This instalment highlights noteworthy high performance liquid chromatography (HPLC) and related products introduced at Pittcon 2015 in New Orleans, Louisiana, USA, as well as in the year prior. It summarizes the technical aspects of new HPLC and supercritical fluid chromatography (SFC) systems, modules, software, and product extensions. The focus is on innovative products, major upgrades, and novel features with substantial user impact. 

	The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is the world's largest conference on laboratory science. The 66th Pittcon was held this year at the Ernest N. Morial Convention Center in New Orleans, Louisiana, USA, from 8 to 12 March. Prior years that Pittcon was held in the same city were 1988, 2001, 2002, and 2008. New Orleans, Chicago, Illinois, and Orlando, Florida, are the three top locations for Pittcon, supplemented by venues in other cities such as Philadelphia, Pennsylvania, and Atlanta, Georgia (1). New Orleans is located on the bank of the Mississippi River and is famous for its Cajun cuisine, jazz and blues heritage, and the annual festivity of Mardi Gras. The subtropical climate at Pittcon this year contrasted greatly with the austere wintry temperatures of Pittcon 2014, which was held in Chicago.

	Pittcon 2015 attracted 14,200+ attendees from more than 90 countries to attend more than 2000 technical presentations, ~150 short courses, and a huge exposition of analytical instruments. Once again, Pittcon 2015 provided a premier meeting place for analytical chemists and a global show case for instrument manufacturers.

	2015 has been a productive year so far for new high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) product introductions. A little more than 10 years after the commercial introduction of the first ultrahigh-pressure liquid chromatography (UHPLC) system in 2004 (2,3), second-generation UHPLC systems are steadily making their debuts. Following are a few of the megatrends in HPLC equipment:

		Improved second-generation UHPLC systems that offer higher operating pressure ratings, lower dispersion, innovative ovens or autosamplers, and faster cycle times (4–6).

		Many manufacturers offer a single instrument platform with multiple-tiered pressure ratings (Agilent 1290 versus 1260 or 1220) or configurations (modular versus integrated). Modular systems typically have higher system performance, while integrated systems generally have lower pricing for quality control applications (for example, Shimadzu Nexera X2 versus Nexera-i).

		New systems can be brand-new platforms (such as the JASCO LC-4000 and Thermo Vanquish) or upgraded versions of existing systems (for example, Agilent 1290 Infinity II). They can also be specialized application systems (for example, two-dimensional (2D)-LC, as exemplified by Shimadzu's Nexera-e system).

		While hardware specifications remain important, purchasing decisions continue to be dominated by considerations of the choice of the chromatography data system (CDS) (7).

	SFC is emerging as a preferred technology for chiral separation and purification with an increasing number of manufacturers appearing in the marketplace (JASCO and Shimadzu). Table 1 lists prominent new product introductions (arranged alphabetically by vendor name) at Pittcon 2015 or during the prior year, followed by more-detailed descriptions and comments for each product.


	Table 1: New HPLC product introductions at Pittcon 2015 or in the prior year.

New HPLC and UHPLC Systems and Line Extensions

	Several new HPLC and UHPLC systems or specialized applications systems and modules are summarized in Table 1 and are described with more details below:

	Agilent 1290 Infinity II UHPLC - Five years after the introduction of the 1290 Infinity UHPLC, Agilent implemented a major upgrade to their flagship UHPLC system. All modules have a similar look and footprint (slightly wider for the column compartment) with significant performance enhancements for the autosampler and the column oven. The Agilent 1290 Infinity II has an upper pressure limit of 1300 bar with reduced system dispersion. Increased system throughput is accomplished with a new multisampler with an optional dual-needle design operating on two separate flow paths to reduce the cycle time to seconds. A higher sample capacity of up to 16 microtitre plates or 6144 samples (or 432 vials) is made possible by an integrated elevator plate feeder. Lower carryover (<9 ppm) can be achieved via external multisolvent wash to the outer needle and additional needle-seat backflush. The larger multicolumn thermostated compartment now fits up to eight short columns and accommodates column lengths of 30 cm with a temperature range of 4–110 °C. Two 1300-bar pumps are available: a high-speed (binary) pump and a flexible (quaternary) pump with improved compositional accuracy. A new diode-array detector has a higher sampling rate of 240 Hz and a smaller flow cell for improved efficiency with 2.1-mm i.d. columns. The Infinity II system uses Instrument Control Frame-work and RapidControl Drivers software to facilitate connection to any Agilent CDS (OpenLab or MassHunter) or other third party CDS. A new quick-connect fitting (fingertight up to 1300 bar) that autoadjusts tubing insertion length to eliminate void space is available.

	Figure 1 shows the reduced cycle time of the Agilent 1290 Infinity II system using the double sampling needle option to eliminate time lags between injections.


	Figure 1: Two comparative signal traces of the reduced cycle time of the Agilent 1290 Infinity II system using the new Multisampler autosampler with a dual-needle design that eliminates time lags between injections.

	Cecil Instruments' Merit HPLC systems - The Merit HPLC systems from Cecil Instruments are low-cost HPLC systems with built-in chromatography software and UV–vis or photodiode-array detection designed for teaching, quality control, column screening, and process development applications.

	JASCO LC-4000 Series HPLC and UHPLC systems - The JASCO LC-4000 series is the latest LC platform consisting of many 300-mm wide, stackable modules. The modules can be configured into conventional (500 bar), rapid analysis HPLC (700 bar), UHPLC (1300 bar), or preparative LC systems (20, 50, or 120 mL/min). The LC-4000 replaces the existing JASCO series of X-LC-3000 or LC-2000 systems. All are controlled by the upgraded JASCO ChromNAV 2.0 CDS.

	More than 30 new modules are available for analytical or preparative-scale applications with various specifications as follows:

		Autosampler: HPLC (300 bar, 60 vial), rapid HPLC (700 bar, 180 vial), UHPLC (1300 bar, 180 vial), and preparative scale (400 bar, 10-mL injection, 40 vials).

		Column ovens: Compact, high capacity (holds six or 10 columns), preparative (temperatures ranging from ambient -15 °C to 100 °C), and a reaction oven for postcolumn derivatization.

		Pumps: Four analytical pumps (binary or quaternary, 700 or 1300 bar) with dynamic or turbulent flow mixers and built-in microdegassers. Three preparative pumps have maximum flow rates of 20, 50, or 120 mL/min rated to 500 bar and have optional solvent degassing and recycling features.

		Detectors: Available UV–vis, photodiode-array, refractive index (analytical or preparative), fluorescence, circular dichroism, and optical rotation detectors.

		ChromNAV 2.0 CDS: Operates under Windows 7 or 8.1 with a graphical user interface and controls up to four JASCO systems. This CDS is 21 

 Part 11 compliant. An optional ChromNAV-FC CDS is available for preparative LC and an optional ChromNAV-GPC add-on is used for molecular weight distribution calculations.					 	

	PerkinElmer Altus HPLC/UPLC - At Pittcon 2015, PerkinElmer launched its Altus HPLC and Altus UPLC systems, controlled through Waters Empower CDS, for the environmental, industrial, and applied markets.

	Shimadzu Nexera-i and Prominence-i systems - Shimadzu introduced two identical looking integrated HPLC (Prominence-i) and UHPLC systems (Nexera-i) to complement their existing Prominence and Nexera XR/X2 modular systems. According to Shimadzu, the "i" in the i-series names stands for "integrated" (6) and also for "innovative", "intuitive", and "intelligent". These integrated units are designed for quality control and academic laboratories and are controlled by Shimadzu's LabSolutions and major third-party CDS. Each system is equipped with a built-in colour touch screen to allow control of all LC parameters, including automatic on/off, manual purging, batch table sequencing functions, and chromatogram display. System status (ready, pretreatment, run, and error) is readily visible from the front panel. One can start an analysis from the instrument's front panel immediately after loading samples. Remote monitoring via smart devices is possible.

