While spectacular developments in chemical imaging and spectroscopy continue to entice the market, separation science remains a key enabling technology. Increasing demands for higher sample throughput and data quality have led to automated methods for solid-phase extraction, dried blood-spot analysis and analytical separations with LC/MS. This review details a selection of separation science products featured at Pittcon 2016, held March 6–10, in Atlanta, Georgia.
Particle size analysis
The CPS Disc centrifuge from CPS Instruments (Prairieville, La.) provides a robust and convenient method for the high-resolution measurement of multi-modal distributions of particles. This is important in drug development, especially in formulation, and in the analysis of nanomaterials. Particles in the 20-nm to 40-μm range that differ in size by less than 5%, and often as little as 1.5%, can be separated via sedimentation velocity into bands and measured photometrically. The output looks like a chromatogram with individual peaks. Monomers are resolved from dimers, trimers, etc.
The centrifuge rotates at ~18,000 rpm. Particles settle in the fluid according to Stokes’ law. A broad polydisperse mixture of sizes smears out across the detection window, but if the sample is a mixture of particle size families, each with narrow dispersity, they focus into narrow bands. The position of the bands is recorded. Run time is 3–40 min, run-to-run precision is ±0.5% and the chamber must be cleaned between runs.
Gas chromatography
Chromatography analyzers from IUT Medical (Berlin, Germany) use photoionization detection (PID) and ion mobility spectroscopy (IMS) detection. Not all applications need separations; IMS alone is often sufficient. Assay of volatile organic compounds (VOCs) is an exception. The preferred approach is GC with PID detection. Since the analytes in a VOC sample have different risk profiles, they need to be treated individually. For example, an infrared detector can be used for the detection of fumigation gases, which are found in containers used for the ocean transport of foods. IUT’s ethylene-oxide analyzer uses GC as an inlet to reject matrix interferences. It is portable and provides real-time results essential to sterilization work.
JEOL (Peabody, Mass.) added a combination electron ionization/photoionization source to the AccuTOF-GCx high-resolution TOF/MS. The instrument provides multi-dimensional GC separations for complex samples such as fuels. Photoionization is particularly useful for the detection of aromatics, including polynuclear aromatic hydrocarbons in environmental samples.
MS detectors for GC
Combining mass spectra with retention time in GC/MS is the most popular way to identify analytes. For unknowns, accurate-mass MS can be supplemented with multiple-reaction monitoring.
Thermo Fisher Scientific (Waltham, Mass.) introduced the Q Exactive GC Orbitrap GC-MS/MS, which combines GC with high-resolution accurate-mass (HRAM) Orbitrap mass spectrometry.
The Orbitrap provides a mass resolving power of 100,000 at m/z 272, sufficient to differentiate between molecular formulas that have the same number of nuclides. In addition, the quadrupole stage transports the ions to a collision cell for fragmentation. The pattern provides further discriminating power between potential isomers. Mass range is 50–3000 m/z. Since the GC is the inlet, this seems more than enough range, especially for multiply charged ions.
As with many other multi-stage MS detector schemes, non-ionized components entering the MS are deflected by bends in the ion path. Ion sources include both positive and negative chemical ionization and electron impact. The Q Exactive should appeal to scientists working with volatile natural products, metabolites, unknowns such as leachables, and to those doing initial environmental surveys.
Element- and group-specific detectors
Detector Engineering & Technology (Walnut Creek, Calif.) presented systems that use catalytic combustion or thermionic emission to detect and often differentiate between isomers. For example, catalytic combustion ionization detection responds selectively to ignition of methylene groups (–CH2 –). This is a low-cost way to check fuel quality. Thermionic emission with electrically heated ceramic beads converts nitrogen-, phosphorus- or oxygen-containing compounds to electrical current. These detectors are robust and quantitative.
Detectors from O.I. Analytical (College Station, Texas), a Xylem brand, include an electrolytic conductivity detector (ELCD), photoionization detector (PID), halogen-specific detector (XSD) and pulsed-flame photometric detector (PFPD) (see below). U.S. EPA Methods 502.2 and 8021 specify tandem PID and ELCD for the assay of volatile aromatic and chlorinated compounds in drinking and wastewater.
