppression in MS and their removal provides improved sensitivity and selectivity. To illustrate the mechanism of phospholipid retention, Figure 2 shows the design of the packed wells of a 96-well plate (left-hand side) along with a depiction of the interactions of a phospholipid with the zirconia-coated silica surface (right-hand side). 

Figure 2: Retention mechanism of hybrid phospholipid SPE sorbent for separating hydrophobic analytes from phospholipid contaminants in biological sample matrices. (Courtesy of Supelco/Sigma-Aldrich.)			

Additional products for QuEChERS were introduced at Pittcon 2014. This method is an emerging technique that is becoming increasingly popular in the area of multiresidue pesticide analysis in food and agricultural products. In QuEChERS, food or agricultural samples are first extracted with an aqueous miscible solvent (such as acetonitrile) in the presence of high amounts of salts (for example, sodium chloride and magnesium sulfate) or buffering agents (such as citrate) to induce liquid phase separation and stabilize acid- and base-labile pesticides. After shaking and centrifugation, an aliquot of the organic phase is subjected to further cleanup using SPE. Unlike traditional methods using SPE tubes, dispersive SPE (dSPE) cleanup is conducted by mixing bulk amounts of SPE sorbent and additional magnesium sulfate with the extract. After sample cleanup, the mixture is centrifuged and the resulting supernatant can either be analyzed directly or subjected to minor further treatment before analysis.  

A most interesting product for dSPE was shown. Pigments in fruit and vegetable samples are mostly removed during the second QuEChERS extraction step by the use of graphitized carbon black (GCB). This substance is rather messy to work with because it sticks to the edges of the extraction container and sometimes particles remain suspended, but a bigger problem is that, besides removing chlorophyll and other pigments, it also removes planar pesticides. An additional step with toluene extraction is required to recover these pesticides, but some of the pigment also gets back into the solution during the process. United Science introduced the CarbonX COF product, where the carbon is deposited on a Florisil substrate. It is denser than GCB and thus settles out of solution, but its main claim to fame is that it will not extract the planar pesticides. In official methods such as the AOAC 2007.01 QuEChERS method, it can be substituted directly for GCB. 

Mycotoxins are particularly harmful in our food supply. Normally, expensive and problematic selective immunosorbents are required for their isolation from food extracts. Two new products appeared at Pittcon that do not require immunoaffinity methods. One is based on molecularly imprinted polymers, which is an area that is getting more attention; these sorbents are among the most very selective (see Table I). To illustrate the simplicity of some of these newer methods for mycotoxins analysis, Figure 3 provides the workflow for the isolation of mycotoxins from food samples using simple flow-through Isolute Myco SPE columns from Biotage. A large number of mycotoxins have been quantified in a variety of matrices. Methods have been developed that meet the maximum residue limits set down by EU and US regulations in terms of detection limits, percent relative standard deviation (%RSD), and recoveries. 

Figure 3: Workflow for solid-phase extraction of mycotoxins from food samples using the Isolute Myco sorbent. (Courtesy of Biotage.)			

Chromatography and Sample Preparation Accessories

New hardware products and accessories designed to make the life of the chromatographer easier are always popular introductions at Pittcon. This year I have separated the columns and sample preparation accessories (Table II) and small, benchtop dedicated instruments that are designed to automate sample preparation and other chromatographic techniques (Table III). Each area is covered individually. 

Airtight, inert containers for frozen samples are important in many laboratories and a whole family of such products were noted. Vials are always in big demand in the chromatography laboratory and an interesting vial closure system has been developed that prevents evaporation of solvent while vials are sitting in the autosampler. 

HPLC, UHPLC, and Ion Chromatography Products

The advent of UHPLC has spurred an increasing number of ultrahigh-pressure products. This year additional high-pressure components included fittings and bio-inert columns (both stainless steel and PEEK), reusable fittings, high-pressure seals, and a high-pressure injection valve. A new guard column holder that allows the user to replace a guard column without disconnecting the inlet and outlet fluid connections was displayed. Eluent suppression in ion chromatography is often required to achieve optimum sensitivity, and a new electrolytically regenerated suppressor was introduced by Thermo Fisher Scientific this year. 

A high-pressure gas switching system was introduced that will automatically switch between two high-pressure lines, useful for a bank of gas cylinders. Nobody wants to work directly with toxic volatiles in the GC laboratory. A gas permeation tube for phosgene was introduced. A permeation tube provides a steady, low concentration of a volatile analyte that can be used for calibration and other purposes. Probably the most practical item introduced in the GC accessory market is Agilent's self-tightening stainless steel column nut. Graphite–polyimide ferrules favored by gas chromatographers using GC–MS are notorious for requiring occasional retightening when they are cycled through repeated temperature programming operations. Otherwise, they leak, which is not good for oxygen-sensitive columns or detection methods such as MS and electron-capture detection. The new column nut has a novel spring-driven piston that continuously applies pressure to the ferrule, which prevents it from becoming loose after temperature programming cycles. 

In addition to the new sample preparation products from Table I, a new series of QuEChERS standards were introduced by Restek. These are mainly popular pesticides that are encountered in fruit and vegetable samples. 

Table III shows 21 new products, mostly for sample preparation. Sample preparation still represents a major bottleneck in the laboratory because it is often a manual operation. Small benchtop instruments that automate some of the manual sample preparation steps can save time, eliminate tedium, and reduce errors. In this section, I cover benchtop instruments that range from robotic designs that perform multiple sample preparation procedures on small sample volumes to instruments that perform a single task such as solvent evaporation or mixing.  

Common sample preparation tasks such as evaporation, grinding, milling, shaking, mixing, cell lysing, and centrifugation can be performed by relatively simple automated instruments that aren't going to break the bank cost-wise. Of course, for some grinding and milling tasks, fairly heavy machinery may be needed to break up rocks, plastics, or other hard substances into a powder form that can be extracted, and these may be costly. Table III covers a number of these dedicated benchtop devices for routine sample preparation operations. 

Among the more sophisticated devices covered in Table III is one that automates SPE by handling as many as eight samples simultaneously. SPE has several experimental steps in its operation, and to automate all of them requires a good bit of manipulation of solvents, cartridges, and gas flows (for drydown). Supercritical fluid extraction (SFE) appears to be making a comeback, and one new device for large-scale SFE was introduced. 

For GC, there are several products covered in Table III. Simple low-cost leak detectors and flowmeters are particularly useful when performing GC analysis. On the more expensive side, a new headspace sampler was shown at Pittcon and another hydrogen generator. The latter are becoming even more popular since the world seems to be going from helium to hydrogen as the sources of helium are drying up. 