	The built-in autosampler can make reproducible injections down to less than 1 μL with a 14-s cycle time and can accommodate up to 216 1.5-mL vials. The large-capacity forced-air column oven fits six 10-cm or three 30-cm columns with a temperature range of ambient -10 °C to 85 °C. The built-in quaternary pump is rated at 440 bar (Prominence-i) or 660 bar (Nexera-i) and uses a parallel double plunger design. An optional low-pressure valve allows selection of up to seven solvent reservoirs. The built-in UV–vis or photodiode-array detector is equipped with TC-Optics and flow cell to reduce signal drift because of ambient temperature changes. Shimadzu's LabSolutions CDS allows many convenient autofunctions such as start-up, purging, column equilibration, baseline check, shutdown, system validation, and email notifications.

	Shimadzu Nexera-e 2D-LC system - Shimadzu Nexera-e (the "e" stands for exponential) is a comprehensive 2D LC system based on the company's Nexera X2 UHPLC system. The system is designed for the analysis of complex samples by coupling two "orthogonal" separating modes with a built-in valve–loop switching system, and detection using photodiode-array or mass spectrometry systems (8). System configuration and setup is accomplished using LC×LC Assist software. Comprehensive 2D qualitative and quantitative analysis using contour graphics is accomplished via ChromSquare software, which can also display mass spectral data. Figure 2 shows a chromatogram of a first-dimensional separation of a complex traditional Chinese medicine sample and the resultant 2D contour display of more than 200 peaks displayed with ChromSquare software.


	Figure 2: (a) Chromatogram in the first dimension of a separation of a complex traditional Chinese medicine sample with UV detection; (b) Resultant 2D contour display of more than 200 peaks with the ChromSquare software shown as spots in the 2D plot from "orthogonal" separations in the comprehensive 2D mode. Coeluting peaks from the first dimension (manifested as a peak shoulder in the inset of 2(a) are separated as distinct spots shown in 2(b).

	Thermo Scientific Vanquish UHPLC system - The Thermo Scientific Vanquish UHPLC system is a new integrated, low-dispersion, biocompatible UHPLC system with an upper system pressure limit of 1500 bar or 22,500 psi (5). The Split Sampler HT is capable of precise injections of 0.01–100 μL. Its injection cycle time can be as short as 15 s, with carryover as low as 4 ppm. The sampler supports injections from HPLC vials as well as from 96- and 384-well plates. The optional charger unit extends sample capacity to 23 plates (8832 samples). Automated barcode reading for rack identification is available as an option. The unique column compartment H (5–120 °C) can operate in two thermostatting modes: a still-air mode to maximize column efficiency by reducing radial thermal gradients inside UHPLC columns or a forced-air mode to mimic column ovens of other manufacturers (9,10). The binary pump H has a unique dual-piston-in-parallel design with variable stroke volume capability. It has a flow accuracy of ±0.1% and pulsation of <0.4%. It supports binary high-pressure gradient mixing of six solvents (in a configuration of 2×3 and a total of nine combinations) with a default mixing volume of 25 μL. The built-in diode-array detector HL uses silica "LightPipe" technology, has a flow cell volume of 2 μL or 13 μL (pathlengths of 10 mm or 60 mm, respectively), and supports programmable slit widths of 1–8 nm spectral bandwidths. The system is controlled by the Chromeleon CDS with integrated mass spectrometry (MS) support.


	Figure 3: The excellent precision of the Vanquish UHPLC (peak area RSD of ~0.03%) in an overlaid chromatogram of 10 repetitive injections of a pharmaceutical sample containing three major drug components.

	Figure 3 shows the excellent precision performance of the Vanquish UHPLC system with an overlaid chromatogram of 10 repetitive injections of a pharmaceutical sample. Figure 4 shows the simulated thermal heat maps inside an UHPLC column operating under a still-air mode (to maximize column efficiency) or a forced-air mode (to mimic operation in other manufacturers' ovens) (9,10).


	Figure 4: Simulated thermal heat maps inside an UHPLC column operating at very high pressure in a Thermo Scientific Vanquish UHPLC system: (a) Using the column oven operating under a still-air mode (with the fan off to maximize column efficiency without any radial thermal gradients); (b) using a forced-air mode with a circulating fan. Note that radial thermal gradients in the column under the forced-air mode can be highly detrimental to column efficiency performance.

	Several new HPLC modules were introduced this year:

	Agilent Infinity II ELSD - An upgraded evaporative light-scattering detection (ELSD) system with an improved linear dynamic range is capable of subambient drift tube operation for detection of thermally labile compounds.

	SofTA 2300 ELSD - The upgraded 2300 ELSD system from SofTA has more than three orders of magnitude in dynamic range and an option of using helium nebulizer gas for improved sensitivity.

	Scientific Systems Next Gen pumps - Scientific Systems, Inc. (SSI) introduced a new family of Next Gen pumps: M1, MX, LS, LU, LD, PR, and CP pumps capable of a wide range of flow rates (0.001–300 mL/min) and pressure limits (50–25,000 psi). The company's product line consists of pumps for analytical and preparative scales, flash, SFC, mini-metering, process, UHPLC, and biocompatible applications.

	A number of new updates in chromatography data systems were introduced this year:

	Agilent OpenLab CDS - The improved OpenLab CDS software has a more intuitive user interface and faster time for data processing, reviewing, and reporting. It is 21 

 11 and European Union Annex 11 compliant and has a new graphical tool for sample entry for the new Infinity II Multisampler autosampler.

	Chromperfect SEVEN CDS - Chromperfect SEVEN CDS v.6.0.10 for Windows 7 and 8 was found to be compatible to a prereleased version of Windows 10. Both Chromperfect SEVEN client-server and small laboratory (SL) CDS are 21 

part 11 compliant and can control most gas chromatography (GC) and many HPLC systems.

	DataApex Clarity v.6 CDS - DataApex introduced an updated version of Clarity v.6 CDS with an enhanced user interface, sequence functions, GC×GC extension, and higher linearity ranges for MS and flame ionization detectors as well as system controls for Hitachi HPLC systems and Advion MS systems.

Preparative LC, SFC, and Other Related Products

	Several new preparative LC, supercritical fluid chromatography (SFC), and other related products were also introduced this year, such as:

	Grace Reveleris Prep system - The Grace Reveleris Prep is a medium-pressure (1700 psi), hybrid flash chromatography–preparative LC purification system, with integrated control software displayed on a 12-in. touch screen. It has a maximum flow rate of 200 mL/min with a built-in automated injector and fraction collector that uses three UV–vis plus evaporative light-scattering detector signals. This simple, yet flexible system is ideally suited for use in an organic synthesis or sample purification laboratory.

	JASCO 4000 Series SFC - JASCO introduced five SF-4000 Series systems for semimicro, analytical, analytical or semipreparative, and semipreparative and preparative SFC. The systems have pumps capable of flow rates ranging from 0.2 mL/min to 150 mL/min at pressures up to 500 bar, variable loop injections of up to 180 samples, multiple column or solvent selection, detection by UV, circular dichroism, photodiode array, flame ionization, ELSD, and MS, and enclosed automated fraction collection.

