This is the first installment of "Perspectives in Modern HPLC," a new column that will be published every quarter on this topic. Here, we highlight prominent high performance liquid chromatography (HPLC) and related products introduced at Pittcon 2013, as well as the previous year. A summary of technical details is provided for these new HPLC systems, modules, and chromatographic software products, and major product extensions are also presented. The focus is on innovations and unique features from a user's perspective.

The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is the world's largest annual conference and exposition on laboratory science. It is organized by The Spectroscopy Society of Pittsburgh and Society for Analytical Chemistry of Pittsburgh and is managed by a committee of volunteers with a small permanent staff. Pittcon 2013 was held at the Pennsylvania Convention Center in Philadelphia from March 17 to 21, the first time in recent memory to be held in the northeastern United States (the last time was 1990 in New York City). Despite a trend of declining participation by exhibitor staff in recent years, Pittcon remains the premier analytical chemistry conference, unmatched in scope or scale by any contenders. This year, Pittcon boasted more than 18,000 attendees from industry, academia, and government agencies from more than 90 countries. There were more than 2000 technical sessions, including plenary lectures and invited, contributed, and award symposia; workshops; posters and networking sessions; ~90 short courses; and a huge exposition with more than 1000 vendors displaying at ~1900 booths.  

Traditionally, Pittcon is the forum for new product introductions and a "must-go-to" conference for laboratory scientists looking for new equipment. However, the many meetings with narrower focuses such as mass spectrometry, high performance liquid chromatography (HPLC), chiral separations, and biotech applications continue to compete for attendees. More importantly, the new information age now offers convenient alternatives for information gathering (such as Google searches, webcasts, virtual meetings, and social media). Nevertheless, the opportunities for face-to-face meetings to renew acquaintances and continuing education from symposia or short courses still hold sufficient motivation for many in this annual pilgrimage. 

In my opinion, the transformation from HPLC to ultrahigh-pressure liquid chromatography (UHPLC) is essentially complete this year. The revolution (or evolution) started with the proof-of-concepts research by Professor James Jorgenson in 1997 (1), followed by the first commercial UHPLC instrument (Waters Acquity UPLC) in 2004 (2). UHPLC is becoming the standard HPLC platform as all major manufacturers have some type of UHPLC system in their product line (3–5) with pressure limits ranging from 15,000 to 19,000 psi (1000–1250 bar). Acceptance by users is pervasive, including the very conservative quality control segment. The dissenting voices of skeptics have disappeared. In a few years, with universal adoption of this new technology, the term "UHPLC" may simply revert back to HPLC. It is noteworthy that Professor Jorgenson predicted faster and higher-resolution HPLC in small-internal-diameter columns packed with very small particles with further development of equipment with higher pressure capability (that is, 50,000 psi) (6): 

Moving to still higher pressures (50,000 psi) will enable the use of smaller particles and/or longer columns, and yield faster and better separations. This will almost certainly require the use of sub-mm bore (capillary) columns due to issues with heat generation and dissipation. This won't be easy, but the separation potential in terms of high speed with high resolution is enticing.  

Table I lists new HPLC product introductions (alphabetically by vendor) at Pittcon 2013 or in the prior year, followed by descriptions and commentaries of each product, categorized as systems, modules, data systems, and software.  

Table I: Summary of new HPLC product introductions at Pittcon 2013			

2013 appears to be a lean year for HPLC introductions. With most manufacturers having UHPLC systems in their product line, vendors are offering product extensions or specialized applications systems. The following are some examples of new systems or system extension offerings. 

Agilent 1290 Infinity Quaternary LC system — Low-pressure mixing quaternary UHPLC pumps are available from many manufacturers and generally have lower pricing but less desirable higher delay volumes. Agilent has closed the gap this year by the introduction of the 1290 Infinity quaternary pump equipped with an innovative inlet Jet Weaver mixer. The pump has a delay volume of ~350 µL and is designed for HPLC method development. This quaternary pump has a lower price than the 1290 Infinity binary pump (~20%) and can operate with a "software-selectable" post-pump mixer for high-sensitivity applications. 

Agilent 1290 Infinity 2D-LC system — Two-dimensional LC (2D-LC) is a useful platform technology for complex samples such as proteomics, metabolomics, biopharmaceuticals, peptide maps, biofluids, plant extracts, food matrices, polymers, and genotoxic impurities. Several manufacturers have 2D-LC offerings and their ranks are joined by Agilent this year. Agilent's 2D-LC system uses 1260/1290 Infinity system hardware in the first dimension and a 1290 Infinity binary pump in the second dimension. It uses specialized valving for comprehensive or heart-cutting operation. The system's multiple detectors can all be configured and controlled by a single Agilent ChemStation chromatography data system (CDS). (See Figure 1 for a screen display of the enhanced OpenLab CDS ChemStation edition software version capable of both system control and data handling for both dimensions from the 2D-LC system). 

Figure 1: Agilent Infinity 2D-LC Solution - OpenLab ChemStation with direct control and data handling for both dimensions (software screen display for dual systems control and valving configuration shown here).			

Bio-Rad NGC chromatography system — This system is a new medium-pressure PEEK-based protein purification system. It is a flexible, modular plug-and-play system with easy-to-use touch-screen software for module configuration, system or method control, and data handling and display. Two pump modules are available (for 10- and 100-mL/min flow rate capabilities with pressure limits of 3650 or 1450 psi, respectively). Optional modules can be plugged into a highly flexible system frame and include sampling pumps, mixers, detectors (UV single or multiple-wavelength, conductivity, pH monitoring), injector or autosampler, column switching valves, and fraction collectors. This system targets biomolecule purification at the research, process development, and laboratory-scale levels. 

Hitachi Primaide HPLC system — Hitachi High Technologies America has focused on the lower-cost HPLC market segment for routine testing and quality control. Primaide (pronounced as "prime aide") is the first 6000-psi system in the series featuring "affordability" for routine testing. Only UV–vis and photodiode-array detectors are available. System control and data handling are performed using Primaide System Manager or Agilent OpenLAB CDS EZChrom Edition software. 

The Shimadzu's Nexera X2 UHPLC system — This second-generation system targets routine analysis in regulated environments demanding complex system setups. New modifications and options include an improved inlet valve, an enhanced degassing unit, a low-delay-volume quaternary pump, and an improved SPD-M30A photodiode-array detector with higher thermal stability, sensitivity (noise = 0.4 × 10

  AU) and spectral resolution. The system's standard flow cell has a volume of 1 µL with a pathlength of 10 mm. A high-sensitivity flow cell with an 85-mm pathlength will be available shortly. A unique iPDeA (intelligent peak deconvolution analysis) software allows for better quantitation of coeluted peaks by using a peak deconvolution algorithm based on spectral characteristics of the analytes. Figure 2 shows comparative baseline data of the SPD-M30A detector vs. that of a competitor's product in varying room temperature environments. 

Figure 2: Comparative detection baseline traces vs. room temperature changes demonstrating the improved thermal stability of the SPD-M30A detector in the  Shimadzu Nexera X2 UHPLC system vs. that of a competitor’s product.			

Thermo Scientific Dionex UltiMate 3000 Biocompatible Rapid Separation (BioRS) system — Bioinert UHPLC systems designed for use in harsh, high-salt mobile phases for analysis of biomolecules have become available in prior years from several vendors. This new titanium-based UHPLC system is designed to support high-resolution separations for biopharmaceutical R&D and quality control applications at pressures as high as 15,000 psi. The system was introduced with a new line of ion-exchange and hydrophilic interaction liquid chromatography (HILIC) columns for the analysis of recombinant biopharmaceuticals, monoclonal antibodies, proteins, peptides, and DNA. Options include a wide selection of pumps, detectors, and fraction collectors. Figure 3 shows an example of its precision performance in high-resolution peptide mapping.  