I would like to thank the manufacturers and distributors that kindly furnished the requested information in advance of Pittcon 2014, thus allowing a timely report on new product introductions. For those manufacturers who would like to be considered for inclusion into Pittcon 2015 coverage, please send the name of the primary company contact, the mailing address, phone number, and e-mail address to Laura Bush, Editorial Director, 

, with the subject line "Pittcon 2015 Column Watch." 

is the editor of "Column Watch," an analytical consultant, and a member of 

's editorial advisory board. Direct correspondence about this column to 

Articles

Column Watch

New GC columns, SFC columns, and products for sample preparation, including small benchtop instruments

New Chromatography Columns and Accessories at Pittcon 2014, Part II

Joseph Jack Kirkland answered questions from Gert Desmet on his pioneering career in high performance liquid chromatography (HPLC).

Gert Desmet: How did you enter the field of chromatography?

 In the late 1950s I began to use gas chromatography (GC) to solve problems relating to agricultural products that were being developed at DuPont. This effort was triggered by interactions with DuPont's Dr. Steve Dal Nogare, who started the early studies in GC, especially in programmed temperature operation. GC analyses proved to be quite useful, but there were many compounds of interest to DuPont that were not volatile and could not be separated by this technique.  

In some cases, I was able to derivatize these materials to produce structures that were sufficiently volatile for GC analysis, but many problems still remained that needed a solution. Around 1960, I attempted liquid chromatography (LC) separations of some nonvolatile compounds using large-particle silica gel columns as the separating medium, a low pressure pump, and a refractometer as a detector. Crude separations resulted, but it was too slow, poorly reproducible, and very frustrating. I really did not know enough to create the desired result. Therefore, this project was not pursued further. However, in 1964, when I was visiting Europe, during a trip to Eindhoven, The Netherlands, I came across Dr. Joseph (J.F.K.) Huber in a laboratory performing what we now call high performance liquid chromatography (HPLC).  

Huber was using a constant-pressure pump (1000 psi), a small diameter column with ~50 µm diatomaceous earth particles, and an ultraviolet (UV) spectrometer that had been converted into a crude detector. With this arrangement, Huber was demonstrating remarkable (for that time) separations of nonvolatile compounds using a liquid–liquid chromatographic technique. This visit was so interesting that on my return to DuPont, I approached my supervisor and pleaded for the time and resources to begin a program into researching HPLC technology. This request was approved and I then started to develop the materials that were needed to make the technique routine. To improve the technology, I believed that a better column packing was required, together with a reliable and highly sensitive UV detector. Both of these items were subsequently developed, put into use, and a patent and papers on them published. 

 Figure 1: Jack Kirkland in his laboratory in 1960.			

GD: Who were the people that most influenced your scientific and professional life and work?

 There have been many important people in my life that have strongly influenced the paths that I have taken and the things that I have done along the way. R.A. Day, professor of analytical chemistry at Emory University in Atlanta, Georgia, opened my eyes to the possibilities of analytical chemistry. He encouraged me in many ways so that I was later able to acquire a doctorate (at the University of Virginia in Charlottesville, Virginia) that better equipped me for research activities.  

Dr. Ralph K. Iler hired me into DuPont, which resulted in a strong personal friendship and a world-class mentor in the science of colloids, especially silica. My efforts in developing silica supports for HPLC have largely grown out of my association with Iler. Dr. Joseph J. DeStefano, longtime friend and coworker, has largely been the driving force for any entrepreneurship that I might have shown, always encouraging basic studies that have resulted in commercial products for chromatography. The publications and friendship with Professor J. Calvin Giddings were key factors in my (partial) understanding of the basics in chromatography and for my research in field-flow fractionation (FFF) during the 1980s. Dr. Lloyd R. Snyder, has been a strong personal friend and coworker for more than 40 years. Snyder and I have participated in research, published papers, written several books, and delivered HPLC training courses together. To this day, we still discuss HPLC results and developments. It is important to say that Snyder has had a crucial influence on my career and on many developments in HPLC that I have made. Finally, my wife, Karin Monson Kirkland, has always been a key factor in my life and my career. Also a chemist, Karin has been a strong advocate, a sharp technical critic, and a patient and tireless reviewer of ideas. 

Figure 2: Left to right: Jack Kirkland, Joseph L. Glajch, and Lloyd R. Snyder in 2008.			

GD: What do you consider as the major breakthrough in the field that happened during your career?

 The development of optimized particles and stationary phases have made HPLC a major analytical technique. This development has occurred slowly but surely over the past 50 years, with the efforts of many diligent scientists. For example, for me, first-of-a-kind commercial developments during this period included Zipax superficially porous particles in 1968, Permaphase (E.I. duPont de Nemours) bonded phase packings in 1970, Zorbax (E.I. duPont de Nemours) porous silica microspheres in 1972, Zorbax Rx (E.I. duPont de Nemours), Type B silica (E.I. duPont de Nemours) in 1989, and sterically protected silane stationary phases in 1990 (with Dr. Joseph L. Glajch). Resurrection of superficially porous particles began with a 1992 production of 7-µm widepore superficially particles. This was followed by 5-µm Poroshell-300 (Agilent Technologies) particles in 1997 and Halo (Advanced Material Technology) sub-3-µm superficially porous particles (often called core–shell, porous shell, or Fused-Core [Advanced Material Technology] particles) in 2006. However, probably the most active user response was after the introduction of Zorbax (E.I. duPont de Nemours) porous silica microspheres, which are still commercial. Many other scientists, both academic and industrial, have made important developments in column packings that resulted in the prominence of HPLC today. 

Figure 3: Jack Kirkland discussing silica chemistry with U.S.S.R. Professor A.V. Kiselev at a conference in Tallin, Estonia, in 1982.			

Equally important in breakthroughs have been developments in the marriage of HPLC with mass spectrometry (MS). These exciting developments (for which I essentially had no input) have crystallized the benefits of HPLC separations and enormously widened the scope of the method so that now the opportunities for utility in many areas are virtually unlimited. 

GD: Which decisions or changes in your career would you certainly make again and which not?

 It would have been an advantage if I had concentrated more on training in mathematics during my schooling. In some ways I focused more on obtaining a general education and this left time too short to develop the mathematical skills that would have made my scientific career more fruitful. Other than that, I am happy with the decisions that I made along the way, and would probably not change any major movements in my career.  

GD: Did your work in industry slow down your scientific career or was it the opposite? Would your work have been much less influential if you had done your work in an academic environment?

 My work in industry actually made it possible to develop my scientific career. Formative years at Hercules Powder Co. and then later at DuPont (after my doctorate) were spent solving difficult practical problems. This sharpened my focus and resulted in my learning how to obtain the required data to successfully meet a particular project goal. During that time, I gained experience in technologies such as polarography, infrared, and UV spectroscopy and colorimetric analysis of trace organic components, all of which broadened my analytical skills. 