	Shimadzu Nexera-UC SFE–SFC system - The Shimadzu Nexera-UC (unified chromatography) SFE–SFC–MS system is an innovative on-line system designed for automated extraction and ultrafast analysis of a variety of analytes including labile compounds in complex samples (11,12). The automated Nexera SFE system can extract 48 samples (in 0.2- or 5-mL extraction vessels, at temperatures up to 80 °C, and pressures as high as 40 MPa) with subsequent on-line SFC analysis with MS and UV detection. The system is uniquely suited for analysis of difficult samples such as multiresidual pesticides and polycyclic aromatic hydrocarbons in foodstuffs or soil, labile lipids or disease biomarkers in biofluids or dried bloods, swabs for cleaning verification (12), and additives in organic polymers. The Nexera SFC system includes the LC-30AD pump (5 mL/min to 66 MPa) with a back-pressure control unit and can be configured for chiral screening (12 columns with four organic modifiers) or as an SFC-UV analytical system. The system's automated extraction, coupled with the direct splitless SFC–MS interface, leads to highly reproducible and sensitive assays of labile or water-sensitive analytes in complex sample matrices.

	Figure 5 shows results from two applications of the SFE–SFC–MS system: automated analysis of a labile analyte (reduced coenzyme Q10 from a dietary supplement) and residual detergent analysis from a cleaning verification swab sample.


	Figure 5: The results of two applications of the Nexera UC SFE–SFC–MS system: (a) An on-line SFE and SFC analysis of reduced coenzyme Q10 extracted from a dietary supplement shown in the bottom chromatogram is preferred over analysis using off-line extraction. (b) SFC chromatogram of residual detergents (alkyl benzenesulfonates) from a cleaning verification swab sample extracted directly from the swab with SFE versus a blank swab.

Chrom is a flash chromatograph using liquid (not supercritical) carbon dioxide as the primary eluent. A standard carbon dioxide cylinder provides the pressure to drive the system. A supplementary pump is used for delivering an eluent modifier (such as methanol). This purification system is equipped to use disposable and reusable columns, and comes with integrated software, a UV detector, and an automated fraction collector.

	Many analytical chemists, including myself, come to Pittcon for professional and personal reasons. My schedule this year (my 16

 consecutive year) included several short courses on HPLC and UHPLC and the drug development process, attending the plenary lecture by Dr. Halas, and two of my own oral presentations and a poster on low-cost MS. My networking events included an 

editorial advisory board lunch meeting and the Chinese American Chromatography Association (CACA) dinner with ~100 attendees (featuring speeches by Professor Yafeng Guan of Dalian Institute and Dr. Richard Henry from Supelco). I spent two days on the exposition floor gathering confirmatory information for this instalment. The excellent organization of Pittcon allows me to learn about emerging analytical trends, meet with old colleagues, and make new acquaintances. I look forward to attending future Pittcon conferences to be held in Atlanta, Chicago, Orlando, Philadelphia, Chicago, and back to the "Big Easy" (New Orleans) in 2021.

	This instalment summarizes new HPLC and SFC product introductions (systems, modules, CDS, software, and related products) launched at Pittcon 2015 and in the prior year. It focuses on innovative features from a user's perspective. Since the coverage is brief and nonexhaustive, the reader is referred to manufacturers for additional technical details. Note that new HPLC columns introduced at Pittcon are presented and discussed separately in 

's annual reviews (13). The opinions expressed in this instalment are the author's own and bear no reflections of those from 

, the Pittsburgh Conference, or any other organizations.

	I would like to thank the Pittcon staff for organizing this global conference so predictably well, where everything runs like clockwork. Also, a special thanks to the marketing personnel of manufacturers for their timely responses to our requests for information. The author is particularly grateful to Drs. Davy Guillarme and Szabolcs Fekete of the University of Geneva, Mengling Wong from Genentech, Dr. Raphael Ornaf of Vertex Pharmaceuticals, and John Batts of IDEX Health & Science for their invaluable editorial and technical input.

is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California, USA. 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 more than 100 publications and a best-seller in chromatography. He is a member of the editorial advisory board of

. Direct correspondence about this column should be e-mailed to the editor-in-chief, Alasdair Matheson, at 

UHPLC: Where We Are Ten Years After Its Commercial Introduction, Trends in Anal. Chem. 

	(4) S. Fekete, I. Kohler, S. Rudaz, and D. Guillarme,

 (Wiley, Hoboken, New Jersey, USA, 2006), Chapter 4.

Handbook of Pharmaceutical Analysis by HPLC

, S. Ahuja and M.W. Dong, Eds. (Elsevier, Amsterdam, Holland, 2005), Chapter 20.

	(8) D.R. Stoll, X. Li, X. Wang, P.W. Carr, S.E.G. Porter, and S.C. Rutan,

	(12) T. Uchikata, A. Matsubara, E. Fukusaki, and T. Bamba, 

Articles

Perspectives in Modern HPLC

This instalment highlights some of the new HPLC systems, modules, accessories, and related technology introduced at Pittcon this year.

New HPLC Systems and Products Introduced at Pittcon 2015: A Brief Review

Environmental sample analysis by large-volume injection (LVI) in combination with liquid chromatography–tandem mass spectrometry (LC–MS–MS) is described for polar and nonpolar analytes in both aqueous samples and organic extracts. LVI is the direct introduction of large sample volumes (for example, 900–1800 µL) into the LC system for separation and subsequent detection. The practical benefits of LVI include faster sample preparation times, lower detection limits, and minimal waste generation.

	Environmental sample analysis by large-volume injection (LVI) in combination with liquid chromatography–tandem mass spectrometry (LC–MS–MS) is the direct introduction of large sample volumes (for example, 900–1800 µL) into an LC system for separation and subsequent detection. The primary advantages of LVI compared to traditional off-line or on-line sample preparation techniques, such as solidâphase extraction (SPE) or liquid–liquid extraction (LLE), include decreased sample preparation, greater analyte mass introduced for detection (that is, increased sensitivity), and less solvent and solid waste. LVI is wellâsuited for the concentration and separation of water-soluble analytes on reversed-phase guard and analytical columns (1), and has also been extended to organic extracts (2). Successful combination of LVI with LC–MS–MS requires an understanding of analyte properties (polarity, stability, and so on) and sample properties (salinity, particulate matter, matrix components, and more) (3,4). The unique elements of several applications are discussed here to highlight the various capabilities and limitations of LVI and are divided between aqueous and organic solvent injections. Finally, a set of loading strategies designed to capture nonionic analytes from large volumes of organic extracts on reversedâphase columns is described and experimental components are listed.

	An Agilent 1100 high performance liquid chromatography (HPLC) system, upgraded with a 900-µL analytical head and an extended-seat capillary sample loop, was used for all methods described below.

	Past examples of aqueous LVI include the analysis of neurotoxins in drinking water, pharmaceuticals in wastewater, and corrosion inhibitors in surface water (4). Here, two LVI methods are presented with a focus on the important technical features required to inject 1800 µL onto C18 guard and analytical columns. The LVI methods highlighted involve the direct injection of raw wastewater for the determination of illicit drugs (5) and seawater with 25% isopropanol for the determination of the surfactant components in the oil dispersant Corexit (Nalco) (6).
	 

	Before injection, raw wastewater is centrifuged to remove particulate matter that can potentially clog columns and other LC components (5). Filtration is a commonly used alternative, but its suitability is method- and analyte-specific because filter membranes can serve as a potential sink or source of analyte contamination (4,7). SPE, however, is not limited by particulate matter and has been used to analyze both the aqueous and particulate bound fraction (8). After centrifugation, the raw wastewater sample is added to an autosampler vial and spiked with isotopically labelled internal standards.
	 