Figure 3: Overlaid chromatograms of 14 peptide maps demonstrating the performance of the Thermo Scientific Dionex 3000 Ultimate BioRS UHPLC system.			

Thermo Scientific Dionex ICS-5000+ Reagent-Free HPIC system — This system is the first PEEK-based reagent-free ion chromatography system capable of continuous operation at pressures as high as 5000 psi that is designed to work with capillary, microbore, or standard columns. Applications supported include high-speed and high-resolution analysis of anions, cations, organic acids, amines, carbohydrates, and amino acids. This system debuted with the new ICS-4000 QD charge detector; charge detection is a low-cost detection technique with universal and linear calibration supplementing the traditional suppressed conductivity detection. 

The Waters Acquity Advanced Polymer Chromatography (APC) system — This low-dispersion gel permeation chromatography (GPC) system is optimized for the analysis of organic polymers in aqueous and organic mobile phases. The system features a new refractive index (RI) detector designed for a new set of sub-3-µm hybrid polymer columns. Key benefits are faster analysis, higher sensitivity, and accurate measurements of molecular weights and distribution. System control and data handling are performed by Waters Empower 3 CDS with optional GPC software. Figure 4 shows the comparative performance of this new system–column combination vs. that of a conventional system in polymer analysis. 

Figure 4: Polymer analysis chromatograms comparing the performance of (a) a conventional GPC system with (b) Waters Acquity APC system with a new set of 3-Âµm hybrid columns. Columns: (a) 300 mm X 7.8 mm Styragel (3X); (b) 150 mm X 4.6 mm Waters APC (3X).			

Several modules introduced as stand-alone units are described here. 

Agilent 1260 and 1290 Infinity evaporative light-scattering detector — This evaporative light-scattering detection (ELSD) system provides sensitive detection and gives a near-universal response for low- or non-chromophoric, nonvolatile compounds. The unit is adaptable to both HPLC and supercritical fluid chromatography (SFC) and has a subambient mode for detection of semivolatiles and a standby mode for reduced nitrogen consumption.  

				PerkinElmer Aurora photodiode-array (PDA) detector — This new detector has improved performance specifications (better wavelength accuracy and low noise, drift, and stray light) and enhanced low-dispersion flow cells (1 and 5 µL with pathlengths of 10 and 50 mm, respectively).			

Scientific Systems, Inc. (SSI) HF Series pump — This pump is designed for preparative LC and column packing operations. The high-performance, relatively low-cost, dual-head pump is capable of delivering precise flow rates as high as 300 mL/min at pressures as high as 10,000 psi. 

Wyatt Technology Optilab UT-rEX RI detector — Wyatt Technology, known for its innovative instrumentation and software for absolute macromolecular characterization, has developed a new RI detector. Wyatt claimed this detector to be the first RI detector designed for UHPLC. It is equipped with a 1.5-µL flow cell. It has excellent sensitivity and linearity performance, and can operate in a temperature range of 4–50 °C. It is compatible with UHPLC systems from all manufacturers.   

Chromeleon 7.2 — Thermo Scientific Dionex introduced a major upgrade to its flagship client–server network CDS, Chromeleon. The new version 7.2 has an improved graphical user interface (GUI) built on the Windows 

 framework. This CDS boasts a more effective mass spectral data interface for viewing data from both LC–mass spectometry (MS) and gas chromatography–mass spectrometry (GC–MS), a common weakness for CDS software. 

HPLC Method Development and Other Software

S-Matrix QbD LC method development software for Agilent ChemStation — S-Matrix Corporation has offered Fusion AE HPLC method development software (FMD) for Waters Empower for several years. It now offers a similar product for Agilent ChemStation/OpenLab. FMD automates method development experiments via principles of design of experiments (DoE) and quality by design (QbD). The new version has enhanced experiment design features to automate the on-line preparation of mobile phases to study variables such as buffer or salt concentration and pH.  

Another software product of interest is the company's LC Method Validation (FMV) software, which automates the time-consuming method validation process required for good manufacturing practices (GMP). FMV provides a full suite of method validation experiments for multiple compounds, including rigorous robustness testing. FMD and FMV now fully automate these experiments on Agilent's Infinity Series or 1100/1200 LC systems. Figure 5 shows a screen display of the Fusion AE software program demonstrating its feature for graphical visualization of design space and proven acceptable ranges of an optimized method from a DoE run. 

Figure 5: Fusion AE LC method development software screen display showing visualization of design space and proven acceptable ranges from an optimized method.			

Spectrus Data Base — ACD's AutoChrom Method Development System software is now available with a new Spectrus Data Base feature to provide enhanced back-end connections for better data management and integration. 

Agilent Intelligent System Emulation Technology (ISET), second release — This software is designed to facilitate method transfer and method robustness evaluations. This new version allows the Agilent Infinity 1290 system to execute legacy methods using Waters Alliance LC systems to obtain similar chromatographic results by operating the 1290 system under an Alliance emulation mode without physically modifying the methods or system hardware.  

This review summarizes new HPLC product introductions (systems, modules, chromatography data systems, and software) at Pittcon 2013 and in the prior year. It highlights innovative features and technical details of these new products or extensions from a user's perspective. The process of collecting information for this review started in December 2012 with questionnaires sent to all HPLC manufacturers to solicit information on pending product introductions. This was followed-up with direct communication to marketing staff and actual visits to the Pittcon exposition booths. My goal was to be inclusive, thorough, and objective, with a focus on relevant innovations of interest to our readership. Note that new HPLC columns introduced at Pittcon are covered by Ron Majors in his "Column Watch" annual Pittcon review series.  

Personally, I continue to be baffled by vendors electing not to showcase their equipment at Pittcon. I do understand the many rationales of alternative marketing strategies, tight budgets, rising travel costs, and return-on-investment considerations. Nevertheless, missing Pittcon means missing the huge opportunities for face-to-face meetings with a vast pool of potential customers, old friends, and new scientists as well as missing the excitement of seeing new competitive products on the exposition floor. After all, Pittcon is the premier global analytical chemistry conference with a tradition of excellence for 64 years. In my view, a continuing presence by a manufacturer, albeit with smaller floor spaces and core staff, is clearly preferred over a sporadic showing or complete absence in this global annual venue. 

The opinions expressed in this article are solely those of the author and bear no reflections on those from 

 magazine, the Pittsburgh Conference, or other organizations. Inaccuracies and inadvertent omissions are the responsibilities of the author alone.    

The author thanks the marketing staff of all manufacturers who provided timely responses to the

questionnaires. The author is grateful to Drs. Davy Guillarme of the University of Geneva and Raphael Ornaf of Vertex Pharmaceuticals, and Marian Nardozzi of Pittcon for providing useful inputs and comments. 

(1) J.E. MacNair, K.C. Lewis, and J.W. Jorgenson, 

, D. Guillarme, J.-L. Veuthey, and R.M Smith, Eds. (Royal Soc. Chem., Cambridge, UK, 2012), Chapter 2. 

D. Guillarme, J.-L. Veuthey, and R.M. Smith, Eds. (Royal Society of Chemistry, Cambridge, UK, 2012).  

(5) M.W. Dong, "UHPLC in Pharmaceutical Analysis: Perspectives, Practice and Potential Issues," presented at The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon), Philadelphia, Pennsylvania, 2013.  