Figure 4: Voges Mountain prior to the HPLC symposium in 1983. Left to right: Professor Goran Schill, Professor Milton Hearn, Karin Kirkland, Jack Kirkland, Professor Klaus Unger, and Johann Kraak.			

I was particularly fortunate to have worked in DuPont during the "golden days" of research. Because of the extraordinary resources at DuPont available for virtually all areas of chemical science, I was able to do things that were only possible at this institution. Within DuPont, I could find experts in almost all areas of chemistry, engineering, and bioscience to assist and mentor me. Much of my research in separations in later years was commercialized by DuPont, which enhanced my experience in the practical, utilitarian world. As mostly a pragmatic, practical-minded person, industry was an excellent fit for me and I did not have to spend time attempting to secure grants and other funds to support my research.  

I was also able to secure and train excellent technicians who were with me for a long time to assist in projects. One technician (Charles Dilks, Jr.) worked with me for 25 years including during my time at DuPont and later when I was with Rockland Technologies, Hewlett Packard, and Agilent Technologies. All of this experience has contributed to my research activities at Advanced Materials Technology, which is my current (and last) challenge. I have also actually sampled the academic world as an adjunct professor at the University of Delaware in Newark, Delaware, and during the 21 years that Snyder and I taught courses in HPLC for the Continuing Education Department of the American Chemical Society (ACS). These experiences were interesting and challenging, but I am better suited, and my interests and experience are better fitted, to the industrial world.  

GD: What are, in your opinion, the most critical elements to produce a good silica particle and column (material purity, coating, column loading, and so on)?

 Several important factors are involved in forming a high-efficiency, stable column. Good columns for many compounds require the use of Type B high-purity silica for good peak shapes and high efficiency. Techniques for reactions needed for the effective bonding of stationary phases are now wellknown and widely published. Once the stationary phase is loaded to this silica support, the column packing method is most critical. Column packing techniques are a combination of science and art. This results from the lack of knowledge of how many unknown factors are involved in forming an efficient, stable packed bed after all these many years of developments. Manufacturers typically do not publish their experiences with column-packing techniques, but a few articles have been published with useful information (5). 

GD: You are the founding father of the porous-shell technology. How proud does that make you?

 As with many developments in science, my efforts in superficially porous (porous shell) particle technology were catalyzed by previous studies. Csaba Horváth and Sandford Lipsky first made pellicular ion-exchange particles by sorbing small ion-exchange particles to the surface of glass beads. These pellicular particles were interesting to me, but had limitations in sample loading properties because of very small surface areas. When I began my studies in HPLC, I recognized that new particles were needed to enable better column performance (at that time, large irregular diatomaceous earth particles used for GC were used to make HPLC columns). Iler had demonstrated the layering of silica sol particles to form porous films that exhibited light refraction. By marrying pellicular technology with the Iler layering method, I was able to come up with ~ 30-µm spherical particles with solid cores and an outer porous shell, which were used and subsequently patented. These were successfully commercialized by DuPont as Zipax (E.I. duPont de Nemours) superficially porous particles (SPPs) as the first column packing materials specifically designed for HPLC. These SPPs were initially used in columns for liquid-liquid chromatography. However, subsequent development of Permaphase-bonded phase packings (with Dr. Paul C. Yates) led to the development of the now widely-used reversed-phase techniques pioneered by DuPont's HPLC Applications Laboratory in the Instrument Products Department. With the development of the much smaller and more efficient porous silica microspheres such as Zorbax (E.I. duPont de Nemours) in the early 1970s, the larger Zipax (E.I. duPont de Nemours) particles quickly lost favor. More than 20 years passed before there was a rebirth of the SPP.  

However, during this period, I was still interested in the potential of porous-shell particles, and was able to do some research in this area that resulted in a patent for making particles by spray drying. An SPP designed for size-exclusion chromatography was also developed in the mid-1970s, disclosed in a patent but not commercialized. In 1992, I published a paper on the properties of a widepore 7-µm SPP made by spray drying and designed for separating peptides and proteins. A similar 5-µm product made by another method was commercialized as Poroshell-300 while working at Agilent in 1997. After beginning research at Advanced Materials Technology in 2005, the vision for how to make a small SPP took place, and as a result, the porousshell (Fused-Core [Advanced Material Technology]) 2.7-µm particle called Halo (Advanced Material Technology) was developed. The success of these SPPs has resulted in the proliferation of these materials by competing manufacturers, and this technology has now been established as a major force in HPLC. This situation gives me great pleasure as it vindicates my long-time interest and belief in the chromatographic capabilities of particles with a nonporous core and a thin porous outer shell. 

GD: What does it take to make a major scientific breakthrough? Perseverance or rather a flash of inspiration? Or just plain luck?

 In my experience, breakthroughs are a combination of perseverance and luck. To be on the right track, one has to have the notion of what is needed and some ideas of how it might be accomplished. The rest is hard work to come up with feasible hypotheses, then devise and run the experiments that will ultimately allow the goal to be reached. In my career, I have had only one "flash inspiration" that resulted in a success. The rest of the time it has been a grind. Even when the work is a scientific success, it may not find utility or a commercial outlet. Therefore, luck and timing plays a large role in obtaining what might be termed as a "breakthrough." I once thought that with my astute coworker, Dr. Wallace W. Yau, we might have a "breakthrough" when we were able to develop a sedimentation field flow apparatus for operation up to 100,000

. This unique apparatus and new exponential force decay technique allowed the gentle separation of a very wide range of particles and soluble materials, including proteins and other such sensitive large molecules with good resolution. Unfortunately, this published development was too early for general acceptance and this high force field technology languishes to this day — no luck! 

GD: You have had a career which is full of entrepreneurship. Did you do this for the thrill of the entrepreneurship or the love of the product that you wanted to make?

 Early work within DuPont was initiated to develop unique technology for solving some of the difficult problems in agricultural and industrial chemistry for which I was presented. I was able to do some basic research during this period, and this continued fulltime when I transferred to DuPont's Central Research Department in 1973. My later work in industrial laboratories was more directed toward developing new products that would have commercial interest. Therefore, my later years involved some entrepreneurship, especially as a partner in Rockland Technologies (formed in 1989 and sold to Hewlett Packard in 1995). However, I still do research because it is my first love, although the hope is that results will justify commercialization of a product with practical utility for scientists. 

GD: Where do you think the R&D departments of the major instrument and column manufacturers should invest most in? What new technology could revolutionize the way that we do our separations and analyses?