	The six-port valve connections in the LC autosampler (Figure 1) are labelled as either mainpass (solid red channel) or bypass mode (dashed blue channel). The mobile phase in mainpass mode flows sequentially through the analytical head, sample loop, needle, extended-seat capillary sample loop, and then onto the columns. Bypass mode directs the flow directly onto the column. Initially, the LC system is operated in bypass mode so that the needle may be inserted in the sample vial and the first 900-µL volume is withdrawn. The needle is returned to its seat and the 900 µL of wastewater is then ejected into a 1400âµL extendedâseat capillary sample loop, which acts as a storage device during the withdrawal of the second 900 µL. Afterwards, the six-port valve is switched to mainpass mode and the cumulative 1800 µL of wastewater sample is chased by the mobile phase out of the sample loops and onto C18 guard and analytical columns. After the sample is out of the sample loops, the sixâport valve switches back to bypass mode. If left in mainpass mode, the gradient would travel through all of the mainpass tubing before reaching the column, requiring that the total run time be extended by several minutes.
	 


	Figure 1: Basic LVI configuration for 1800-μL injections onto a reversed-phase system. The six-port valve and injection assembly was adapted from Agilent autosampler schematics (16).

	Both the wastewater sample and initial mobile-phase solvents are aqueous with characteristically low eluotropic strengths on reversed-phase columns, such that methamphetamine and other illicit drugs and biomarkers are focused on the head of the C18 guard column. The guard column protects the analytical column and is preemptively replaced every 50 sample injections. This is ultimately more cost and time effective than using individual SPE cartridges for sample cleanup and analytical column protection. After loading analytes onto the C18 guard and analytical columns, a traditional reversed-phase gradient is used to separate and elute all analytes.
	 

	Neither reversed-phase LVI or SPE can selectively remove matrix components that are coeluted with the analytes of interest. With LVI, a postcolumn divert valve can be precisely controlled to divert column effluent containing sample matrix components away from the detector. The control of divert valve timing is achieved with greater precision than the analogous wash steps used in SPE and can send all unfavourable effluent to waste. As will be discussed below, the postcolumn divert valve is of even greater importance with the direct injection of seawater.

	
	An LVI-based method was developed by Place and colleagues (6) using the direct injection of seawater to quantify the surfactant components of Corexit: dioctyl sulphosuccinate (DOSS), Tween 80, Tween 85, and Span 80. Liquid standards of sorbitan monooleate (Span 80; purity: 70.5%), sorbitan monooleate polyethoxylate (Tween 80; purity: 74%), and sorbitan monooleate polyethoxylate (Tween 85; purity: 67%) were obtained from Sigma Aldrich. The direct injection of 1800 µL followed similar steps as described above for wastewater with two crucial adjustments. Open seawater does not require centrifugation and filtration was shown to remove the analytes of interest from solution. In addition, the Corexit surfactants undergo hydrophobic exclusion from aqueous solutions and exhibit significant loss (>50%) to polypropylene or glass container walls within 8 h of sampling at ambient temperature. To prevent analyte loss before analysis, seawater is diluted with isopropanol at the time of collection to give a final composition of 1:3 isopropanol–seawater. Injections of 1800 µL of the isopropanol-stabilized matrix resulted in wellâresolved peaks for anionic and nonionic surfactant classes. At higher additions of isopropanol, there is no improvement of analyte stability in aqueous samples and peak shape begins to suffer.

	
	Direct injection of seawater into the LC–MS–MS system is possible without damaging the mass spectrometer source by using the divert valve to direct the nonvolatile salts contained in seawater to waste. The 1800-µL injection volume (3.6 min loading at 0.5 mL/min) is followed by a 5.9-min “wash” under the primary mobile-phase conditions (5% acetonitrile and 0.5 mM ammonium acetate in water). This wash is diverted to waste and is sufficient to flush the nonvolatile salts from the column and away from the mass spectrometer before elution of the analytes of interest. The time (5.9 min) used to wash the salts from the seawater from the columns was evaluated by testing effluent fractions collected at 30-s intervals with the addition of 100 µL of 1 M silver nitrate–0.6 M nitric acid. The presence of chloride in the collected effluent was determined by the formation of a white silver chloride precipitate, visible in aqueous solutions containing more than 0.1% seawater. No silver chloride was observed in the effluent 7.5 min after injection and a precautionary period of 2 min was added to extend the loading and wash period to 9.5 min. For this application, guard columns are replaced approximately every 100 injections and the analytical column was replaced after approximately 1500 injections. Analysis over two years and more than 5000 injections did not lead to salt accumulation or corrosion in the LC–MS–MS system.
	
	 

Organic Extracts: Orthogonal Chromatography 

	Sample treatment to create organic extracts is desirable if analytes are unstable in aqueous solution, because of hydrolysis or hydrophobic exclusion, or if the sample matrices (for example, landfill leachate) are sufficiently complex that significant sample cleanup is required before injection. In addition, some analytes do not focus well on reversedâphase columns, even under high aqueous conditions, and require a different retention mechanism. For example, per- and polyfluorinated alkyl substances (PFAS) present in landfill leachate are commonly concentrated by SPE before injection (8) because of leachate matrix complexity. Direct aqueous injection for PFAS analysis is additionally disadvantageous because polar PFAS are not retained well on reversed-phase columns and the more hydrophobic PFAS are lost from aqueous matrices onto autosampler vials and to the air–water interface. The two new strategies discussed here for the LVI of PFAS in an organic solvent extract from leachate include orthogonal chromatography (9) and on-line extract dilution.

	In-line orthogonal chromatography was developed for PFAS in ethyl acetate–trifluoroethanol extracts of landfill leachates (9). The orthogonal columns system consisted of two zirconiumâmodified diol (Zr-diol) guard columns (6 µm, 150 Å, 12.5 mm × 4.6 mm, Agilent) placed in-line with a Zorbax Eclipse Plus C18 analytical column (3.5 µm, 95 Å, 75 mm × 4.6 mm, Agilent) (Figure 2). The system proved capable of capturing 70 anionic PFAS from 900âµL injections of the 1200-µL ethyl acetate–trifluoroethanol extract, which is 75% of the extract volume generated.


	Figure 2: Schematic of configuration for PFAS analysis in leachate extracts.

	The PFAS are captured by the Zr-diol columns through interactions between the anionic PFAS and the zirconium d-orbitals during the 900-µL injection of the ethyl acetate–trifluoroethanol extract. The trapped PFAS are then eluted from the Zr-diol guard columns by a mobile phase containing 50% methanol and 5 mM ammonium acetate that then refocuses the majority of analytes on the C18 analytical column. PFAS are then separated by a traditional reversedâphase gradient with an increasing percentage of organic solvent (Figure 3). Without LVI, additional sample preparation steps like solvent transfer and blowâdown are necessary to introduce sufficient analyte mass in the small injection volumes typically used (such as 10–20 µL). Moreover, an injection of 10–20 µL of an extract concentrated into about 100 µL is, at most, 20% of the total extract volume generated. Comparatively, LVI capitalizes on the time, effort, and labour costs expended during the creation of organic extracts by analyzing a greater percentage, while also eliminating additional steps that increase the possibility of analyte loss.


	Figure 3: Typical leachate chromatogram for select PFAS using the Zr-diol guard column configuration. Peaks: 1 = PFBA, 2 = PFHxS, 3 = PFOS, 4 = MeFBSAA, 5 = 6:2 DiPAP, 6 = PFHxDA.