The second-generation model 5383 PFPD from O.I. Analytical boasts a tenfold improvement in detection sensitivity for sulfur (<1 pg/sec) and phosphorus (<100 fg/sec). Most PFPDs are used to detect sulfur- or phosphorus-containing analytes—the 5383 can detect both in the same run. Applications include the assay of fragrances and essential oils, which often contain sulfurous analytes, and assay of pesticide residues.
Model 5383 can be configured to detect 26 more elements, including tin, gallium, arsenic, nickel and vanadium, which are important in electronics manufacturing and material science. The detector has been designed to be self-cleaning, which reduces maintenance needed for coke removal.
Methanizer for pesticide residues
Activated Research Company (Eden Prairie, Minn.) introduced the Polyarc Reactor, a microreactor that integrates into existing GC/FID systems after the column outlet (prior to the FID inlet) and sits on the top of a GC. The Polyarc catalytically combusts and then hydrogenates all carbons in organic compounds to methane. Since the reactor converts all carbon compounds to methane molecules, the FID response factor is 1.0 for all organic compounds. Similar to a traditional FID, reduced noncarbon compounds pass through the FID and are not seen. In one experiment, using methanol as the internal standard, the response factors for all analytes were 1.0 ± 0.04. The mean deviation between the response factors and unity was 1.8%.1 Larger deviations can indicate instrument malfunction, such as contaminated injection port liners or inlet discrimination.
MilliporeSigma (Billerica, Mass.) extended its ionic-liquid polar-GC column phase range with the SLB-IL111i, SLB-IL76i and SLB-IL60i. The SLB-IL111i is the most polar of the series, which gives the most unique elution patterns, especially for alcohols and aromatics. For a polar phase, it is remarkably stable, with a Tmax of 270 °C. The SLB-IL76i is slightly less polar and the SLB-IL60i is even less so. The latter is similar in polarity to pegylated (PEG)/wax columns but has a Tmax of 280 °C. MilliporeSigma recommends the SLB-IL60i for GC × GC applications where the first column is nonpolar.
Assay of trace water by GC is unusual, as water often destroys the stationary phase. The choice of appropriate instruments is limited to thermal conductivity detectors, which are not very sensitive. Even with these limitations, MilliporeSigma introduced Supelco Watercol ionic-liquid stationary phases in capillary-column format (see Figure 1). A range of specific ionic liquids can be used, and selection is sample-specific.
Figure 1 – This data shows the linearity obtained from a >5-point calibration curve (0.01–0.5%) for water on Watercol 1910. (Image courtesy of MilliporeSigma.)Sample processors for GC
Figure 2 – O.I. Analytical’s 4100 Water/Soil Sample Processor is shown with two of the new O.I. Eclipse 4760 purge-and-trap sample concentrators. The 4760 TruColour Indicator changes color as the status of the unit changes: the blue unit is in the “purge end” mode; the red unit is in “bake” mode. (Image courtesy of O.I. Analytical.)The O.I. Analytical Eclipse 4760 purge-and-trap sample concentrator, for the analysis of VOCs by gas chromatography, features patented foam sensor technology that aborts the purge cycle should foam develop (see Figure 2). TruColour LED illumination of the purge chamber (the color changes as the purge-and-trap cycle proceeds) allows the status to be checked from across the room. Patented cyclone water management removes more than 96% of the adsorbed water to protect the GC column.
CDS Analytical’s (Oxford, Penn.) CDS 7000 E purge-and-trap concentrator is compatible with all GC and GC/MS instruments. The upgraded instrument incorporates a wet trap that eliminates more than 96% of moisture from the sample. A patented foam sensor is standard. Pneumatic functions are separated from the electronics to improve durability. Method change from purge and trap to thermal desorption is quick and easy.
Autosamplers
Figure 3 – HT2800T autosampler for GC. (Image courtesy of HTA s.r.l.)HTA s.r.l. (Brescia, Italy) displayed the HT2800T autosampler for GC (see Figure 3) and HT4000L autosampler for HPLC. Starting with the injection mode, the HT4000L performs full- and partial-loop injection with precision. Sample capacity is 176 2-mL vials. Users can also select from 4- to 40-mL containers as well as multiple-well plates. Sample preparation is automated, including dilution, derivatization and internal standard addition. Injection valves for ultrahigh-performance liquid chromatography (UHPLC) and large sample volumes are available and can be user-installed. Biocompatible flow paths and sample cooling are other options.