(6) J. Jorgenson, "Future Trends in UHPLC," presented at The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon), Philadelphia, Pennsylvania, 2013. 

 is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. He holds a PhD in analytical chemistry from the City University of New York and a certificate in Biotechnology from U.C. Santa Cruz. He has conducted numerous courses on HPLC/UHPLC, pharmaceutical analysis, HPLC method development, drug development process, and drug quality fundamentals. He is the author of Modern HPLC for Practicing Scientists and a co-editor of Handbook of Pharmaceutical Analysis by HPLC. He is a member of the editorial advisory board of 

Articles

Perspectives in Modern HPLC

A summary of new HPLC systems, modules, and software, focusing on innovations and unique features from a user's perspective.

HPLC Systems and Modules Introduced at Pittcon 2013: A Brief Review

Part I of our yearly report on new products introduced at Pittcon. This month, columns are highlighted, covering all modes of high performance liquid chromatography (HPLC), ultrahigh-pressure liquid chromatography (UHPLC), and liquid-phase separations. 

The Pittcon 2013 Conference and Expo (less formally known as the 64th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy) was held for the first time in the city of Philadelphia, Pennsylvania, on March 17–21, 2013. This year's event hosted over 1011 instrument manufacturers and laboratory suppliers in more than 1925 booths. In addition to attending the exposition, the conferees listened to more than 2200 technical presentations (orals, posters, workshops, invited and contributed talks, and award symposia), checked on numerous company seminar rooms, or attended one of 100 short courses. 

Although the attendance was slightly up this year compared to last year, the average attendance had been shrinking over the last several years. Still, Pittcon remains one of the most important international analytical exhibitions where companies introduce their latest instruments, instrument accessories, software, columns, and sample preparation and other consumable products. Several notable companies were missing again this year, but enough were present such that visitors had plenty of opportunities to investigate new (and old) technologies. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2013. In some cases, products that were introduced during 2012 but after Pittcon 2012 (1,2) may be included for reasons of completeness. The information is based on manufacturers' responses to a questionnaire mailed in early 2013. 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 Philadelphia. 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 may be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples.   

In this month's coverage, I will describe new column introductions in the areas of HPLC, reversed-phase LC, normal and bonded-phase LC, hydrophilic interaction liquid chromatography (HILIC), supercritical fluid chromatography (SFC), ion-exchange and ion chromatography, size-exclusion chromatography (SEC), and specialty chromatography. Next month, I will look at gas chromatography (GC) columns, sample preparation products, and hardware, accessories, and small tabletop instruments, mainly for sample preparation. 

 This year, I observed that there was a continuing trend in high-efficiency chromatography driven by dramatic increases in chromatographic efficiency, especially in HPLC or ultrahigh-pressure liquid chromatography (UHPLC). In addition, the introduction of more products with inert surfaces driven by instrumental increases in sensitivity especially through increasing use of tandem mass spectrometry (MS) techniques in both GC and LC. The development of systems with more inert flow paths including more PEEK-lined tubing, newer deactivated packing materials, deactivated surfaces such as fused-silica columns, vials, metal tubing, and GC injection liners are all being addressed to improve recovery of very low levels of analytes. 

In HPLC, there was a further increase for instrumental enhancements, hardware components, and accessories designed to work with UHPLC. Many application-specific columns for HPLC and GC that are tested with "real world" samples before shipment were noted. Again at Pittcon, a considerable number of new accessories, hardware, and devices were introduced that are designed to make the chromatographic laboratory more productive. 

 Overall, there were no real breakthroughs in HPLC column technology, but a refinement in particle sizes, stationary-phase technologies, and column dimensions was noted. Pittcon 2013 saw the introduction of a few more sub-2-µm columns for UHPLC, but not to the extent of several years ago. However, many new superficially porous particles (SPP, also known as core–shell), both particle sizes and phases, made their appearance including particles as small as 1.3 µm. UHPLC has now established itself as a viable solution to today's separation problems; at least nine major instrument vendors now address this market with instruments capable of operation at pressures as high as 19,000 psi. This year a few more HILIC columns appeared, but alternative reversed-phase selectivities, especially phenyl-hexyl and penta-fluorophenyl phases, were evident at the booths of a number of column companies. Still, in the number of introductions, reversed-phase HPLC maintained its dominance. Application-wise, chiral, protein (particularly monoclonal antibody columns), and ion chromatography or ion-exchange columns dominated. 

 GC column introductions were dominated by new stationary phases, especially with new levels of inertness. However, chromatographers in the food, environmental, and forensic areas are finding that there is more than column inertness that must be addressed. Thus, ultra-inert total flow systems are under development, driven by the increased sensitivity of mass spectrometers. Porous layer open tubular (PLOT) columns with built-in particle traps to capture any tiny solid phases flaking off the columns made their appearance. Specifically, chiral compounds, polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), semivolatiles, and fatty acid analyses were addressed in the applications' domain. A new polar ionic liquid column with much higher temperature capability than typical wax columns was shown for the first time. 

 Sample preparation products for automation, such as 96-well filtration and solid-phase extraction (SPE) plates and plate seals and SPE micropipette tips, seemed to be in vogue. Because of the popularity of protein-crash approaches for the analysis of drugs and metabolites in biological fluids, filters (96-well plates and single tubes) designed to remove both precipitated protein and lipids or phospholipids were introduced. Affinity SPE phases for proteins and aflatoxins as well as a family of mycotoxins removal cartridges were noted. QuEChERS (quick, easy, cheap, effective, rugged, and safe) is still "catching on" as the favored sample preparation technique for pesticides in fruits and vegetables. Some interesting phase separation columns might bring renewed interest to liquid–liquid extraction. 

 More than 40 new hardware products, accessories, and benchtop instruments for LC, GC, and sample preparation were noted. This year I broke the hardware and accessories introductions from the benchtop instrumental introductions into two separate tables (see the upcoming May installment). UHPLC-specific products (tubing, fittings, column hardware, and in-line filters) with higher pressure ratings and decreased dead volumes were introduced. Several automated sample preparation instruments contribute to minimize the labor-intensiveness and drudgery of manually preparing samples for chromatography were introduced by smaller instrument vendors. This year a number of workstations that do some (but not all) of the sample preparation steps were evident. Products for preparing solid materials for further workup included homogenizers, shakers, and solvent evaporation stations. A unique sample card for direct analysis in real time (DART) MS was introduced that did away with lengthy sample preparation steps. 

Each year manufacturers introduce entire new families of HPLC columns at Pittcon. For the sake of brevity, I have elected to cover those series here instead of listing each column under individual headings in the modes' tables. In some cases, the series are extensions of existing products but cover additional phases; in other cases the families are entirely new to the market. 

New series of HPLC columns were shown by several companies (Table I). With a wide range of particle sizes (made by the same process and chemistry) from UHPLC to preparative, seamless scaleup and scaledown within a family is possible. Method development can be performed on a short, fast analytical column and then can be scaled up to preparative dimensions with easy method transfer. Most introductions of new series included both sub-2-µm and larger particle versions. 

It seems like UHPLC is here to stay. Most new column introductions throughout this article have at least one sub-2-µm particle size in its family. The obvious advantage of such small particles is better column efficiency so that shorter columns can be used to accomplish the same separation as achieved on a longer column. The end result is that separation times are greatly decreased in proportion to the column length reduction. In addition, shorter columns with the same internal diameter run at the same flow rate save a proportional amount of solvent, which is always important these days with the cost of everything on the rise.  

But the big news is that the introduction of SPP columns continues with a wild expansion of more phases and particle sizes from existing suppliers. These columns have caught on not only with those who have older, 400-bar systems, but also with those using UHPLC systems. In Table I, several companies have greatly expanded their lines of SPP well beyond the normal C8 and C18 reversed phases. In addition, you will find later in this report of individual LC modes, additional SPP columns from other vendors. Truly, this new type of column seems to be catching on with the masses. 