 Some believe that research in HPLC is becoming too redundant and that there is little to gain from continuing efforts. Actually, there is still much left to do, although true "breakthroughs" are becoming more difficult. Most of any new developments in new HPLC technology will probably be in the life sciences for large molecules, for here is where many important challenges remain. I am still enamored with the possibilities for FFF methods, for this family of separation methods are really best suited for large macromolecules, both natural and synthetic. I felt this in the 1980s, during which I largely spent my time researching these methods, and this feeling has not subsided. The problem was that my research (and the even more extensive research by Cal Giddings) was just too early; the macromolecular, especially the bioscience community, was not ready for these developments. FFF has not reached the expected important utility because the larger instrument companies have not applied the required long-term effort and resources to make these methods into a broad commercial success. Smaller FFF instrument manufacturers have created a resurgence which gives hope that this technology will finally attain the prominence that it deserves.  

(2) L.R. Snyder, J.J. Kirkland, and J.L. Glajch, 

, second edition, (John Wiley and Sons, New York, 1997). 

(3) L.R. Snyder, J.J. Kirkland, and J.W. Dolan, 

Introduction to Modern Liquid Chromatography

, third edition, (John Wiley and Sons, Hoboken, New Jersey, 2010). 

(4) A.M. Striegel, W.W. Yay, J.J. Kirkland, and D.D. Bly, 

Modern Size-Exclusion Liquid Chromatography

, second edition, (John Wiley and Sons, Hoboken, New Jersey, 2009). 

 obtained his PhD in chemical engineering in fermentation technology from the Free University of Brussels in Brussels, Belgium, where he currently is a full professor in chemical engineering and heads the department of chemical engineering. In this capacity, he teaches courses on bioreactor design, chemical reactor engineering, nano- and microbiotechnology, and chromatography in both the faculty of sciences and bioengineering and the faculty of engineering. 

His research focuses on the miniaturization of separation methods and on the investigation and the modelling of flow effects in chromatographic systems. 

History of Chromatography

An interview with Jack Kirkland on his pioneering career in HPLC

Joseph Jack Kirkland: HPLC Particle Pioneer

In this "GC Connections" installment, I review gas chromatography (GC) instruments and accessories that were newly on display at the Pittsburgh Conference in March 2014, or were introduced to the marketplace in the preceding year.

The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) returned to Chicago's McCormick Place for its 65th annual meeting on March 2–6, 2014. This year, more than 16,200 conferees and exhibitor personnel attended Pittcon and the cohosted Food Labs Conference. More than 36% were listed as first-time attendees, but the published statistics did not differentiate between Pittcon and Food Labs registrants. The exposition floor sited 935 exhibitors in a total of 1763 booths; 32 countries were represented. As was the case in 2013 (in Philadelphia, Pennsylvania), the exhibition floor proved easier to navigate than in earlier conferences. This did not seem to be related to the size of the conference, which has not varied by more than about 10% in recent years, but rather the layout was well organized with many booths positioned in logical groups. 

 Table I: Companies introducing new GC products			

The technical program was of high quality with 73 symposia, 10 awards, 84 oral sessions, plus posters and workshops making up a total of 2000 technical sessions. Emphasis was clearly on life science topics, which constituted 40% of the presentations. The short course program continued its high activity with 116 offerings. 

Pittcon will journey to New Orleans, Louisiana, in 2015 — a welcome respite from two consecutive years in colder northern locations. Heading to Atlanta, Georgia, in 2016, conferees will now enjoy a couple years of Southern hospitality until the conference swings back to Chicago in 2017. 

This annual "GC Connections" installment reviews gas chromatography (GC) instrumentation and accessories shown at this year's Pittcon or introduced during the previous year. For a review of new GC and liquid chromatography (LC) columns and related accessories, please see "Column Watch" in the April and May 2014 (1,2) issues of 

The information presented here is based on manufacturers' replies to questionnaires, as well as on additional information from manufacturers' press releases, web sites, and product literature on the past year's products. It is not based on actual use or experience of the author. During Pittcon, I took time to stroll around the convention aisles and see some of the new products first-hand as well as to discover a number of items that weren't covered by the questionnaires. Every effort has been made to collect accurate information, but because of the preliminary nature of some of the material 

cannot be responsible for errors or omissions. This column installment cannot be considered a complete record of all new GC products introduced this year at Pittcon or elsewhere because not all manufacturers chose to respond to the questionnaire or attend the conference, nor is all of the submitted information necessarily included here because of the limited available space and the editors' judgment as to its suitability.  

Table III: New autosamplers and accessories			

GC again displayed renewed vigor at this year's Pittcon technical sessions. As in previous years, work in comprehensive GC×GC continues to yield significant advances, while the older technique of moving thermal-gradient GC (TGGC) is finding a new niche. Of particular interest were two contributions in separate sessions by members of Milton Lee's group from Brigham Young University on the theory and practical applications of TGGC for microfabricated GC and for GC×GC (3,4) Originally investigated decades ago, TGGC helps focus and control the passage of analytes through a column to produce improved temperature-programmed separations. While the technique has not been adopted for commercialization in recent years, perhaps a new TGGC instrument will appear sometime in the future.			

Two other GC topics stood out: alternative carrier gases and ionic liquid phases for GC. With the continuing uncertainty that surrounds helium supplies, the use of hydrogen or nitrogen as alternatives still garners much attention. Interest in hydrogen was evident throughout the exhibition as well as in a number of papers and posters. Recent developments seem to indicate that new supplies of helium from areas such as Qatar will ensure availability for the foreseeable future, but prices may remain high. Ionic liquid GC phases provide some unique selectivities and interest remains strong while applications of these materials expand to liquid–liquid and solid-phase micoextraction. See the related article on chromatography columns at Pittcon 2014 (2) for more details. 

In the product realm, there were a few GC instruments introduced since Pittcon 2013. From Agilent Technologies, introduced at ASMS in 2013, the model 7000C triple-quadrupole GC–MS-MS system represents the high end of GC-MS with its advanced spectrometric capabilities and high sensitivity. During the past year, Agilent also introduced a hydrogen sensor and a large heated valve oven for its model 7890B GC system. 

The 9100 Series gas chromatograph from Baseline Mocon is an on-line instrum ent that is reported to achieve laboratory-quality analysis for gas analysis across a wide range of applications. The model 5500 Fixed Gas Analyzer from CDS Analytical is an adjunct to the company's trapping pyrolysis systems for the analysis of low-molecular-weight gaseous by-products. 			

Bruker showed the GC-APCI II GC–MS interface, which can be combined with any Bruker ESI mass spectrometer, including the impact HD LC-QTOF system

introduced at the 2013 ASMS conference. The interface makes the impact HD's unique LC–QTOF-MS capabilities — such as accurate mass screening or determination of elemental compositions — accessible for GC–QTOF-MS. 