HT2800T can be mounted on any gas chromatograph. A vial heater or cooler and vortex station support pre-injection sample prep. Other options include headspace assay with vial-leak check and solid-phase microextraction (SPME). A high-resolution touchscreen provides a quick, intuitive human interface. Automation protocols are compatible with modern gas chromatographs. External control and data reporting are compliant with CFR21 Part 11.
Sample processing modules
New sample prep stations from GERSTEL (Linthicum, Md.) include the QuickMix for stirring samples in vials from 2 to 20 mL, with an optional vial heater. The CCD cryostatic cooling station is designed to quickly cool samples and traps as low as –40 °C. Actual temperature is reported to the software for recordkeeping. A dual-camera system images barcodes on vials and plates in the SID barcode reader.
GERSTEL focused on new stations for its MPS roboticpro workstation, such as an SPME package that includes a new injection liner which fits into the GERSTEL CIS inlet, thus avoiding septa-associated artifacts. The MPS roboticpro can also derivatize a sample on an SPME needle and is controlled with an intuitive graphical human interface.
Gas generators for GC and GC/MS
Figure 4 – GASTRAP helium purifier and GC column from Quadrex Corp. (Image courtesy of Quadrex Corp.)GCs may require a variety of gases for the carrier gas and detectors. Quadrex Corp.’s (Bethany, Conn.) GASTRAP self-regenerating gas purifiers for GC improve purity by a factor of 10, e.g., 15 ppm to <1 ppm (or 99.999%) (see Figure 4). Thus, it is possible to start with lower-cost gases and upgrade them to 5 nines (99.999%) while eliminating gas line filters entirely. The purification utilizes pressure swing adsorption to adsorb gases at high pressure and differentially release them as the pressure is reduced. Sorbents include zeolites and activated carbon.
The Hydrogen and Hydrogen + Leak unit deliver five nines hydrogen by removing oxygen, nitrogen, carbon dioxide, hydrocarbons and moisture. The + Leak accessory terminates hydrogen flow exceeding 450 mL/min for 5 minutes. This also triggers an audible warning. If the flow is higher than 1 L/min, it is stopped immediately.
For nitrogen, the Quadrex GT-N2 and the GTZN2 selectively trap oxygen, water, carbon dioxide and hydrocarbons from an existing N2 supply. These gas purifiers operate silently to deliver 5 nines nitrogen at 300 mL/min and 175 psi. The GT-HE removes the same impurities. A color LED allows operation to be monitored, even from across the room.
Air and zero-air GASTRAP models include the GT-ZAIR (zero-air) version, which uses a catalyst to reduce methane to less than 0.05 ppm. The GT-AR selectively removes oxygen, moisture and CO2 from an existing argon gas supply.
VICI DBS Instruments (Vigonza, Italy), which recently joined VICI Instruments (Houston, Texas), introduced the FID Station Plus. It uses the latest polymer membrane technology to produce hydrogen at a purity of 6–7 nines hydrogen at a maximum flow of 600 cc/min. This is sufficient for using hydrogen as a carrier gas and FID detection. The zero A side produces 1.5 L of air with maximum hydrocarbon content lower than 0.5 ppm. An integrated microprocessor controls all functions of the gas station, including the LCD touchscreen. Leak detectors and water-level monitors provide continuous monitoring of production and leaks. The hydrogen gas volume is less than 50 mL.
Liquid chromatography
The Vanquish Flex UHPLC system from Thermo Fisher Scientific offers full biocompatibility over a pH range of 2–12. Chloride compatibility is up to 1 M. The unit’s serial, dual-piston pump, with a Pmax of 15,000 psi, has automated compressibility compensation, which does not depend on the composition of the mobile phase. Dwell volume is 670 μL, with the mixer contributing 400 μL. The dual-stage mixer couples a proprietary capillary unit with a 50-μL volume mated to a 350-μL static mixer.
The ACQUITY UPLC, introduced by Waters (Milford, Mass.) in 2004, reduced column internal diameters to about 2 mm, and dwell volume became important. The rigid requirements associated with validated methods often conflicted with and sometimes precluded adoption of clearly superior separations technology, particularly when separations required gradient elution.
Even within the ACQUITY brand, the larger dwell volume of the ACQUITY H class, with its pre-pump quaternary gradient (400 μL dwell volume), did not exactly mimic the dual-pump gradient of the original ACQUITY (120 μL dwell volume), especially using gradient elution. Even larger differences were noted for conversion between instruments such as the Waters Alliance (<650 μL dwell volume) and chromatographs from different vendors.