The SPP boom started out with particles sizes in the 2.6–2.7 µm range. These products gave efficiencies equivalent to the sub-2-µm columns that have been introduced over the last several years, but with about half of the pressure drop when compared to sub-2-µm columns with the same dimensions. If one compares columns operating at the same pressure, the SPP columns provide twice as many theoretical plates. Three years ago, Phenomenex came out with a 1.7-µm SPP (Kinetex) that gave higher plate counts than equivalent porous particles. Unfortunately, the instruments at the time were not of low enough band dispersion to provide the plates that such a small SPP should present. This year, Phenomenex has even gone lower to a 1.3-µm particle diameter with an array of their bonded phases. On the opposite side, other SPP companies have now gone to larger particles, 5 µm. The same concept holds true here. A 5-µm SPP gives the same efficiency as a 3-µm porous particle but the pressure drop is dramatically lower that the 3-µm column (of the same dimensions) and equivalent to a 5-µm porous particle column. It will be of interest to see if chromatographers gravitate to the larger-particle SPP columns as replacement for their 5-µm columns. Presumably, the United States Pharmacopeia will permit the use of these columns in validated methods. 

As is usual, many new reversed-phase chromatography columns were introduced at Pittcon 2012 (see Table II). If one also adds all the columns capable of reversed-phase chromatography interactions covered in the new series (Table I) and in specialty columns (Table III), one can clearly see the dominance of this operational mode. This year at Pittcon more than 64 new reversed-phase chromatography columns were added to the huge numbers that already exist from past introductions. Silica-based bonded phase columns dominated the introductions again this year (~95%), but three organic–silica hybrids and one polymeric column were also on the scene.  

Table II: Reversed-phase chromatography  			

The C18 (octadecylsilane) functionality proved again to be the most popular with nearly 30% of all reversed-phase column introductions being this alkyl phase. However, this phase isn't always the optimum for all separations and a great number of other hydrophobic stationary phases were introduced. Besides the normal C8, C4, and phenyl phases, C18 with mixed functionality (such as di-isopropyl sterically protecting groups), phenyl-hexyl, diphenyl, embedded polar, pentafluorophenyl (PFP), cyanopropyl, AQ, and C30 phases were made available by various suppliers to present selectivity alternatives. HILIC columns that have some hydrophobic interaction possibilities are found later in this report. 

A number of PFP columns were introduced this year. This phase has pentafluorophenyl groups bound to the silica surface and provides a unique aromatic selectivity because of highly electronegative fluorine atoms on the periphery of the phenyl ring ligand. It is recommended for the separation of aromatic and halogenated compounds and offers a different selectivity compared to C8 and C18 columns. To illustrate the separation possibilities, Figure 1 shows the rapid baseline separation of six catecholamines on the Tokyo Chemical Industries 5-µm Stella PFP column using gradient elution. 

Figure 1: Separation of catecholamines on a TCI Stella PFP column. Column: 150 mm Ã 4.6 mm, 5-Âµm dp TCI Stella PFP; mobile-phase A: 5 mM formamide, adjusted to pH 3.5 with formic acid; mobile-phase B: methanol; gradient: 5â20% B in 10 min; flow rate: 1.0 mL/min; detection: UV absorbance at 254 nm; temperature: 25 Â°C. Peaks: 1 = noradrenaline, 2 = adrenaline, 3 = dopamine, 4 = tyramine, 5 = isoproterenol, 6 = serotonin. (Courtesy of Tokyo Chemical Industries.) 			

In recent years, there has been a trend in introductions of bonded silica-based columns with wider pH limits. Most bonded phase silicas are recommended to be used in the 2–8 pH range. At low pH (<2), the bonded phase, especially those with short chain lengths, will hydrolyze, leaving behind bare silica. Consequently, retention times will generally change and peak tailing becomes a big problem. At high pH (>8), the silica gel itself will begin to solubilize; this process is dependent on the solvent, type of buffer, and temperature. By bonding carbon phases with sterically protecting groups, with high coverage of long alkyl groups, or by coating the silica with polymeric material, the lower pH range can be extended. By using organosilica hybrid chemistry, bidentate bonding, and polymer coatings or encapsulation, the range can be further extended to pH 12. This year YMC America, Inc., introduced Triart Phenyl hybrid columns with an encapsulation design that provides an extended range of pH 1–10. Advanced Chemical Technologies introduced a C18 column with a pH range of 1.5–12.  

To show the value of having a selection of HPLC columns with different reversed-phase stationary phases in one's column inventory, Figure 2 provides a nice comparison of six different YMC reversed-phase columns run isocratically with the same phosphate buffer–methanol mobile phase. One can clearly see different elution orders as a function of the types of interactions these different test analytes (acid, basic, and neutral) showed. Most chromatographers think of C18 first when it comes to choosing a column, but this study shows that an alternative selectivity might actually be faster or provide more selectivity for analytes of interest. 

Figure 2: Selectivity comparisons of a family of Triart columns using the same isocratic conditions: (a) C18, (b) C8, (c) Phenyl, (d) PFP, (e) YMC-Pack Ph, (f) YMC-Pack CN. Column dimensions: 150 mm Ã 3.0 mm, 5-Âµm  d p; eluent: 25:75 20 mM H3PO4âKH2PO4 (pH 3.1)âmethanol; flow rate: 0.425 mL/min; temperature: 40 Â°C; detection: UV absorbance at 265 nm; injection volume: 4 ÂµL. (Courtesy of YMC America, Inc.) 			

Columns for Supercritical Fluid Chromatography

Recently, there has been a resurgence of interest in SFC. With major instrument companies now pushing the technology, more users are trying out the technique to see what it might do for them. Columns companies are correspondingly addressing the column needs. Most columns for SFC are based on HPLC-like packings but often they contain different phase chemistries than those normally used in HPLC. Although normal bonded-phase materials have been used, phases like 2-ethylpyridine, which are not found in the LC laboratory, are popular for the separation of basic compounds. At Pittcon this year, Nacalai USA/ChromaNik Technologies introduced its SunShell 2-ethylpyridine SFC column bonded onto a 2.6-µm SPP silica base material. Because supercritical fluid carbon dioxide used as a mobile phase already has a low viscosity when it is run on an SPP, rapid separations at very low pressure are a result.  

The structural similarities between polar lipids such as cholesterol and related analogs have posed chromatographic challenges to analysts interested in quantifying these potential biomarkers. CosmosilQuinoline is another SFC phase from Nacalai Tesque, based on 5-µm porous (120-Å) silica, that has been developed to improve the separation of these structural isomers by using the π-π interactions and structural rigidity of the naphthylethyl phase and the hydrogen bonding of the pyridine phase.  

ES Industries, who recognized early that SFC would catch on, has been making SFC phases for a few years. Their product offering is called GreenSep because SFC with carbon dioxide mobile phase is considered biofriendly. GreenSep ODS-P is a silica-based product (120-Å pore size) with a C18 bonded phase but with special polar endcapping. Columns are available in particle sizes ranging from 1.8 to 20 µm and with dimensions from nano to preparative scale. For the ODS-P column, the phase coverage is 10% carbon. The column is said to be the first C18 phase specifically designed for SFC separations. The GreenSep DS-8 column is a rather special phase consisting of a mixture of phenyl and imidazole. Thus, it exhibits strong π-π interactions and provides an alternative selectivity to the ODS-P column. The phase has been found to be highly selective for the separation of diastereomers containing π electrons. Except for the stationary phase, the base silica, particle size, and column dimensions are the same as the ODS-P. 