	Orthogonal chromatography (as described above) is not capable of capturing nonionic PFAS from a large volume of organic extract. Dilution of organic extracts with water is one approach to perform LVI for the analysis of nonionics in organic extracts (for example, pesticides in vegetable [10] or soil extracts [11]). The traditional use of small-volume injections with organic solvent extracts of high eluotropic strength achieve focusing of analytes on reversed-phase columns because of dilution of the small organic extract volume with the larger volume of initial aqueous mobile phase. In the case of PFAS in landfill leachates, an eightfold dilution with water would be needed to retain the most water-soluble, nonionic PFAS on C18 columns (for example, MeFBSA). For comparable levels of sensitivity using off-line dilution, the 900-µL extract, injected previously, would become at least 7.1 mL and require multiple large autosampler vials, larger extended sample loops, and a longer injection programme. However, as previously mentioned, many PFAS can partition out of the bulk solution under high aqueous conditions. Therefore, on-line dilution strategies are explored here.
	 

	The objective of the following dilution-based LVI method is to use the ethyl acetate–trifluoroethanol leachate extracts and the orthogonal Zr-diol–C18 column system for the analysis of nonionic PFAS. Thus, the first alternative to offâline dilution was to inject 900 µL in small volume “packets” segmented by defined volumes (determined by flow rate and time) of the initial aqueous mobile phase. To minimize the overhead time required by the autosampler transport assembly to inject multiple small volumes, one 900-µL volume was drawn up into the sample loop and loaded onto the column in small packets by switching back and forth between mainpass and bypass mode (Figure 2). The volume of packets is determined by the flow rate and time spent in mainpass mode. The flow rate while loading packets onto the Zr-diol guard columns and C18 analytical column was 2 mL/min and the times spent in mainpass and bypass mode were programmed as 0.01 and 0.3 min, respectively (Figure 4). Mainpass and bypass were switched back and forth 18 times over the first 7.5 min where the sixâport valve was left in mainpass mode for 1 min the last time. At 8.3 min the gradient was started in bypass mode and the flow rate was decreased to 0.5 mL/min.


	Figure 4: Pressure isotherm and chromatogram for mainpass and bypass packet injections. Landfill leachate peaks for ionic PFAS: 1 = PFBA, 4 = PFOcDA. Nonionic PFAS peaks: 2 = MeFBSA, 3 = EtFOSA.

	The solvents in the mainpass assembly are pressurized when the six-port valve is in mainpass mode. But when in bypass mode, small volumes are lost to the waste line on the six-port valve as the mainpass assembly solvents depressurize and expand. Consequently, the six-port valve leading to waste (Figure 2) was plugged with a blanking nut to avoid losing small volumes of sample while switching between mainpass and bypass mode. Because the waste line was plugged, the 900 µL of mainpass solvent normally ejected to waste in preparation for drawing up sample were instead programmed to eject into an empty sample vial.
	
	When samples are loaded using this on-line, packetâdilution configuration, both ionic and nonionic PFAS are captured and separated from landfill extracts (Figure 4). The most water-soluble ionic (PFBA) and nonionic (MeFBSA) PFAS are successfully retained after 18 packet injections of leachate extract.
	 

	The LC pressure isotherm (Figure 4) is an important tool to aid method development. Pure and mixed solvents have different viscosities and therefore exert different corresponding back pressures that can be used to track gradient and sample delivery. The sawtooth pressure profile during the first 7.5 min is evidence of extract solvent packets mixing with the aqueous mobile phase in the Zr-diol guard columns. While programmed as 0.01 and 0.3 min, the actual times spent in mainpass and bypass mode were calculated using the average frequency of packet mixing seen in the pressure isotherm and were closer to 0.028 and 0.35 min, respectively. So at 2 mL/min the average packet was 56 µL with 700 µL of aqueous mobile phase on either side. The volume of mobile phase on both sides of the organic packets effectively dilutes the extract solvent inside the Zr-diol guard columns, thus reducing the eluotropic strength of the sample solvent so that focusing of the nonionic PFAS occurs on the C18 analytical column. The Zr-diol guard columns were more effective mixing chambers than other guard columns (such as amino propyl, C18) that required smaller packet volumes to focus nonionic PFAS on the head of the C18 analytical column. After loading nonionic PFAS the analytes were eluted from the Zr-diol guard columns and C18 analytical column separately; the anionic PFAS were focused on the Zr-diol columns and nonionic PFAS were focused on the C18 column.
	 

	The second alternative strategy is to trap nonionic PFAS from an organic extract on reversed-phase columns using a variation of at-column dilution that divides the flow from a single pump. At-column dilution typically uses two pumps that either pushes the sample at a slow flow rate to the tee junction or pumps a weak solvent at a high flow rate that dilutes the sample when the two flows combine at the tee (12). The amount of sample dilution is a function of the two flow rates. At-column dilution has been applied to load larger sample volumes onto columns during preparative LC (13) or analytical LC (for example, pesticides in river water using on-line SPE with LC–MS–MS) (14). The advantage of dividedâflow at-column dilution is that a second pump does not need to be purchased and at-column dilution is achieved using only two tee junctions and tubing of different internal diameters (Figure 5).
	 


	Figure 5: Divided-flow at-column dilution schematic. The tubing internal diameters for tubing 1 and 2 were changed from 0.175 mm to 0.125 and 0.76 mm, respectively.

	Chromatographic separation was accomplished using a Zorbax Eclipse Plus C18 guard column (12.5 mm × 4.6 mm, 5-µm Agilent) and a Zorbax Eclipse Plus C18 analytical column (75 mm × 4.6 mm, 3.5-µm Agilent). The internal diameter of the tubing between the tee and the autosampler six-port valve (Figure 5) was decreased from 0.175 to 0.125 mm. The internal diameter of the tubing connecting the two tees was 0.175 mm and the internal diameter of the 3-cm length of tubing on the outflow of the second tee and before the C18 guard column was large (0.76 mm) to ensure complete mixing of the two recombined flowpaths. Mobileâphase A consisted of 3% methanol in deionized water and mobile phase B consisted of 100% methanol and 10 mM ammonium acetate. The initial aqueous mobile phase flow rate was 3 mL/min for 3.5 min, during which time the 900âµL leachate extract was loaded onto the C18 guard column. The C18 guard column effluent was sent to waste for the first 3.5 min to keep the pressure low during the high flow rate. After 3.5 min the flow rate dropped to 0.5 mL/min and the guard column effluent was directed onto the analytical column. The gradient was then increased to 100% over the next 10 min and held for 5 min. PFAS were detected using a Quattro Premier XE MS–MS system (Waters Corporation), operated in multireaction monitoring mode.

	Adequate dilution of the organic extract before the C18 guard column is evidenced by nonionic peak retention and symmetry (Figure 6). More polar PFAS (such as PFBA) were not retained using only a C18 guard column. To capture anionic PFAS additional guard columns with orthogonal retention mechanisms (for example, Zr-diol) can be added before or after the C18 guard column with minimal increases to the back pressure.


	Figure 6: Divided-flow at-column dilution pressure isotherm and chromatogram. Peaks 1 = PFHxS, 2 = MeFBSA, 3 = EtFOSA, 4 = PFOcDA.