Markes International's Select-eV variable-energy ion-source technology for GC–MS allows the energy used for electron ionization to be changed on a sliding scale without loss of sensitivity. This allows the production of both classical mass spectra and repeatable soft-ionization spectra with reduced fragmentation and an enhanced molecular ion. 

VUV Analytics displayed a new type of GC detector that collects vacuum-ultraviolet spectra of peaks as they are eluted for peak identification, deconvolution, and selectivity. The detector operates from ~120 to 240 nm, down to shorter wavelengths than the ~190-nm capability of routine diode-array LC detectors. 			

A number of autosamplers and related devices debuted this year. The model 4100 Water/Soil sample processor from OI Analytical automates the handling and processing of samples in up to 100 40-mL volatile organic analysis (VOA) vials for purge-and-trap analysis in conjunction with one or two of the company's Eclipse purge-and-trap instruments. The sample processor is equipped with a unique pneumatically actuated cylindrical vial gripper. Also from OI Analytical, the model LV-20 standards addition module is an option for the company's model 4551A water autosampler. The standards addition module adds internal standards, surrogate, or matrix spike standards for GC and GC–MS analyses. 

The Flex autosampler from EST Analytical can perform a wide variety of liquid injections as well as some basic sample preparation on-board. This system can be upgraded to perform headspace sampling as well. And from CDS Analytical, the model 7500 thermal desorption autosampler is an extension of the company's model 7400 purge-and-trap system that offers the ability for the user to change between air-sample thermal desorption (TD) tubes and VOA vials for soil or water analysis.  

Thermo Scientific's programmable Virtuoso vial printing system prints indelible, detailed information directly on sample vials in three text fields with a mini quick response (QR) barcode. Vial labeling takes approximately 5 s and the identification data can be input using the unit's touchscreen or directly from the lab network.  

Gerstel's Hot Injection and Trapping (HIT) technique combines analytes from several headspace injections in a trapping or desorption procedure for single GC–MS runs. The technique integrates the capabilities of the company's MultiPurpose Sampler (MPS), Thermal Desorption Unit (TDU), and Cooled Injection System (CIS) inlet. 

From Hamilton, the new S-line syringe for CTC PAL Autosampler applications complements the company's existing C-Line and X-Type CTC PAL autosampler syringes. S-Line syringes are built for economic everyday use for GC and high performance liquid chromatography (HPLC) injection applications.  

I would like to thank the manufacturers and distributors that kindly furnished the requested information, which allowed a timely report on new product introductions over the past year. For those manufacturers who did not receive a preconference questionnaire this year and would like to receive one and be considered for early inclusion into the 2014–2015 new GC and related product introductions review, please send the name of the primary company contact, the mailing address, fax number, and e-mail address to Laura Bush, Editorial Director, 

(3) H.D. Tolley, S.E. Tolley, A. Wang, M.C. Asplund, and M.L. Lee, "Properties of Thermal Gradient GC Separations," presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon 2014), Chicago, Illinois, 2014. 

(4) A. Wang, A.R. Hawkins, H.D. Tolley, and M.L. Lee, "Enhancing Separation Performance of Microfabricated Gas Chromatography Using Temperature Gradients," presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon 2014), Chicago, Illinois, 2014. 

 "GC Connections" editor John V. Hinshaw is a Senior Scientist at Serveron Corporation in Beaverton, Oregon, and a member of 

s editorial advisory board. Direct correspondence about this column to the author via e-mail: 

GC Connections

A review of GC instruments and accessories that were new at Pittcon or introduced to the marketplace in the preceding year

New Gas Chromatography Products 2014

This month's "LC Troubleshooting" looks at two reader-submitted questions regarding method calibration.

One of the parts of being a column editor for 

 that I enjoy is interacting with readers, primarily by e-mail. I try to answer each reader's question promptly. Some questions have a wider interest to the liquid chromatography (LC) community, so I occasionally pick a question or two to use as the centerpiece of my "LC Troubleshooting" columns. Reader questions also help me keep my finger on the pulse of reader interests so that I can pick topics that will be useful to a wide variety of reader needs. This month I've picked two topics related to method calibration that came in recent e-mail messages. If you would like to submit a question, contact me via my e-mail address listed at the end of this article. 

: I am running a method to verify that the product we produce contains 100±5% of the label claim of the active ingredient. The method has worked well for our product containing 50 mg of active ingredient. We are just introducing a lower potency product that contains 28 mg of the active ingredient, and I can't get the product to pass the specifications. I weighed out 28 mg of reference standard to check the method and this reference standard will not pass either. 

My method calls for making two weighings of ~50 mg of my reference standard and making two standard solutions. Six replicate injections of the first solution (STD1) are made and two of the second (STD2). The response factor is calculated as the average area of the six injections divided by the weight of the standard. System suitability requires that all injections are within ±2% of the nominal amount. I have shown the results of a typical test in Table I. When I make a lower concentration of reference standard by weighing 28 mg, repeating the injections, and checking the concentration using the response factor, however, I am unable to get the 28 mg standards to pass system suitability. What am I doing wrong? Additionally, the method calls for the two weighings, but after the initial check of the second standard (

 = 2), it is not used again. It always seems to agree with the first standard, so why is this required? 

 As is my usual practice when reviewing data, I first examine it to see if I can establish any indicators of data quality. One easy way to do this is to look at the repeatability. At the bottom of Table I I've shown the average, standard deviation, and percent relative standard deviation (%RSD) for the 

 = 6 replicate injection sets (as is usual for these discussions, I've rounded the numbers for display convenience). You can see that the 50-mg set comes in at 0.12% RSD and the 28-mg set at 0.23%. Both of these are reasonable and show that the performance of LC system, especially the autosampler and data system, is adequately reproducible. In our laboratory, we typically see ≤0.3% RSD for six 10-µL injections, and these data meet those criteria. 

My next step is to examine the individual and pooled results to see if I notice any patterns. I've reorganized the data slightly in Table II, where the first column of data lists the weighed amounts and the second column lists the amount of compound calculated from the area for each injection using the response factor (RF= 357, bottom of Table I) based on the weight divided by the average response for the 50 mg, 

= 6 sample. You can see that all of the individual 50 mg injections as well as their averages are within the ±2% limit (50.2 mg × 2% = 1.0 mg), so all is well there. However, most of the 28-mg injections and all the averages are outside the limits (28 mg × 2% = 0.56 mg). Because the averages for both 28-mg samples agree (102.1% and 102.6%), it is unlikely to be a weighing error (more on the use of duplicate weighings later). 