In the past few years, only a few new SEC columns have been introduced at Pittcon. That doesn't mean that SEC is not a highly practiced separation mode. It is a standard technique for the separation and characterization of organic polymers, both organic- and water-soluble. In addition, the mode is widely used for the separation of proteins and other biopolymers, sometimes known as gel filtration chromatography. Four companies (Phenomenex, Showa Denko, Waters, and Thermo Fisher Scientific) introduced columns for SEC at Pittcon 2013. Three of the introductions were earmarked for aqueous size separation of biomolecules and water-soluble polymers, and a new multipore column and small-particle hybrid particle columns for gel permeation chromatography were also seen. 

The Phenomenex offering was a family of 3-µm silica-based aqueous SEC columns called Yarra. The family consists of three different pore sizes: 145-Å Yarra SEC-2000, 290-Å Yarra SEC-3000, and 500-Å Yarra SEC-4000. Biocompatibility is achieved by bonding a proprietary hydrophilic ligand to the base silica. The stainless steel column dimensions were 300 mm × 4.6 and 7.8 mm and 150 mm × 7.8 mm. To protect the analytical columns, SecurityGuard cartridges are available for all three phases. The pH range for all three columns is 2.5–7.5, and the maximum flow rate for the two lowest pore sizes is 1.5 mL/min and for the 500 Å it is 1.2 mL/min. Suggested applications are proteins; peptides; PEGylated proteins, peptides, and IgG; biosimilars; antibodies; IgG aggregrates; and other biomolecules. 

Thermo Fisher Scientific's Acclaim SEC columns for aqueous SEC are the company's new entry into this field. Instead of silica, the packing in the Acclaim column is a spherical, hydrophilic, monodispersed multipore polymethacrylate resin with a pH range of 2–12. Two pore sizes are available: the popular 300-Å size (Acclaim SEC-300, 5-µm 

) and a wide-pore 1000-Å size (Acclaim SEC-1000, 7-µm 

). Together these two pore sizes cover a calibration range of 100–1,000,000 Da. Analytical and semipreparative columns (300 mm × 4.6 mm, and 300 and 150 mm × 7.8 mm) along with matching guard columns are available. The recommended flow rate range is 0.2–1.0 mL/min. The Thermo phases are designed for the separation of water-soluble polymers and oligomers. The multipore resin provides a wide calibration curve without inflection points. The stable surface bonding provides low column bleed and is thus compatible with UV detection, refractive index (RI) detection, MS detection, evaporative light-scattering detection (ELSD), and charged aerosol detection. 

The Showa Denko Shodex GPC-LF-806 column is packed with a 9-µm styrene–divinylbenzene copolymer gel that is also a multipore type of packing material. It offers an extended molecular weight operating range compared to the GPC-LC-806 column; its exclusion limit is 15,000,000 Da (polystyrene as a standard). Thus, it is especially suitable for molecular weight distribution analyses of polymers that have a very large molecular weight (over 2 MDa) and for the separation or characterization of polymers in a wide molecular weight range. The linear calibration curve for this column shows no inflexion points. Typical solvents that are compatible with the column include tetrahydrofuran, dimethylformamide, chloroform, dimethylacetamide, and dimethyl sulfoxide. The column has a working temperature range of 20–60 °C. 

Waters' entry consisted of new sub-3-µm Acquity APC (Advanced Polymer Chromatography) columns that were tied to a low-dispersion SEC instrument to take full advantage of the more efficient particle size. The columns are based on ethylene bridged hybrid (BEH) chemistry, which forms a stable bed that resists the swelling and shrinking that occurs with conventional polymeric stationary phases. The APC XT range of columns is designed for organic-based separations, and the APC AQ columns are compatible with aqueous-based separations. 

Ion-exchange- and ion-chromatography are interchangeable in that they both use the same technology and often the same phases. Dionex, now firmly established under the Thermo Fisher Scientific umbrella, has long been an innovator in the separation of ionic species. Capillary columns are typically columns with internal diameters under 0.5 mm. They have two main advantages. The first is higher sensitivity in a sample-mass-limited situation. If the same mass of sample is injected onto a column with a smaller internal diameter, the peak height will increase and perhaps will enable the measurement of smaller analyte concentrations. Second, to maintain the same separation time, the linear velocity must be the same. For a column with a smaller internal diameter, this means that the flow rate must be decreased proportionally to the inverse radius-ratio squared. This result is lower usage of solvent. 

Last year, Dionex introduced a new family of 250 mm × 0.4 mm capillary ion chromatography columns. This year the company has expanded that capillary line to include the CarboPac PA-10 phase, a quaternary amine anion-exchange column, recommended for the separation of mono- and disaccharides in foods, drugs, and plants as well as those found in mammalian glycoproteins. Separation is accomplished with a simple isocratic eluent pulsed amperometric detection (PAD) without any derivatization required. A matching guard column (50 mm× 4.6 mm) is available. Figure 3 shows an isocratic baseline separation of saccharides using a 250 mm × 0.4 mm version of this column. The two chromatograms show the effect of adding a guard column to the separation system. Because of the added length (an additional 50 mm), retention time is increased proportionally, in this case by about 20%. Using a guard column is a very good way to protect the expensive analytical column but one should make sure that the added dead volume doesn't affect peak efficiency. By good hardware design, the guard column should have little or no effect on the peak widths or tailing factors. 

Figure 3: Separation of saccharides on a CarboPac PA10 column (a) with and (b) without a guard column. Column: Dionex CarboPac PA10 capillary; eluent: 100 mM potassium hydroxide; eluent concentration: -40 min 100 mM (strong wash), -25 min 100 mM, -24.9 min 18 mM (equilibration), 0 min 18 mm (loadâinject), 20 min 18 mM; eluent source: EGC-KOH cartridge; temperature: 22 Â°C; flow rate: 0.01 mL/min; injection volume: 0.4 ÂµL/ detection: integrated amperometry, quadruple pulse waveform; working electrode: PTFE Gold, disposable; reference electrode: AgâAgCl. Peaks: 1 = fucose, 2 = galactosamine, 3 = glucosamine, 4 = galactose, 5 = glucose, 6 = mannose. (Courtesy of Thermo Fisher Scientific.) 			

The other capillary column to join this family is the IonPac AS16 column, recommended for analyzing trace perchlorate in drinking water in accordance with US EPA Methods 314.0 and 314.1 (Primary Method). The Dionex IonPac AS16 column simplifies the determination of polarizable anions, including thiosulfate, iodide, thiocyanate, and perchlorate using an isocratic hydroxide eluent. The column has the same dimensions as the CarboPack PA-10 column but has alkanol quaternary ammonium functionality.  

For both columns, the capillary format is packed with the same material as the equivalent standard-bore and microbore versions (providing the same performance as 4-mm and 2-mm columns, respectively), but requires only 1/100th the eluent flow rate. The capillary format provides the advantage of significantly reduced eluent consumption, which helps reduce operating costs. The IonPac AS16 capillary column is also used in the second dimension of the two-dimensional ion chromatography method for the analysis of trace perchlorate (sub-parts-per-billion levels) in drinking water. 

Specialty columns are HPLC columns that have been developed for specific separations that are difficult to achieve on a standard column. However, sometimes manufacturers will use a standard column but will test it specifically for a certain class or compounds and provide a recommended set of chromatographic conditions. In some cases, the specialty column comes as part of a "total solution" kit with reagents, standards, and a method. Most specialty columns will be delivered with a test chromatogram from an analysis performed at the factory before shipment, and some are guaranteed for a specific separation. Table III shows the 18 specialty columns that were presented at Pittcon 2013. Silica gel–based columns dominated the introductions, but polymer-based specialty columns and packings were also introduced.  