This may not appear to be much of a problem, but it is a huge issue in comparing, troubleshooting and method transfer between different laboratories. The originating laboratory doing method development is often blessed with a research-grade instrument, while the QC or contract research organization (CRO) lab may have conventional HPLCs, often with large dwell volumes. Once a method is transferred, the results do not match. Such situations are addressed with the ACQUITY Arc from Waters and Nexera-i MT from Shimadzu (Columbia, Md.).
The Nexera-i MT (intelligent method transfer) has two flow paths on the same chassis, one for conventional HPLC and the other for UHPLC. Newly developed Analytical Conditions Transfer and Optimization software helps mitigate the effect of dwell volume on chromatograms obtained on different models. The ACQUITY Arc also has dual-flow paths supported by Empower software.
In addition to method transfer, chromatographers are finding it useful to have a modern and legacy instrument in the same bench space. The Nexera-i MT and ACQUITY Arc mimic other brands of HPLC, which improves scheduling flexibility.
Multiplexed LC/MS
Shimadzu demonstrated a way to double throughput in LC/MS using staggered injection with two flow columns and flow paths feeding a high-speed mass spectrometer. Nexera MX Dual Stream Technology automates the entire LC/MS process, from column wash and equilibration to data collection from the MS. Samples are managed and injected as fast as every 20 seconds from the SIL-30ACMP autosampler, which holds six 384-well plates for a total of 2304 samples. Carryover is an incredibly low 0.0004% without rinsing. Four protein biomarkers can be separated in the Cytochrome P450 family every 38 seconds versus every 82 seconds for a conventional, nonmultiplexed workflow.
Preparative liquid chromatograph
With conventional preparative LC, fractions are isolated in a fraction collector. Then the mobile phase is evaporated. Most often, water is the least volatile liquid. Wet materials tend to form cakes with a surface of low permeability. Thus, mobile phase and mobile-phase additives (buffer and trifluoroacetic acid, etc.) are trapped in the residue. The mobile phase can eventually be removed, but the process takes hours.
Shimadzu scientists came up with a better idea—the Crude2Pure automated purification/powderization system. The process has been extended to the Prominence UFPLC (ultrafast preparative and purification liquid chromatograph). In 90 minutes, nonexpert users can obtain up to five fractions of highly purified sample as a dried powder.
After the initial chromatographic separation on the Prominence, up to five fractions are collected on Shim-pack C2P-H trapping columns, which capture and retain the target analytes during the subsequent wash to remove mobile-phase contaminants. The trap column is washed with aqueous ammonia, which converts the salts to freebase that is collected in a volatile organic solvent. Dry-down time to powder is reduced by up to 90%. Prominence UFPLC integrates and automates the entire process in a single, simple-to-operate instrument. It also reduces bench space by 70% compared to the Crude2Pure system.
Dalian Elite Analytical Instruments Company (DEAIC, Dalian, PRC) has emerged as the largest vendor of HPLCs produced in China. For years, I’d wondered when DEAIC would seek to export its instruments. DEAIC had a booth at Pittcon 2016, looking to work with distributors and OEMs. The company also packs and markets HPLC columns. Sales are approaching 10,000 columns per year.
HPLC analyzers
The BioEthanol analyzer from Shimadzu is a Prominence-i integrated HPLC equipped with a refractive index detector that measures ethanol production from bioethanol plants and breweries. The analyzer is useful in method development, process control and lot release.
High-resolution cross-fractionation chromatograph
Polymer Char (Valencia, Spain), specialists in chromatographic analysis of polyolefins, introduced a system for automated high-resolution cross-fractionation chromatography (CFC). CFC is a two-dimensional chromatographic process in which the sample is fractionated using temperature-rising elution fractionation (TREF) in the first dimension. The sample is heated in a solvent and the resulting solution is transferred to a gel permeation chromatography (GPC) column and eluted. While the first sample is eluted, TREF is programmed up by 1–3 °C. The newly dissolved material is passed to a second identical GPC column for elution in parallel. Then the first column is ready for the third sample from the TREF. This process continues over several hours with fractions 1, 3, 5 ... (odd) chromatographed on column 1 and the even-numbered samples chromatographed on column 2. The result is a 3-D plot of GPC response as a function of log[M] and temperature, which can be related to sample structure and mechanical properties.