Table II: Comparison of results with two calibration methods			

So, what's going wrong here? I suspect that it is a problem with the calibration procedure. This method uses what we call 

, where a reference standard is made at one concentration and a response factor is determined based on the peak response (area) and the standard concentration. Then sample concentrations are calculated by dividing the sample peak area by the response factor. This is a simple and straightforward technique, but it assumes that the calibration plot goes through zero (

 = 0). The response factor is simply the slope of the calibration line, and to establish the calibration line, you need two points. One point is the response of the standard and the other is assumed to be zero — that is, zero response for zero concentration — a very logical assumption. However, this assumption must be demonstrated during method validation to justify single-point calibration.  

To illustrate the problem, I've assumed that all the weighings at the 50-mg and 28-mg levels are accurate as shown in Table I. By pooling all 16 injections for both concentrations, we can perform a linear regression on the data set. In this case, we get a formula for the calibration curve of 

= 0.9999. On the other hand, if we only use the 50-mg points and force the origin through zero, we get 

= 1.0000. It is simple to determine which formula is appropriate for the data set, as was discussed in an earlier "LC Troubleshooting" column (1). In the regression data report for the full data set, there is a value called "standard error of y" (SE

), which can be thought of as the normal error, or uncertainty around the value of 

, the curve can be forced through zero, and in the present case, this would justify a single-point calibration. If the reported value is greater than the standard error of 

, forcing the curve through 0,0 is not justified, and a multipoint calibration is required. In the present case SE

 = 34. In other words, if the calibration line crosses the 

 axis at 0 ±34, it is within 1 standard deviation of zero and not statistically different from zero, so the use of 0,0 as a calibration point is reasonable. Here, though, the calibration curve 

 = 530, which is more than 10 times larger than SE

, so a single-point calibration is not justified. 

To illustrate how much better the data appear when we do a multipoint calibration, I have selected just the second weighings of the 50.2-mg and 28-mg data and used the 

 = 2 injections of each (four total injections) to generate the calibration curve (

 = 1.0000). I've used this calibration to back-calculate the concentration of each injection, as shown in the right-hand column of Table II. You can see that the 50-mg data points are about the same as for the response-factor approach, but the 28-mg points all fit within approximately 0.5% of the target values. This is definitely a better choice for calibration of the current method. 

So, why was the problem not noticed with the 50-mg product, whereas it shows up with the 28-mg one? I've sketched a greatly exaggerated calibration plot in Figure 1 with arbitrary slopes to illustrate the problem. With the single-point curve, we have a real data point at 50 mg and an assumed one through the origin that generates the dashed line. By definition, the 50-mg point will be on the line. Any concentration of product that gives a response within ±5% of the 50 mg response will pass the 100±5% specification. Even with the wrong slope of the calibration line, some product concentration variations will pass. I calculate that a product that varied by as much as ±2.5% of real concentration would still appear to fit the ±5% allowance based on the single-point curve. So, if the manufacturing process generated very accurate concentrations (±2.5%), the product would pass specifications. However, product that deviated more than ±2.5% would appear to exceed the ±5% limits. The net result of this condition would be that all "passing" product would be within the specifications and therefore safe to use, but some product that was actually within the ±5% specification would appear to fail and would be scrapped or reworked, adding expense to the manufacturing process. 

Figure 1: Hypothetical calibration plots. The dashed line represents 50-mg reference standard and calibration line assumed to pass through the origin (0,0). The solid line represents calibration with 50- and 28-mg reference standards. The open circle shows error in apparent concentration when improper calibration plot is used. See text for details.			

When we move to the 28-mg product, however, the story changes. I've drawn the solid curve in Figure 1 to represent the true calibration curve when both the 50- and 28-mg points are used to generate the plot (again, the difference in slopes is exaggerated for illustration). The 28-mg point is shown on this solid line as a solid dot. However, if this same response is plotted on the single-point curve (dashed line), the point is moved to the right (open circle) to fit the curve and gives a reported value that is higher than it should be. This is exactly what we see with the data of Tables I and II when the response-factor (single-point) approach is taken — the 28-mg values are larger than expected by a little more than 2% (the drawing of Figure 1 greatly exaggerates the difference). With this understanding of the problem, it is not at all surprising that the 28-mg points all fail with the single-point calibration at 50 mg. If another single-point calibration curve were made using 28-mg calibrators, those calibrators would be expected to pass system suitability, and it is likely that at least some of the product batches would also pass specifications for the same reason that the 50-mg ones did with a 50-mg calibration. That is, a single-point calibration can appear to work properly if the test values are close to the calibration point, even if the wrong calibration scheme is used. 

I don't know the history of the method or if the single-point calibration was ever validated properly. In any event, when a method is transferred as a pharmacopeial method or an internally developed method, it is always wise to make several concentrations of reference standards over the range of zero to the proposed single-point concentration (and likely to 25–50% higher than the single-point value). Make a calibration curve using all the data points and compare it to a single-point calibration. If they give the same results, easily checked by testing whether the calibration curve can be forced through zero or not, then a single-point calibration is justified. 

The final part of the reader's question pertained to the requirement for making two equal weighings of the reference standard and comparing the response of these. Technically, if you are positive that your weighings are always correct, this practice is a waste of time. However, there is a normal amount of uncertainty (error) in the laboratory, and sometimes we just make a mistake. This is the reason for making duplicate weighings — to double-check that a mistake was not made. As a consumer, I am certainly more comfortable depending on a certain dose of a pharmaceutical product if I know the concentration has been double-checked. This is the same reason that duplicate preparations of sample often are called for in an analysis. In some methods, both of the standards are used for calibration. For example, one might have a sequence of STD1, STD2, SPL1a, SPL1a, SPL1b, SPL1b, STD1, STD2, where STD1 and STD2 are the two reference standards, and sample 1 (SPL1) is prepared in duplicate (SPL1a and SPL1b) and each preparation is injected twice. Perhaps all four bracketing standards are averaged to get the response factor for that set of samples. The design of the injection sequence, calibration method, and number of replicates will depend on the analytical method, laboratory policy, and regulatory requirements. 

When Should an Internal Standard Be Used?

: I'm new to LC and am confused about the difference between an internal and external standard. When should an internal standard be used? 

: I discussed the use of various standardization techniques in a couple of earlier columns (2,3), but let's review the highlights here. External standardization receives its name because the reference standard is external to the sample. A single-point standard or a series of reference standards at different concentrations are prepared in a solution, often containing everything that is expected to be in the sample except the active ingredient. That is, known amounts of analyte are added to a placebo or other blank matrix. These are then analyzed using the LC method to establish a calibration curve (also referred to as a standard curve). This curve can be described by a mathematical relationship, the most common of which is a linear fit, 

 = 0). Samples of unknown concentration are then injected and the calibration curve is used to determine how much analyte is present based on the response for the sample, as discussed in the previous example. Additional calculations may need to be made to adjust for the purity of the reference standard, dilution effects, and so forth. Most of the time, external standardization is the simplest way to calibrate a method. It is best when sample preparation is a simple process, such as weighing, filtering, and dilution. 