	While in mainpass mode the flow was divided at the first tee following the pump (Figure 5), with less mobile phase being directed through the sample loop. The reduced flow of mobile phase in the sample loop slowly moved the sample volume to where it combined with the rest of the flow at the second tee. The pressure isotherm (Figure 6) increased 1 min after switching to mainpass mode when the diluted 900-µL organic solvent extract first reached the guard column. After 3.5 min the entire sample had passed through the guard column and the C18 guard column was once again filled with the aqueous mobile phase. For 900 µL to pass through the guard column in under 2.5 min the flow rate through the autosampler was at least 0.36 mL/min, which corresponds to a 8.3-fold dilution when combined at the second tee with the rest of the 3 mL/min. Reducing the internal diameter of the tubing leading to the six-port valve from the first tee from 0.175 to 0.125 mm effectively reduced the percent flow to the six-port valve from 20% to 12% to achieve the eightfold dilution necessary for nonionic PFAS retention.
	 

	The incidental introduction of foreign matter can adversely change the internal diameter and by extension the percent flow to the six-port valve will also change (15). Should the internal diameter change significantly, samples may not be sufficiently diluted or may take longer to load onto the column. The risk of foreign matter introduction can be reduced by centrifuging or filtering the samples and mobile phases. Any change of the flow division at the first tee will be evident in the pressure isotherm profile, and clogged tubing can be cleared with extended flushing (15) or easily replaced; the divided-flow at-column dilution tubing used is relatively short (<5 cm) and therefore inexpensive. Overall, divided-flow at-column dilution has a simple, more universal configuration compared to “packet injections,” and it exerts lower back pressures, is faster, and can be used to inject more than 900 µL by using the extending capillary (Figure 1) used in the wastewater and seawater methods described above.
	 

	LVI combined with LC–MS–MS offers several advantages over traditional small-volume injection and off-line sample cleanup techniques without diminished method performance. Direct injection LVI often requires little to no sample preparation and reduces solid and solvent waste generation associated with traditional off-line sample preparation. The injection of large sample volumes is analogous to the concentration step of SPE and increases the sensitivity of a given analysis by introducing more mass of the analytes of interest to the detector. Matrix components can be separated from the analytes of interest with greater specificity using LVI because of the control afforded by gradient elution and waste divert valves. Centrifugation or filtration is necessary before the direct injection of aqueous samples with suspended particulate matter, whereas some SPE-based methods simultaneously analyze the dissolved and particulate phases.
	 

	When matrix complexity or analyte stability require the generation of an organic extract, LVI capitalizes on the time, effort, and labour costs expended by injecting a larger percentage of the total extract volume. LVI of organic extracts also shortens sample preparation without a loss in sensitivity by eliminating additional steps associated with small volume injections (such as solvent evaporation) and the corresponding opportunities for analyte loss. Direct injection of organic extracts onto orthogonal columns was sensitive and robust, but was limited to ionic analytes. On-line dilution of organic extracts enables both ionic and nonionic analyte retention on reversedâphase columns and is preferable to off-line dilution for analytes unstable in aqueous solution. Divided-flow at-column dilution required only one pump, tee junctions, and tubing and efficiently focused anionic and nonionic analytes onto a reversed-phased system.
	 

	(1) T. Reemtsma, L. Alder, and U. Banasiak, 

	(2) W.J. Backe, T.C. Day, and J.A. Field, 

	(4) F. Busetti, W.J. Backe, N. Bendixen, U. Maier, B. Place, W. Giger, and J.A. Field, 

	(5) A.C. Chiaia, C. Banta-Green, and J. Field, 

	(6) B.J. Place, M.J. Perkins, E. Sinclair, A.L. Barsamian, P.R. Blakemore, and J.A. Field, 

http://dx.doi.org/10.1016/j.dsr2.2014.01.015i

	(7) J.W. Martin, K. Kannan, U. Berger, P.D. Voogt, J. Field, J. Franklin, J.P. Giesy, T. Harner, D.C.G. Muir, B. Scott, M. Kaiser, U. Järnberg, K.C. Jones, S.A. Mabury, H. Schroeder, M. Simcik, C. Sottani, B.V. Bavel, A. Kärrman, G. Lindström, and S.V. Leeuwen, 

	(8) J.P. Benskin, M.G. Ikonomou, M.B. Woudneh, and J.R. Cosgrove, 

	(9) B.M. Allred, J.R. Lang, M.A. Barlaz, and J.A. Field, 

	(10) A.C. Hogenboom, M.P. Hofman, S.J. Kok, W.M.A. Niessen, and U.A.T. Brinkman, 

	(12) "At-Column Dilution Application Notes," Waters Corp., Milford, Massachusetts, 2003, pp. 1–22.

	(14) H. Sasaki, J. Yonekubo, and K. Hayakawa, 

	(16) Agilent Technologies, Agilent 1100 Series Standard Micro and Preparative Autosamplers, Agilent Technologies, Germany, 2001, pp. 205–206.

Matthew J. Perkins and Jennifer A. Field 

are with the Department of Environmental and Molecular Toxicology, all at Oregon State University, in Corvallis, Oregon. Direct correspondence to: 

Cover Story

Environmental sample analysis by large-volume injection (LVI) in combination with liquid chromatography–tandem mass spectrometry (LC–MS-MS) is described for polar and nonpolar analytes in both aqueous samples and organic extracts.

Large-Volume Injection LC–MS-MS Methods for Aqueous Samples and Organic Extracts

In this instalment, John Hinshaw reviews gas chromatography (GC) instruments, columns, and accessories that were newly on display at the Pittsburgh Conference in New Orleans, Louisiana, USA, during March 2015, or were introduced to the marketplace in the preceding year.

From 8–12 March 2015 the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) returned to the Morial Convention Center in New Orleans, Louisiana, USA, for its 66th annual meeting. This was the first time since 2008 that the conference had taken place in New Orleans, and the city welcomed the 14,272 registered attendees with open arms, brass bands, and beignets. There were 919 exhibitors in 1690 booths, and this year 90 countries were represented. Although I did not attend many of the technical sessions, they were of as high a quality and as well attended as in previous years. Of particular interest to the readers of 

 was the half-day session devoted to the presentation of the 2015 LCGC Lifetime Achievement in Chromatography Award to Jack Kirkland (Advanced Materials Technology) and the 

 Emerging Leader Award in Chromatography to Caroline West (Université d’Orléans, France). Please see the February 2015 issue of 

 (1) for more information about this year’s awards.

	Pittcon will head to Atlanta in 2016, where conferees will enjoy a second consecutive year of Southern hospitality. In 2017, the conference returns to Chicago.

	
	This annual instalment reviews gas chromatography (GC) instrumentation, columns, and accessories shown at this year’s Pittcon or introduced during the previous year. For a review of new products in other areas of chromatography, columns, and related accessories, please see the additional coverage in the April issue as well as this issue of

(2–4), which are also available on-line at 

	The information presented here is based on manufacturers’ replies to questionnaires, as well as additional information from manufacturers’ press releases, websites, and product literature about the past year’s products, and not 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 firsthand as well as 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 instalment cannot be considered to be 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.

	Gas chromatography again displayed renewed vigour in the past year, which certainly was evident at the 2015 Pittcon conference. Comprehensive GC×GC continues to yield significant advances, in particular when combined with mass spectrometry (MS) detection. MS detectors for GC alone experienced no fewer than six new or enhanced product introductions. Advances in fast mini- and micro-sized GC systems were evident, too, as well as a nice assortment of valves, fittings, syringes, gas accessories, and columns.