Detectors for LC
Wyatt Technologies (Santa Barbara, Calif.) exhibited the μDAWN multi-angle laser light scattering (MALS) detector for UHPLC. The flow-cell volume on previous MALS models had a volume of about 60 μL, which is very large for UHPLC instruments. For the μDAWN, the volume was reduced to 6 μL, reducing band broadening by about 25×. Faster electronics and improved light isolation are included, as is the Comet ultrasonic flow-cell cleaner. μDawn can be combined with the Optilab UT-rEX high-sensitivity differential refractive index detector to provide a complete μSEC (size exclusion chromatography)-MALS detection system for natural and synthetic polymers.
Solid-phase extraction systems
In response to the demand for higher sample throughput, solid-phase extraction (SPE) is migrating from individual tubes to multiple-well plates. Several companies introduced liquid handlers specifically optimized for SPE in ANSI 96- and 384-well formats.
Figure 5 – VERSA SPE workstations fully automate SPE workflow from cartridge conditioning to sample derivatization on a single deck and ensure efficient flow of samples and reagents through cartridges with modules such as ReagentDrop, Heater-Shaker, Gripper and Nitrogen Dryer. (Image courtesy of Aurora Biomed.)Aurora Biomed, Inc. (Vancouver, B.C.) introduced an updated version of the VERSA 1100 SPE workstation. In addition to 96-well plates, the VERSA can handle racks of 1-, 3- and 6-mL tubes (see Figure 5). Both positive pressure and vacuum can be applied to accelerate liquid movement. A typical protocol involves linking subroutines for conditioning, sample loading, washing, elution, fraction collection, derivatization and plate loading for LC/MS. Optional modules enable liquid/liquid extraction, shakers and temperature-control stations on a 15-position deck.
Horizon Technology’s (Salem, N.H.) new SmartPrep Extractor uses positive-pressure sample and solvent flow for high recovery and precision. The automated process facilitates recovery of up to four fractions from each SPE tube. Compatible tube sizes range from 1 to 6 mL; sample sizes are from 1 to 1000 mL.
Horizon’s SPE-DEX 4790 extracts semi- and nonvolatile analytes from drinking water. The control module can manage between one and eight extraction modules.
Hamilton Co. (Reno, Nev.) introduced a positive-pressure extraction and evaporation module that sits on the deck of the VANTAGE, STAR and NIMBUS liquid-handling platforms. The Hamilton MPE2 automated extraction and evaporation module can be used to dry the liquid sample that is eluted from an SPE process. Once the elution liquid is removed, the sample may be taken up in an organic solvent that is more compatible with the intended assay technology. The drying gas can be either compressed nitrogen or air. Heating the gas is an option.
Biotage (Charlotte, N.C.) presented a poster on interwell contamination during the loading and drying of plates. This led to the development of the Biotage ACT Plate Adapter (patent pending), which sits atop a multiple-well plate. It significantly reduces cross-contamination, even with volatile analytes.
QuEChERS methods and products
In addition to conventional QuEChERS, UCT (Bristol, Penn.) has developed protocols called AOAC QuEChERs (AOAC 2007.01) and Buffered QuEChERS (EN 15662). Carbon black is replaced with a proprietary polymeric sorbent called Chlorofiltr to remove chlorophyll without significant loss of planar analytes. UCT QuEChers products show very low background compared to packed tubes prepared in the lab.
Dried-blood-spot processors
GERSTEL’s DBSA (dried-blood-spot analyzer) automates sample processing, from card to injector. Evaluation of the analyzer with samples of dried bovine and rat blood spiked with ketamine and amitriptyline and other drugs using an LC/MS/MS analyzer showed that the blood spots produced signals that were about 110% of target for bovine blood, with RSDs of 4–6%.
The CAMAG (Wilmington, N.C.) DBS-MS 500 dried-blood-spot sample-prep workstation supplies liquid samples for assay by LC/MS. Its spacious XY platform provides ample room for 2000 DBS cards. Cards are identified and tracked with inputs from a barcode reader. The spot is punched and added to the vial, and the card is then returned to its tray. Internal standard is added just before extraction. Appropriate wash solvents dissolve the analytes, which are separated from the card matrix by centrifugation. Supernatant is recovered and prepared for injection into the LC/MS. Percent coefficient of variation for peak area ratios is better than 3%, and carryover is insignificant.