With the internal standard technique, a known amount of a second standard (the internal standard) that is not present in any of the samples is added to each sample. For calibration, a set of reference standard calibrators is prepared, each of which contains the same concentration of the internal standard, but different concentrations of reference standard. Instead of plotting the concentration of analyte versus the response, the concentration ratio of the internal standard to analyte is plotted against the ratio of the area response of the internal standard and analyte. The resulting relationship can then be used to find the concentration of analyte in an unknown sample that contains a known concentration of internal standard.  

Sounds complicated, so why go to all this extra work? Internal standardization is especially good when there is a lot of sample manipulation during sample preparation. These might include operations such as liquid–liquid extraction, solid-phase extraction, evaporation to dryness, and other steps where the total volume recovery of the sample may vary. Because the ratio of the analyte to internal standard is tracked, it doesn't matter if only 90% of the sample reaches the injection vial in one case and 80% in the next — such variations are canceled out with the internal standard technique. 

Pharmaceutical drug substances (pure drugs) and drug products (formulated product) usually involve simple sample preparation processes, so external standardization is the norm. On the other hand, bioanalytical analysis (drugs in plasma), often comprises many sample preparation steps, so internal standardization is more common for this type of sample. Environmental, biological, herbal, chemical, and other sample types usually will use one or the other of these techniques, depending on the complexity of the sample and how much sample preparation is required. 

We have examined some different aspects of calibration. These included the comparison of single-point and multipoint calibration curves, when it is appropriate to force the curve through zero, and when to choose a different calibration method. References 1 and 2 are part of a five-part series of columns about calibration that I wrote several years ago. This series was so popular that 

 decided to publish it as a stand-alone e-book for ready reference in the laboratory. If you are interested in getting a copy, information is included in reference 4. 

http://www.nxtbook.com/nxtbooks/lcgcebooks/lcgc_092013/#/0

"LC Troubleshooting" Editor John Dolan has been writing "LC Troubleshooting" for LCGC for more than 30 years. One of the industry's most respected professionals, John is currently the Vice President of and a principal instructor for LC Resources, Walnut Creek, California. He is also a member of LCGC's editorial advisory board. Direct correspondence about this column via e-mail to 

LC Troubleshooting

A look at two reader-submitted questions regarding method calibration

Readers' Questions: Calibration

In this study, the quality-by-design principle is applied instead of trial-and-error in the development of a liquid chromatography method. With few measurements, the appropriate stationary phase and chromatographic conditions such as the composition of mobile phase, gradient time, temperature, and pH can be determined. A mixture of an active pharmaceutical ingredient and its 13 impurities was analyzed on a short narrow-bore column (50 mm × 2.1 mm, packed with sub-2-µm particles) providing short analysis times. The performance of commercial modeling software for robustness testing was systematically compared to experimental measurements and design-of-experiment–based predictions.

The separation of an active pharmaceutical ingredient (API) and its impurities is a necessary step in the control of drugs. In most cases, the structures of the impurities vary widely. Some impurities are very similar to the API and others are built very differently. Reversed-phase chromatography is mainly used for their separation. In reversed-phase chromatography several parameters influence the quality of the separation. One of them is the stationary phase, where several interactions determine the separation: Hydrophobic interactions are the most dominant in terms of retention, and silanol groups influence the "polar selectivity" (1). If their ratio changes, the separation might change also. Other parameters include the composition of the mobile phase (%B or time of gradient [

]), pH, and temperature. Both selectivity and working parameters must be in strict control if we transfer the method from laboratory to laboratory, or from high performance liquid chromatography (HPLC) to ultrahigh-pressure liquid chromatography (UHPLC) or vice versa. In the early stage of HPLC this was realized and method development was based on scientific recognitions in the field. In 1976 Horváth, Melander, and Molnár established the fundamentals of reversed-phase chromatography based on a design-of-experiments (DoE) approach (2). In 2002, regulatory authorities in the pharmaceutical industry, such as the Food and Drug Administration (FDA), recognized that analytical chemistry, in our case HPLC, is an integral part of the production of an API. In their guidelines, they highlighted that every parameter that can affect the analytical results must be forecasted. Today, this approach is called analytical quality by design (QbD). 

The aim of this work was the implementation of analytical QbD in LC using intelligent modeling software (3–6). The modeling software, which was originally developed in 1986 in cooperation with Snyder and his colleagues (7–9) and Molnár, looking at one and two dimensions, has now reached a three-dimensional (3D) stage (3,4,10,11). This intelligent software is based on the solvophobic theory of Horváth and colleagues (2) and the gradient elution theory of Snyder and Dolan (12). Further additions such as an improved peak tracking tool, the cube, and a robustness device help decrease the number of experiments needed for the development of a method and establish QbD-compatible UHPLC methods. This is particularly true for the 3D design (3).  

In 2004, a new age started in LC with the introduction of a new instrument with low extracolumn peak broadening (13). To get high efficiency, sub-2-µm particles were introduced. The columns packed with these small particles needed higher pressure compared to conventional HPLC. Today, the pressure limit is above 1000 bar or 14,500 psi. This new type of LC technique is called 

Some considerations should be mentioned about the interconnection of QbD and intelligent software packages. To get a robust method, we need to know the influence of parameters and their combined action on a separation. In QbD this means we have to establish the 

, a region where we have baseline separation. In a classical trial-and-error approach one needed thousands of experiments and the final result might have been robust or not. It was hard to predict the resolution outside of an investigated area. Using intelligent software approaches, the number of measurements can be reduced by two or three orders of magnitude down to 12 runs (14,15). The critical point is the robustness of the separation. In trial-and-error approaches it is impossible to run hundreds of experiments; therefore the robustness is always under debate. Using intelligent software, 12 measurements are enough to get a robust method and indicate its limits (4). 

This article investigates the development of an HPLC method for a pharmaceutical drug and its impurities using modeling software with multifactorial optimization of three measured critical HPLC method parameters — gradient time (

), and pH — as well as further calculation of three factors: the flow rate, %B start, and %B end using UHPLC.  

The mobile phase used in this work was a mixture of acetonitrile and 5 mM citrate buffer. Acetonitrile (gradient grade), citric acid, sodium hydroxide, standard reference buffers (pH 2.00, 4.01, and 7.00) were purchased from Merck. For the measurements, water was prepared fresh using ELGA Purelab UHQ water (ELGA). The buffer was filtered before use on regenerated cellulose filter membrane with a pore size of 0.2 µm (Sartorius). 