As depicted in Table III, the three main application areas that dominated the specialty column introductions were proteins, particularly antibodies; chiral compounds; and peptides. The protein columns were based on multiple separation modes (cation-exchange, anion-exchange, reversed-phase, size-exclusion, affinity, and mixed mechanisms). For many years, the study of monoclonal antibodies has been a hot area in the potential development of biopharmaceuticals for diagnostics and clinical uses. A number of monoclonal antibody–based drugs have been approved by the U.S. Food and Drug Administration. For the purification and polishing of very large biomolecules like viruses, the Capto Core 700 from GE Healthcare uses a size-exclusion mechanism to keep the biomolecules from entering the rather large pores while small contaminants will enter the pores and interact with either hydrophobic groups or anion-exchange functional groups and be trapped.  

Chiral separations are still in the forefront especially in the pharmaceutical industry. Six new chiral HPLC columns were introduced at Pittcon 2013 (see Table III), based on modified cellulose, amylose (polysaccharide), and protein phases. An interesting zwitterionic quinidine-derived phase that separates underivatized amino acid- and peptide-enantiomers was shown by Chiral Technologies. In addition, Chiral Technologies expanded its offering of immobilized polysaccharide-derived chiral stationary phases, first introduced in 2008 at Pittcon. These immobilized phases are more stable than the coated phases and have found use in both HPLC and SFC. Being immobilized, they can be used with a wider variety of mobile phases and pH values. Since many analytical separations are scaled up to obtain larger quantities of purified enantiomers, preparative particle sizes are frequently part of column introductions, hence some particles as large as 20 µm were displayed. 

Column Watch

Part I of our yearly report, covering columns for all modes of HPLC, UHPLC, and liquid-phase separations.

New Chromatography Columns and Accessories at Pittcon 2013, Part I

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Is it possible to get the same separation under isocratic and gradient conditions?

In last month's "LC Troubleshooting" (1), we began our series of discussions on gradient elution. We saw that liquid chromatography (LC) separations under isocratic and gradient conditions had some of the same properties when operating conditions are changed. For example, when run times are made longer, resolution tends to improve, peaks broaden, and peak height drops. Longer run times are obtained in reversed-phase LC by reducing the concentration of the organic solvent (%B) in isocratic separations or increasing the gradient time (reducing the gradient steepness) with gradients. In this installment, we'll see how to compare gradient and isocratic separations under "equivalent" conditions.  

In Figure 1a, we see a chromatogram for the isocratic separation of 15 compounds at 60% B. Several deficiencies are seen. The peaks early in the chromatogram are narrow and tall, but the first two are poorly resolved. At the end of the chromatogram, the peaks are well separated, but they are broadened excessively. The run takes nearly 30 min. If we try to improve the separation at the beginning of the chromatogram, we would reduce the %B in the mobile phase. This would increase the retention of the early peaks, and in general is expected to improve the resolution. However, the peaks at the end of the run would broaden more and move to even longer retention times — neither of which is desirable. Alternatively, we could improve the separation at the end of the chromatogram by increasing the %B, which would reduce the retention times, close up some of the gaps between the peaks, and sharpen the peaks. But the higher %B would compress the chromatogram at the beginning, making it even worse.

Samples such as in Figure 1a are a good example of 

. In such cases, it is difficult or impossible to get a satisfactory separation for all the peaks under a single isocratic condition. We can improve the beginning or end of the chromatogram, but not both at the same time. The reason for this is that the polarity range of the sample is too large. Recall that under the most desirable conditions, we'd like the retention factor, 

 < 20 can be obtained, we often can find acceptable separation conditions. In the present case, 0.9 < 

 range (34/0.9 ≈ 38) is approximately twice the maximum we would like (20/1 = 20). When samples such as this are encountered, a gradient is likely to give a better separation. 

When the sample of Figure 1a is run under gradient conditions, we get the separation of Figure 1b. We can quickly see that the separation is improved. The resolution between peaks is increased at the beginning of the run and the excess time between peaks at the end of the run in Figure 1a is reduced. Thus, the peaks are spread out more evenly across the chromatogram. Also, note that the peak widths throughout the run are approximately the same. Narrower peaks at the end of the run translate into taller peaks, so detection is improved in this part of the chromatogram. Finally, for the present sample, the run time is reduced by a factor of four. All these improvements make the gradient a better choice for this sample. (Gradients, however, are not always better, as we saw in last month's "LC Troubleshooting" [1] in which an isocratic separation was a better choice.) 

Figure 1: Simulated chromatograms for the separation of a 15-component sample on a 150 mm Ã 4.6 mm column operated at 2 mL/min: (a) isocratic separation at 60% B, (b) gradient separation of 50â100% in 5 min, and (c) isocratic separation of subsets of 15-component sample such that k â 3 for each group. Peak identity and %B are noted on chromatograms. 			

Comparing Isocratic and Gradient Separation

Next, let's look at isocratic and gradient separation under so-called equivalent conditions. By this we mean mobile-phase conditions under which the retention and peak properties are roughly the same with both isocratic and gradient runs. 

It should be obvious that if we consider only a single peak, we should be able to find isocratic and gradient conditions that will give approximately the same retention time and peak width. To illustrate this, I've broken the 15-component sample into five subsamples in Figure 1c. In each case, I've adjusted the isocratic %B so that 

 ≈ 3 is obtained for the peaks. For example, at 49% B in Figure 1c, peak 1 has a retention time, 

 = 3.2 min. Under these conditions, the column dead time, 

 value at 49% B is 3.07 ≈ 3. In a similar manner, we find that 61% B gives 

 ≈ 3 for peaks 3–7, 72% for peaks 8–9, 80% for peaks 10–12, and 88% for peaks 13–15. In each case, the retention times of the peaks in each group are approximately the same, so the peak widths are approximately the same, as we'd expect under isocratic conditions.  

Next, compare the isocratic peaks in Figure 1c with the appropriate section of the gradient in Figure 1b. Notice that in both separation modes all the peaks are approximately the same width. Also, the peak spacing between adjacent peak pairs is nearly the same in both cases. You can mentally connect the isocratic chromatograms of Figure 1c and get a separation that is very close to that of Figure 1b. 

Because we see the same peak behavior in Figures 1b and 1c, we say that these are equivalent conditions. The only catch is that we have to have several different isocratic conditions to match a single gradient. But this gives us another way to visualize what is happening during a gradient. If we could program the LC system to run a series of isocratic steps that would maintain the separations of Figure 1c, we would start at 49% B until peak 2 was eluted, then step to 61% until peak 7 comes out, and so forth. Of course steps like this won't give us exactly the same separation, because the peaks of each segment are influenced by the conditions of the prior segment, but it serves as a useful mental model. After all, a gradient can be thought of as a series of isocratic steps where the step size is infinitely small. Under such conditions we get the results of Figure 1b. 

With the isocratic separations of Figure 1c, all the peaks traveled through the column with 

, is a useful tool for developing isocratic separations and describing isocratic retention. When equivalent gradient conditions are used, Figures 1b and 1c suggest that the peak characteristics are approximately the same, so the retention factors also should be similar. To avoid confusion between isocratic and gradient retention factors, the gradient retention factor is abbreviated as 

*, which can be thought of as the average 

Another way to think about how gradient elution works is shown in Figure 2. In Figure 2b, we see a chromatogram overlaid with the gradient program (dashed line). For discussion purposes, let's assume that the gradient is a linear gradient of 5–100% B over 20 min. In Figure 2a, the left-hand 

-axis tracks the band migration as it moves through the column (in the direction of the arrow on the axis); the band position is shown by the solid curved lines. The isocratic 

 value at any instant during the run is plotted on the right-hand 

 value is shown as the curved dashed lines. 