	In the instrument system area, Agilent Technologies introduced the 7200B quadrupole time-of-flight (QTOF) GC–MS system plus the 7010 Triple–Quad GC–MS system with an improved electron ionization (EI) source. Both of these offerings work with the company’s gas chromatographs. Also in the GC–MS area, the AccuTOF–GCx TOF GC–MS system from JEOL works with comprehensive two dimensional (2D) GC to make a powerful GC×GC–MS analyzer. LECO displayed its newest GC×GC–MS system, the Pegasus GC-HRT 4D. Also coming in with a new product in this area, Shimadzu introduced the GCMSâTQ8040 tripleâquadrupole GC–MS system. Finally, Thermo Scientific had their TSQ Duo tripleâquadrupole GC–MS–MS system on-hand. Please see Table 2 for the details and specifications supplied by the manufacturers for these highâend hyphenated GC instruments.

	The AQMAlert Ozone Precursor system from Baseline Mocon combines two of the company’s model 9100 GC systems and a preconcentrator into a photochemical assessment monitoring station. From Gow-Mac, the Series 8100 programmable GC system represents a new customizable application-specific laboratory system with multiple inlets, detectors, and other options. In miniature and micro GC systems, Defiant Technologies showed the TOCAM toxic organic chemical monitor, based on a microconcentrator and micro-GC column for rapid screening and more detailed analyses. A new entry to micro-GC, QMicro brought examples of its GC cartridge with integrated sampling valve, detector, and backflushing options, to be made available to original equipment manufacturers (OEMs).

	Table 3 lists new GC accessories such as autosamplers, detectors, and more. From DANI, the Peakblade 77 GC×GC modulator is a liquidânitrogen-free device with rapid and programmable thermal modulation. Two fast GC accessories were shown at Pittcon: the fast GC conversion kit from VICI that integrates the company’s resistively heated columns and controller with Agilent GC systems, and a fast GC add-on for Ionicon’s PTR-TOF gas analyzer. Analytical Flow Products (AFP) introduced a modular multipurpose valve oven, a miniature version of the company’s multiport valve, and a new design for zeroâdeadâvolume fittings. Shimadzu introduced two accessory products: the AOC–6000 autosampler, which automates calibration sample preparation, and the ECD-2010 Exceed electronâcapture detector. SGE introduced a new version of its Diamond headspace syringe line. The EZGC Software Suite Online from Restek has been expanded with some additional calculation and translation capabilities.

	Only two companies submitted information about new GC columns, as shown in Table 4. Restek has two new columns, the Rt-Silica BOND porous-layer openâtubular (PLOT) column for permanent gas separations and the Rxi-1301Sil MS column, which targets solvent analyses with MS detection. Phenomenex introduced a two-column set that consists of the company’s ZB-CLPesticides-1 and ZB-CLPesticides-2 columns and is targeted for multiple polychlorinated biphenyl (PCB) US Environmental Protection Agency (EPA) methods that use electron–capture detection.

	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 “New Products” questionnaire this year and would like to receive one and be considered for early inclusion into the 2015–2016 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, 

, lbush@advanstar.com, with the subject line “2016 New GC Products”. The questionnaire will be sent out in December 2015.

 is a senior scientist at Serveron Corporation in Beaverton, Oregon, USA, and is a member of the 

 editorial advisory board. Direct correspondence about this column should be addressed to “GC Connections”, LCGC Europe, Honeycomb West, Chester Business Park, Chester, CH4 9QH, UK, or e-mail the editor-in-chief, Alasdair Matheson, at amatheson@advanstar.com

GC Connections

New Gas Chromatography Products, 2014–2015

Unexpected results from calibration standards create confusion in a clinical liquid chromatography (LC) method.

	Recently, I received an inquiry from a reader regarding a problem he encountered with a routine liquid chromatography (LC) method in his clinical laboratory. He had prepared a fresh calibration standard (check sample) for the analyte of interest (I’ll call it “X” to keep the reader’s laboratory anonymous), yet when he assayed a blank sample spiked with 160 ppm of X, he found an indicated 400 ppm. This was puzzling and not a problem normally encountered, so he sent the sample to another laboratory that was analyzing the same compound by gas chromatography (GC), and their results showed that the spiked sample indeed contained 160 ppm of X. At this point he contacted me to help figure out what was happening. As we look at possible causes for and solutions to this problem, we can use this as a specific example to which we can apply general troubleshooting principles.

	Before we get further, let’s take a look at the method, which is designed for the analysis of X in serum. Samples are prepared by taking an aliquot of serum, adding an aliquot of internal standard (IS), and a small amount of hydrochloric acid to acidify it. The solution is vortexed to mix, then an aliquot of dichloromethane is added, the solution is vortexed again, and then centrifuged to separate the two phases. The dichloromethane phase is removed, evaporated to dryness, and reconstituted in the injection solvent. The separation conditions comprise a reversed-phase column (size, stationary phase, and flow rate were not mentioned) with an isocratic mobile phase of acetonitrile, water, and trifluoroacetic acid. Ultraviolet (UV) detection is used. The chromatographic conditions give typical retention times of 9 min (IS) and 12 min (X), and the chromatogram is normally free of any other peaks. Calibration standards are prepared by spiking a stock solution of X into serum at 40, 120, and 160 ppm; these spiked calibrators are then extracted in the same manner as samples. A three-point calibration curve is run and if the regression is acceptable, this calibration curve is used for three months. With each batch of samples, a single injection of blank serum spiked to 160 ppm is made as a system suitability test; if this check sample assays at 160 ppm, the system is deemed stable and samples are run.

	The method had been running acceptably until he ran out of the 1000 ppm stock of X used for spiking the check sample. When the new stock was prepared, the problem of a 400 ppm assay for the 160 ppm sample appeared.


	In a case like this, I like to divide the issue up into several possible problem areas, then see how many of these possibilities I can eliminate with the data at hand. This helps to focus my attention on the source of the problem so that it can be investigated further, if necessary, and corrected. We can broadly, and somewhat arbitrarily, divide the possible problem areas into chemistry, hardware, sample-related, and calibration. Let’s look at each of these in more detail.
	

By chemistry, here I mean the chromatographically related chemical influences. These are the nature of the sample, the column, the mobile phase, and the column temperature. We can quickly eliminate these as the likely sources of the problem. If the column chemistry, mobile-phase chemistry, or column temperature had changed, we would expect a shift in retention for X and the IS, but this was not observed. The sample chemistry, or identity, is unlikely to have changed, because the check sample had no apparent retention problems in either the LC or GC assay.
	

 LC system hardware could malfunction in terms of flow rate, injection problems, or detection. The flow rate must be correct or the retention times would shift for both X and the IS. It is possible that the autosampler is not working properly, but this is unlikely to cause the noted problem, because any volume error in the autosampler would be compensated by the use of the IS. The purpose of the IS is to add it early in the sample preparation process so that any loss of sample volume or injection error would not matter, because it is the ratio of X/IS that is used in the calibration process, not the absolute response of either compound.

	
	Problems related to the detector are a possible source of error, and should be checked. Two obvious possibilities are that the wrong wavelength was selected or that there is something wrong with the detector lamp. The response of X and the IS would be expected to change if the detection wavelength was changed, and a change in the relative response of X and the IS would be likely. This would generate a different X/IS ratio for a given concentration, which in turn would change the assay value for X in the check sample. A change in lamp energy as the detector lamp aged could also cause a change in response, and although I would expect that such intensity would affect X and the IS similarly, that is not a certainty. The proper wavelength should be verified and the lamp energy should be compared to normal values to determine if either of these items could be the problem source.