Sample extraction system
The Waters Oasis PRiME HLB uses a new, patented polymer to reduce the steps in SPE workflow. Conditioning of the sorbent bed and washing are eliminated, leading to two- or three-step protocols.
LC columns
Chemists at MilliporeSigma looked for a surface chemistry that provided slightly different selectivity than conventional alkanes, particularly when screening for a pain panel. They found that the added aromaticity of a biphenyl phase provided a separation of eight target analytes in 2.2 minutes—hence Ascentis Express Biphenyl U/HPLC columns. Biphenyl is bonded to porous silica on a 2.7-μm-diameter Fused-Core silica. This format provides UHPLC column efficiency with pressure drop compatible with legacy HPLC instruments.
COSMOCORE core-shell particles (Nacalai Tesque, Kyoto, Japan) are 2.6 μm in diameter with a 0.5-μm silica shell. Bonded surface chemistries include C18, cholester and pentabromophenyl (PBr).
Akzo Nobel (Pasadena, Calif.) added phenylhexyl surface chemistry to the Kromasil line of columns. This phase seems to be particularly well-suited for separation of histamines under reversed-phase liquid chromatography (RPLC) conditions. It is available on either 1.8-μm or 2.5-μm Kromasil particles as Eternity XT 1.8 or Eternity XT 2.5. The UHPLC version provides very short run times.
GPC LF Series columns from Shodex (Tokyo, Japan) are packed with a 6-μm-diameter packing with a maximum pore size of 3000 Å, which has an exclusion limit of 2,000,000 for polystyrene. The columns offer a wide operating range. For more resolution, two columns are connected in series. This provides a more uniform and convenient way to construct a linear calibration plot than having to tune the column chain by connecting a series of narrow-range-porosity columns.
In the name of Shodex Asahipak NH2P-50 series columns, the “P” designates polyvinyl alcohol polymer bead, which permits ligand attachment via ether linkages, which are more durable, especially at high pH.
Four new SEC columns in the KW400 series from Shodex have protein exclusion limits of 150, 500, 600 and 20,000 kD. The stationary phase is porous silica with a hydrophilic polymer coating. Specifications include Pmax: 1450 psi, Fmax: 0.35 mL/min, temperature range: 5–45 °C, pH range: 3–7.5, particle size: 3 μm for the 150- and 600-kD phases and 5 μm for the larger pore sizes.
Shodex narrow-pore ODP2 HP series columns have reversed-phase selectivity for small molecules, but the pores of the 5-μm-diameter poly(hydroxymethacrylate) particles are narrow enough to exclude large proteins. The columns show strong retention of highly polar metabolites. High salt concentration is not required to get good peak shapes. Recommended range is 1–50 mM. The columns are thus well-suited for chromatography of drugs and metabolites in biological fluids with MS detection using conventional RPLC mobile phases, even with basic pH.
Building on the success of HILICpac VG-50 columns, Shodex introduced the HILICpac VT-50 for more polar analytes such as phosphorylated saccharides and organic acids. OHpak LB-800 columns, for aqueous SEC and lower baseline noise, were also exhibited by Shodex.
MAbPac RP columns from Thermo Fisher Scientific are designed for top-down proteomics. The particle is a 4-μm-diameter polymer with a 1500-Å pore, wide enough for antibodies and antibody–drug conjugates. The column packing can withstand 0.8 M NaOH at 80 °C, which is used as a regenerant solution to clean protein residue from the stationary phase. The polymeric bead is compatible with all common mobile phases used in LC/MS of proteins.
Tosoh (Tokyo, Japan) featured Toyopearl AFrProtein A HC-650F resin for the capture and release of antibodies for sample prep, process research and production-scale chromatography. Protein A can cost as much as $17,000 per liter. Tosoh notes that the high-capacity resin, indicated by the “HC” in the column’s name, often provides the lowest cost per gram of purified target.
Welch Materials Inc. (Austin, Texas) introduced Boltimate core-shell HPLC columns. Packed with 2.7-diameter particles with a 0.5-μm porous shell encasing a 1.7-μm solid silica core, the columns have a sub-2-μm column efficiency (~200,000 plates per meter) while avoiding the ultrahigh-pressure region of UHPLC. A direct-connect cartridge-style guard column is also offered for the Boltimate. For UHPLC, Welch offers columns packed with totally porous particles with nominal particle sizes of 1.8, 3.0 and 5 μm. Surface chemistries include C18, phenylhexyl, perfluorophenyl and hydrophobic interaction liquid chromatography (HILIC). Inline filters protect the columns from particulate contamination, often cited as a cause of pump and injector seal wear.