Figure 2: Peak migration of peaks 1 and 2 during gradient elution. (a) Plot of peak position in the column, x1 and x2 shown by solid curves and left-hand axis; plot of instantaneous isocratic k value, kiso, at any time during the separation shown by dashed curves and right-hand axis; solid dots representing kiso at the midpoint of the column, x = 0.5, equivalent to k*; (b) gradient program, dashed line; chromatogram for peaks 1 and 2 when they reach the end of the column, x = 1.0. Adapted from Figure 9.3 of reference 2. 			

We'll consider two bands, 1 and 2, as they travel through the column. Under the initial conditions (5% B), peak 1 is very strongly retained, so it appears to sit at the head of the column (migration position of 0), and the isocratic 

 value is very high (dashed line off-scale). As the gradient progresses, stronger solvent runs through the column, and eventually the solvent is strong enough to move the band off the head of the column, and it begins to migrate through the column. Meanwhile, the 

 value drops (downward arrow on the right-hand 

-axis). With each increase of %B, the solvent is stronger, which means that the band travels faster, accelerating through the column. Meanwhile, the stronger solvent results in a smaller instantaneous isocratic 

 continues to drop. This acceleration of the sample band and drop in 

 continue until the band reaches the end of the column (migration = 1.0), and a peak appears in the chromatogram.  

In a similar manner, band 2 moves through the column. But because 2 is less polar than 1, it stays at the head of the column longer before a strong enough solvent arrives to begin carrying it through the column. However, its migration pattern through the column (the curvature of the migration plot) is very similar to band 1, but offset to higher %B values. The value of 

 also drops during the run in the same manner as 

If we ignore the time the sample bands sit on the head of the column doing nothing, and only consider the time that they actually migrate through the column, you can see that this migration time is about the same for both bands. In isocratic separation, two bands that take the same amount of time to go through the column will have the same retention times, and thus the same peak widths. This is another way of understanding why all peaks in a gradient run are approximately the same width — they take the same amount of time to migrate through the column. 

*, can be thought of as the equivalent of the isocratic 

 value when a band has migrated halfway through the column. For band 1, the band reaches the midpoint of the column (

 = 0.5, black dot in Figure 2a) at 5 min. Tracing this 

* = 3. Similarly, band 2 reaches the midpoint of the column at ~15 min, which also corresponds to 

*. Both bands reach the midpoint of the column with the same 

* is the same for both peaks. And as we noted above, if the effective migration time of each peak is about the same, we would expect the same isocratic 

* is approximately the same for all peaks in a gradient run.

Figure 2 also helps us understand one oversimplified description of gradient elution. This states that sample compounds sit at the top of the column until a strong enough solvent comes along to wash them off, then they travel quickly through the column. This is a bit simplified, and doesn't give the column much credit for the separation, but it gives us a basic understanding of how bands behave under gradient conditions. 

Let's review what we've covered here. We saw that if the range of isocratic 

 values is too large, a single isocratic separation will be very difficult without sacrificing peak height and run time. In such cases, where the ratio of 

 values for the first and last peaks exceeds ~20, a gradient usually will give better results. Figure 1c was used to demonstrate that if the number of peaks in a sample is limited, we usually can get ideal isocratic chromatography (2 < 

 < 10) by choosing the right %B. And by breaking up a complex sample into small sections, we can obtain similar 

 values for all peaks in the sample, albeit under different conditions. 

When the series of isocratic separations with 

 ≈ 3 for all peaks (Figure 1c) was compared with the gradient separation of the same sample (Figure 1b), the results were surprisingly similar. Further dividing the chromatogram into an infinite number of isocratic steps is equivalent to running a linear gradient, in this case obtaining 

 value at the point a band has moved halfway through the column. It also showed that all peaks tend to migrate through the column in a very similar manner, with the primary difference being how long they sit at the head of the column before significant band migration occurs.

Although not discussed here, Figure 2 also can be used to determine the influence on retention of changing the initial or final %B in a gradient, as well as the gradient steepness (or gradient time) and the shape of the gradient. 

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

 Introduction to Modern Liquid Chromatography, 3rd ed.

"LC Troubleshooting" Editor John Dolan has been writing "LC Troubleshooting" for 

 for more than 25 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 

's editorial advisory board. Direct correspondence about this column via e-mail to 

LC Troubleshooting

Gradient Elution, Part II: Equivalent Separations

Gas chromatography (GC) column preparation, installation, and care are of the utmost importance to ensure good chromatographic performance. Here, we will outline some of the common issues that we come across from CHROMacademy members so that you can avoid these pitfalls. 

GC columns need to be properly prepared to avoid issues such as high bleed and poor peak shape. If your column was shipped with flame-sealed ends, be sure to trim at least 10 cm from each end to avoid high column bleed associated with the flame-sealing process. Be sure that the column cuts are straight (that is, at 90° to the column outside wall), and not jagged. Angled cuts with rough column edges lead to peak tailing or splitting caused by polar analyte interactions with the excess of silanol groups exposed and the formation of turbulent eddies at the inlet, which holds up analytes from entering the column. Be sure to check the quality of the cut with a magnifying glass (a microscope is better), because the quality of the cut directly affects peak shape and therefore the reproducibility of your integration or quantitative data. 

Column installation is critical, and poor installation can lead to reduced efficiency (broad peaks), peak shouldering or splitting, and poor quantitative reproducibility. After you have the column cut properly, follow the manufacturer's instruction regarding insertion distances into both the inlet and detector. A column installed too high in the inlet risks poor quantitative reproducibility, and when a column is installed too low, peak broadening can occur because of the extra dead volume created. At the detector, a column inserted too high will give rise to high baseline noise and spikes with flame-based detectors (as the polyimide column coating burns in the flame!) or again to broad peaks if installed too low. Each column type has a dedicated ferrule size or internal diameter, and gas chromatography–mass spectrometry (GC–MS) ferrules are made from different materials — are you using the correct type? Check with your manufacturer. Oh — and that funny little cup with insulation inside that stops you from getting to the column coupling so you just removed it— well that's probably just lost you some potentially vital plates and degraded the peak shape slightly, so make sure you put it back. 

Column conditioning parameters differ with stationary-phase polarity and film thickness, so always check the side of the box for the maximum isothermal and gradient temperatures. But here's a tip to reduce your conditioning effort and save stationary phase — connect the column to the inlet (not the detector) and allow your carrier gas to flow through at ambient temperature for 10–15 min — this helps to purge dissolved oxygen from the stationary phase and significantly reduces conditioning time and subsequent phase loss. Then condition the column by raising the oven temperature at 10 °C/min to the gradient maximum and hold for the required period of time to subsequently obtain a stable baseline — this is almost never longer than 1 h in our experience and may be as short as 

10 min for less-polar, thinner-film columns. Fitting carrier-gas traps close to the instrument (to remove oxygen and moisture) is a great way to improve column lifetime and detection limits. Use the plate count of a test analyte to monitor column efficiency and set a column discard limit based on your knowledge of required plate counts for your types of analysis. Columns can often be miraculously restored to life by trimming 5% of the total column length from the inlet end — note that retention times may shift slightly after this operation, but efficiency may increase and peak shapes will improve. Consider using a guard column if your samples are particularly dirty. 

Detailed advice on troubleshooting all of the various GC detectors isn't possible in this short column. However, some of the general advice we give to CHROMacademy members follows. 

Always ensure you have the correct gases and gas stoichiometry — and measure gas flows manually from time to time to ensure your instrument isn't misleading you. Be aware, for instance, that nitrogen is the best makeup gas for flame ionization detection (FID) and that the detector sensitivity can often be optimized by tweaking makeup gas flow. Another "for instance" in this category is the necessity to change from nitrogen to argon or methane mixtures for certain applications using electron-capture detection (ECD). 