We know that the identity of the sample is correct and that the standard was made at the proper concentration because the sample assayed at 160 ppm by GC. The reader did not state if a new batch of IS stock was made at the same time, but if we consider the method, either the new batch of IS was made correctly or the old one was still good and was used. The check sample is made by spiking serum, and serum would never be injected directly, so it follows that the check sample was spiked with IS and extracted in the normal manner. One of the reasons for adding IS is to account for the inevitable changes in sample volume that take place during sample preparation.
	
	Let’s review the sample cleanup procedure: 300 µL of serum is combined with 50 µL of IS and 200 µL of dilute hydrochloric acid (550 µL total), centrifuged, and extracted with 600 µL of dichloromethane. All of X and the IS should transfer into the dichloromethane, so the concentration of X and IS is 550/600 of its concentration in the original diluted serum. Next, 400 µL of the dichloromethane is removed, evaporated to dryness, and reconstituted in 50 µL of methanol. This concentrates the dichloromethane extract by 400/50 or eightfold. With the extraction, evaporation, and reconstitution steps, there will be inevitable volumetric errors introduced, which is why the IS is added - the same losses of X and IS should occur, so the X/IS ratio should stay constant. All this leads me to conclude that the GC method would be very unlikely to give an assay value of 160 ppm of X by an external standard method, even if the results were adjusted for the theoretical changes in concentration. Instead, I conclude that the IS method was used for GC, as well, and because the assay was as expected, it tells me that the check sample was made correctly, even though it doesn’t assay properly by the LC method. The bottom line here is that it is unlikely that the current problem lies with the sample or sample preparation.

At this point we’ve eliminated chemistry problems, hardware problems (assuming the detector wavelength is set correctly and the detector lamp is in acceptable condition), and sample-related problems. This leaves calibration problems as the most likely problem source (assuming that we haven’t overlooked something else obvious, which is always a possibility).

	
	My initial interaction with the reader simply indicated that the check sample did not assay correctly by LC, but gave the expected answer by GC. When I requested more information about the method, I learned of the practice of calibrating every three months and using the system suitability check sample to verify that the method was working properly. Although the rules are a bit different in the clinical laboratory industry, this goes strongly against the analysis of the same drugs in serum or plasma to support drug development in the pharmaceutical industry. The latter techniques fall under guidelines from the United States Food and Drug Administration (FDA). The FDA’s “Guidance for Industry: Bioanalytical Method Validation” (1) discusses validation of methods for the analysis of small molecular weight drugs in plasma and other tissues (generally called “bioanalytical” methods, as opposed to methods for the analysis of biological compounds). In this document in the section titled “Application Of Validated Method To Routine Drug Analysis” (pp. 13–14), it is stated:
	 

A calibration curve should be generated for each analyte to assay samples in each analytical run and should be used to calculate the concentration of the analyte in the unknown samples in the run . . . . The calibration (standard) curve should cover the expected unknown sample concentration range in addition to a calibrator sample at LLOQ [lower limit of quantification].

Once the analytical method has been validated for routine use, its accuracy and precision should be monitored regularly to ensure that the method continues to perform satisfactorily. To achieve this objective, a number of QC [quality control] samples prepared separately should be analyzed with processed test samples at intervals based on the total number of samples. . . . The QC samples in duplicate at three concentrations . . .

A matrix-based standard curve should consist of a minimum of six standard points, excluding blanks (either single or replicate), covering the entire range.


	
	This says that the calibration curve should be run with each batch of samples, not once every three months. The calibration curve should cover the expected sample concentration range, and include the LLOQ. Furthermore, QC samples should be run at three concentrations that fall within the range of sample concentrations. These guidelines also make good sense from an analytical chemistry standpoint.

	
	There are just too many potential problems that can occur which might cause the calibration curve to be different on different days. I have been involved with research and development (R&D) studies where the reference standards were so rare and valuable that it was not possible to run them every day, but a surrogate standard was found to verify that the original calibration was still adequate. That may seem to align with the current problem, but in fact the drug X and its IS are very common compounds that can be purchased in reference standard grade for reasonable prices, so it is hard to justify trimonthly calibration on economic grounds.

	
	The fact that the check sample was formulated at 160 ppm and verified by GC underlines the probability that the source of the problem lies with the calibration curve. My best guess is that something in the LC system has drifted over time, most likely the detector response (or an improper wavelength setting), and has caused the current response to the X/IS ratio to be much larger than it was when the calibration curve was run originally.

	I recommend that the proper wavelength setting and detector lamp performance be verified before proceeding. After these are found to be satisfactory, I would generate a new calibration curve using freshly prepared standards of X spiked into blank serum and extracted normally. I believe that the check sample will now assay correctly, closing the loop on identifying the problem source.

	
	Technically, the check sample has done exactly what it was intended for - it has alerted the operator to a problem with the assay before valuable patient samples were run. However, I would modify the method to comply more with the industry standard of the FDA guidelines (1). This would require running a calibration curve, containing samples with at least six concentrations, each day with each batch of samples run. In addition, a set of check samples, or QCs, should be prepared and included in each sample batch to show that during the analysis, the method gives the expected results for samples of known concentration. There will be some documentation required to make these changes, but the method reliability will be much improved and should justify this extra work. The quality of the results produced should improve as well. Finally, should the laboratory be audited by a regulatory agency, there will be much less likelihood of negative findings by the auditors.

	
	In terms of day-to-day added work, there should be only a small impact on the total batch run time for a potentially large improvement in data quality. The calibration and check samples can be quickly spiked with known amounts of X and extracted with QC samples and samples to be analyzed. A total of six calibrators and six QC samples (duplicates at three concentrations) would add 12 samples to the day’s run. At a 12-min retention time for X, this would increase the run time for the batch by about 2.5 h. It may be very easy to compensate for this increase in run time by increasing the flow rate; with an isocratic run, the separation should not be affected by the flow rate. The pressure would rise in proportion to the increase in flow rate, but it is fairly rare with conventional LC runs that pressure is a limiting condition, so the added pressure is unlikely to be an issue.

	We have used a specific example of a method problem to illustrate how to break down the problem into several potential problem sources. Most of these sources could be eliminated by careful consideration of the method and how the results deviated from the expected ones. This left us with two likely problem sources. First, a problem with the detector wavelength setting or detector lamp energy. These could be quickly checked by examining the instrument. The second potential problem source was that the instrument response to X or the IS had drifted between the time the original calibration curve was run and the problem was noted.

	
	The recommended solution was to first check for detector problems, and second rerun the calibration curve. A more permanent fix to the problem would be to change the method to comply better with current FDA guidelines and general analytical chemistry practices of running calibrators contemporaneously with samples.

	1. United Stated Food and Drug Administration, 

Guidance for Industry: Bioanalytical Method Validation

 (FDA, Rockville, Maryland, USA, 2001).
	
	
	“LC Troubleshooting” Editor 

 has been writing “LC Troubleshooting” for 

 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 in Lafayette, California, USA. He is also a member of 

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

. To contact the editor-in-chief, Alasdair Matheson, please e-mail: 

LC Troubleshooting

Calibration Problems — A Case Study

Issue PDF

Click the title above to open the LCGC Asia Pacific June 2015 regular issue, Vol 18, No 2, in an interactive PDF format. 

LCGC Asia Pacific-06-01-2015

New HPLC Systems and Products Introduced at Pittcon 2015: A Brief Review

Large-Volume Injection LC–MS-MS Methods for Aqueous Samples and Organic Extracts

New Gas Chromatography Products, 2014–2015

Calibration Problems — A Case Study

Vol 18 No 2 LCGC Asia Pacific June 2015 Regular Issue PDF