				The sample used throughout the study was an API and its starting materials (Stm), intermediates (Int), and known and unknown impurities (Imp).			

UHPLC was performed using a Waters Acquity UPLC system equipped with a binary solvent delivery pump, an autosampler, a photodiode-array detector, a 5-µL injection loop and 500-nL flow cell, and Empower software. The column was a 50 mm × 2.1 mm, 1.7-µm 

 Acquity BEH C18 UPLC column, which was selected from a large database with more than 500 reversed-phase columns (1). The dwell volume of the system was measured to be 0.125 mL. 

An MP 225 pH meter was purchased from Mettler-Toledo. 

Modeling was carried out using DryLab v. 4.1.5.3 software, and the quantitative robustness evaluation of generated models was performed with the latest DryLab Robustness module v.1.0. (Molnár-Institute). 

MarvinSketch v. 6.0.2 software was purchased from ChemAxon. 

Our study examined an API and its intermediates that were formed during the API manufacturing process. The flowchart of the synthesis can be seen in Figure 1. Both intermediates and newly formed impurities (Stm1, Stm2, and Int1–Int7) can be found in the final product. Two impurities (Imp1 and Imp2) were identified. They were synthesized and used for their identification in the reaction mixture. The identification of two other impurities (Imp3 and Imp4) is in progress. 

 Figure 1: Flowchart of the API preparation.			

Determining the starting points for prediction of retention and resolution by the modeling software is important. There are three ways to do this. The first is to run a reversed-phase gradient from 0 to 100% acetonitrile or methanol on a C18 column. The second is based on previous scientific knowledge, the "Knowledge Space," if available. The third is to predict some physicochemical parameters using intelligent modeling software, if possible. However, if there are unknown components then all three approaches are of limited use. Because we had two unknown impurities, we decided to use a reversed-phase gradient. The gradient time was established with a so called "scouting gradient," where we tried to find all possible unknowns before validating the method. Therefore, we tried to accommodate all known and unknown impurities in the increasing part of the gradient, which in our case was 10–80% B. As gradient times, we selected 1.5 min and 4.5 min. The temperature range tested in two different models between 20 °C and 80 °C.  

In Figure 2 the logD–pH function is given for the distribution of the investigated solutes between 

-octanol and water as a function of the eluent pH.  

Figure 2: LogD prediction by the MarvinSketch software.			

From the logD–pH diagram the following conclusion can be made: At any pH there are big differences in the nonpolarity of the various solutes and, therefore, gradient elution has to be selected. In the QbD concept, the gradient parameters will play an important role. The acidity of an eluent will influence retention and, from Figure 2, it can be concluded that the pH will have a great influence on the separation. The third parameter is the temperature. Unfortunately, silica-based reversed-phase columns have an upper temperature limit between 60 °C and 90 °C. However, the temperature influence is not negligible and must always be measured. 

Explanations of how the modeling software 3D model was developed has been published numerous times (3–11,14,15). Experimental design for the simultaneous optimization of gradient time, temperature, and pH requires 12 experiments, as illustrated in Figure 3. Two basic gradients with different rates (1.5 and 4.5 min, 10–80% B) were carried out at three column temperatures (20, 50, and 80 °C). Mobile-phase A was 5 mM citrate buffer at seven pH values (2.8, 3.4, 4.0, 4.6, 5.2, 5.8, and 6.4); mobile-phase B was acetonitrile. Citric acid was chosen as the buffer because it has high buffer capacity between pH 2.8 and pH 6.4 (p

 = 5.4). Acetonitrile was selected as the organic modifier because of its low viscosity. The flow rate was at 0.8 mL/min. The injection volume was 1 µL.  

Experimental points were designed using the parameters above. Using the three temperature values two temperature ranges were determined: We called 20–50 °C the low temperature range and 50–80 °C the high temperature range. Using the seven pH values, three pH ranges could be determined: pH = 2.8–3.4–4.0 was called the low pH range; pH = 4.0–4.6–5.2 was called the middle pH range; and pH = 5.2–5.8–6.4 was called the high pH range. The gradient time was 1.5 and 4.5 min on every temperature and pH value. Six cubes were prepared using these parameters (see Figure 3). 

Figure 3: Design-of-experiments (DoE) cubes for the simultaneous optimization of gradient time (tG), temperature (T), and pH of eluent A. Circles represent the input experiments for the 3D models.			

 = 1.5 min, 10–80% B), experiments are at points 1, 5, 9, 13, 17, 21, 25, 3, 7, 11, 15, 19, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45. The long gradient time (

 = 4.5 min, 10–80% B) experiments are at points 2, 6 ,10, 14, 18, 22, 26, 4, 8, 12, 16, 20, 24, 28, 30, 32, 34, 36, 38, 40, and 42. The low temperature (

 = 20 °C) experiments are at 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, and 26; the middle temperature runs (

 = 50 °C) are at 3, 4, 7, 8, 11, 12 15, 16, 19, 20, 23, 24, 27, and 28; and the high temperature runs (

= 80 °C) are at 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. Using the three pH ranges that we determined, the low pH range = 2.8 (1, 2, 3, 4, 29, and 30)–3.4 (5, 6, 7, 8, 31, and 32)–4.0 (9, 10, 11, 12, 33, and 34); the middle pH range = 4.0 (9, 10, 11, 12, 33, and 34)–4.6 (13, 14, 15, 16, 35, and 36)–5.2 (17, 18, 19, 20, 37, and 38); and the high pH range = 5.2 (17, 18, 19, 20, 37, and 38)–5.8 (21, 22, 23, 24, 39, and 40)–6.4 (25, 26, 27, 28, 41, and 42). The low temperature range (

) and low pH range are shown in Figure 4a. The low temperature range (

) and middle pH range are shown in Figure 4b. The low temperature range (

) and high pH range are shown in Figure 4c. The high temperature range (

) and low pH range are shown in Figure 4d. The high temperature range (

) and middle pH range are shown in Figure 4e. The high temperature range (

) and high pH range are shown in Figure 4f. The measured parameters are given in Table I. 

In this study, the quality-by-design principle is applied instead of trial-and-error in the development of an LC method.

LCGC North America-05-01-2014

New Chromatography Columns and Accessories at Pittcon 2014, Part II

Joseph Jack Kirkland: HPLC Particle Pioneer

New Gas Chromatography Products 2014

Readers' Questions: Calibration

Quality by Design in Pharmaceutical Analysis Using Computer Simulation with UHPLC

Assessing the Suitability of a Green Solvent for GPC and TREF Analyses of Polyethylene

Triple-Quadrupole Mass Spectrometers for GC–MS

Vol 32 No 5 LCGC North America May 2014 Regular Issue PDF