Detector noise is almost always associated with dirt, so be sure to have a regular maintenance schedule to check the detector and jet or orifice for cleanliness. Blocked detector jets can lead to horrendous peak tailing. Nitrogen–phosphorus detection noise increases with the age of the bead and ECD noise with aging sources that are no longer producing the same amount of beta particles per unit time or current as they used to. Aging ECD detectors often give a negative dip after analyte peaks. 

Baseline drift can often be compensated for by operating in constant flow rather than constant pressure modes. If this isn't the case then consider that either the response or gas flows are changing over time. Is your bottled gas supply running low? Is there an issue with your gas generator?  

Happy troubleshooting — and for more information and the world's most advanced interactive GC troubleshooting tool, visit CHROMacademy (

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Troubleshooting GC Columns and Detectors

Nontargeted metabolite profiling by ultrahigh-pressure liquid chromatography (UHPLC)coupled with mass spectrometry (MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests and novel therapies, and further our understanding of disease processes. Other fields such as ecology, plant biology, and food chemistry can also benefit from this approach. Here, the power of nontargeted metabolite profiling is illustrated in a study focused on assessing the impact of storage conditions on flavor stability in beer. 

 broadly describes an approach rather than a particular technique. For example, choosing among a variety of instrument platforms, you can perform a metabolomics experiment in a targeted or nontargeted manner or a combination of both (1). Historically, metabolite analysis was performed in a targeted manner focusing on a few known metabolites important to the biological question at hand. In such experiments, the biological hypothesis dictates the choice of metabolites targeted, and the analytical steps are optimized to detect those molecules. In recent years, metabolite research has transitioned to a more "omics" approach. Thus, experiments are performed in a broad and unbiased manner, enabling the detection of as many metabolites as possible. The results from a nontargeted metabolomics experiment can generate new hypotheses and drive the next step of research, which often involves switching to a targeted metabolomics workflow. It is also possible — as well as powerful — to combine the two approaches, performing the experiment in a nontargeted manner while at the same time monitoring a panel of known molecules. 

State-of-the-art mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most frequently used techniques for nontargeted metabolite profiling. NMR excels at identifying unknown compounds. Its applicability is limited, however, because it requires large sample volumes and high concentrations, is a lower throughput technique, and has a narrow depth of coverage (2). MS offers the significant advantages of high-throughput analysis, minimal sample requirements (microliter volumes), and a broad depth of coverage. Yet its ability to confidently identify and confirm unknown compounds is substantially limited.  

Structural elucidation of small molecules by MS is accomplished through the acquisition of tandem mass spectra (MS-MS), which involves the fragmentation of ionized molecules within the mass spectrometer. Small-molecule spectral libraries are well developed for gas chromatography–mass spectrometry (GC–MS) applications utilizing electron ionization, but have been slow to develop for liquid chromatography–mass spectrometry (LC–MS) applications. Unlike peptides, which fragment predictably, small-molecule metabolites are structurally diverse, making their behavior in the mass spectrometer less predictable. This unpredictability has greatly inhibited the generation of theoretical spectral databases as used in proteomic applications (3). Recently, some progress has been made in generating small-molecule LC–MS spectral libraries through the analysis of authentic standards (4,5), and these libraries continue to grow. However, given the complexity of the metabolome, its variation among biological species, and the variation in molecular fragmentation with different MS conditions, generating comprehensive spectral libraries through the analysis of authentic standards is a slow and tedious process. For those reasons, the process of molecular identification remains a significant bottleneck in nontargeted, LC–MS-based metabolite profiling studies (6,7).   

Most MS-based, nontargeted metabolite-profiling workflows exploit the fast separations and narrow peak widths obtained by coupling a mass spectrometer with an ultrahigh-pressure liquid chromatography (UHPLC) system. Traditionally, in such a workflow (see Figure 1), only the MS signal (that is, no fragmentation) is collected for each detected molecular feature, optimizing the number of points per peak over the narrow UHPLC elution profiles. This approach ensures the validity of the quantitative data obtained for each feature. The quantitative data are used in downstream statistical analysis to determine molecular features of interest (such as potential biomarkers). Molecular identification is then attempted using a combination of accurate-mass measurements, isotopic distribution, and additional experimental sample analysis to obtain the MS-MS fragmentation spectrum for each feature of interest. The accurate mass and isotopic distribution can be used to predict the compound's molecular formula. The experimental MS-MS spectrum can be used to infer structural information or, in the best-case scenario, to compare with the MS-MS spectrum of an authentic standard (1,7). 

Figure 1: UHPLCâMS-based nontargeted metabolite profiling workflow. Compound identification involves the targeted acquisition of MS-MS spectra for compounds of interest (for example, potential biomarkers) and comparison with authentic standards.			

Nontargeted metabolomic profiling offers an in-depth biochemical analysis applicable to any biological organism or tissue. For example, it is used in many studies that focus on human health to advance our understanding of disease processes and ultimately to enable the development of diagnostic tests and novel therapies. Indeed, the National Institutes of Health (NIH) recognizes the potential impact of the metabolomics field and has shown its commitment to metabolomics by recently establishing the Common Fund Metabolomics Program. 

Nontargeted metabolomics can be applied in other fields that may be less obvious targets for this application. For example, a recent ecological study pursued a nontargeted metabolite-profiling approach to show that the rate of decomposition for leaf litter varied according to the decomposer microbial community. The finding could have important implications in the rate and efficiency of the formation of organic matter in soil and carbon sequestration in soil (8).   

Food chemistry represents another field in which the nontargeted approach has been successfully applied (9). We recently presented the results of a study that adopted this approach to analyze beer. The goal of the study was to assess the effect on flavor stability of storage length and conditions, particularly as they relate to the nonvolatile small-molecule profile (10).  

Why Study Beer with Non-targeted Metabolite Profiling? 

An important metric in the brewing industry is "beer quality," defined by attributes of beer such as flavor, mouth-feel, aroma, and appearance. Unfortunately, storage conditions (including time, temperature, and light exposure) can influence beer quality and result in a limited shelf life for commercial products, after which they are no longer considered true to brand. A better understanding of the molecular cause and effect of the aging process can lead to improved brewing methods that will increase beer stability over a longer time and range of storage conditions. 

Traditionally, analytical work in the brewing industry has focused on volatile compounds known to influence flavor and taste. Yet nonvolatile compounds can also be factors that contribute to — or inhibit — the staling process. In our study, we used a standard nontargeted UHPLC–MS metabolite-profiling workflow to determine whether changes in nonvolatile metabolites during storage could reflect beer quality. Thus, this study incorporated both a molecular-level analysis and a qualitative, sensory analysis (Figure 2). 

Figure 3: Quantitative scale used in sensory analysis to score "oxidation" of beer.			

This analytical approach can be applied in many fields; the example illustrates its use in a food chemistry application.

LCGC North America-04-01-2013

HPLC Systems and Modules Introduced at Pittcon 2013: A Brief Review

New Chromatography Columns and Accessories at Pittcon 2013, Part I

Short Courses

Gradient Elution, Part II: Equivalent Separations

Troubleshooting GC Columns and Detectors

Nontargeted Metabolite Profiling by UHPLC–MS: A Case Study in Beer Reveals Effect of Temperature on Nonvolatile Small Molecules During Storage

Peaks of Interest

Influence of Sample Solvent Composition for SFC Separations

Market Profile: Automated DNA Sequencers

Vol 31 No 4 LCGC North America April 2013 Regular Issue PDF