Waters extended the CORTECS solid-core LC column line by adding CORTECS C8 and CORTECS Phenyl surface chemistry bonded to 1.6- or 2.7-μm particles. The C8 product fills the gap created by some compendial methods that specify C8 surface chemistry. The phenyl phase provides useful selectivity for aromatic analytes.
Osaka Soda Co. Ltd. (Tokyo, Japan) (formerly Daiso Co.) markets silica-based LC column packings with nominal pore sizes ranging from 60 to 120, 200, 300, 1000 and 2000 Å.
Particle sizes start at 1.7 μm and extend up to 40/60 μm for preparative separations. A wide selection of bonded-phase surface chemistries is also available.
N-glycan kit
Precise control is needed during the production of biotherapeutics, as the function of many proteins depends on the structure and location of glycosylation. Generally, the glycol groups are cleaved from the protein, fluorescently labeled and measured by LC. MS is also used.
Regeneron Pharmaceuticals (Tarrytown, N.Y.) compared its existing biotherapeutic production process with one using the new GlycoWorks RapiFluor-MS Kit from Waters. Regeneron reports the current workflow has turnaround times of tens of hours; the RapiFluor kit reduces turnaround time to about 45 minutes. New cleavage and derivatization protocols greatly accelerate the assay. Regeneron expects to use the RapiFluor along with Waters UPLC with QDa detection in the QA workflow for glycoprotein therapeutics. The RapiFluor-MS kit has reportedly been adopted by the top 15-biopharmaceutical firms.
Ion chromatography
Figure 6 – Thermo Scientific Dionex Integrion HPIC System. (Image courtesy of Thermo Fisher Scientific.)The Thermo Fisher Scientific Dionex Integrion HPIC (high-pressure ion chromatograph) includes columns packed with 4-μm particles, which improves chromatographic resolution and speed (see Figure 6). A new electrochemical detector is optional, previously available only with the Dionex ICS-5000. The plumbing of the Integrion has been simplified to provide reliable, leak-free operation with Dionex Viper fittings. A thermally regulated detector compartment is included as well. The Chromeleon Chromatography Data System (CDS) controls the instrument and provides data reduction and report generation. Whole instrument smart monitoring detects deviations before they become problematic.
A new compact ion chromatograph, the Dionex Aquion, replaces the Dionex ICS-1100 platform. The new platform offers electrolytic suppression to provide high-sensitivity detection and convenience. The IC will probably be used as a dedicated analyzer that can run the same assay for years. A column heater and vacuum degasser are optional.
The most distinguished feature of the System S 150 ion chromatograph from Sykam Chromatography (Ersing, Germany) is the Sykam Auto-Suppressor, which recirculates regenerant solution through cation exchange in the hydrogen form. This is the source of hydronium ions that are responsible for the pH shift in the carbonate-containing eluent.
Supercritical fluid chromatography (SFC)
Sepiatec GmbH (Berlin, Germany) introduced the Prep SFC basic system for preparative SFC with small (analytical-scale) columns. Dual independent pumps deliver CO2 and modifier (usually methanol) with a maximum flow of 20 mL/min with a Pmax of 6000 psi. This is compatible with conventional analytical-scale columns with internal diameters ranging from 4 to 10 mm and lengths up to 250 mm.
Injector, columns and the UV detector flow cell are housed in a heated compartment with a Tmax of 70 °C. Separated fractions are collected in one of up to eight gas–liquid separator vials. These vials contain the product and release the CO2 without contaminating the lab. The software has the same user interface as the company’s larger Prep SFC 100 and 360.
SFC columns
Initial columns for SFC were often first developed and optimized for HPLC. ES Industries (West Berlin, Germany) showed that SFC is much better with phases optimized for SFC. For example, about a third of the small-molecule drugs in the development pipeline contain fluorine, and the company’s GreenSep Fluorobasic phases are ideal for SFC of fluoroamines. Peak shapes are excellent, even without adding trifluoracetic acid or alkyl amines.
For a chiral selector, ES chemists focused on 2-fluoro-5-methylphenyl cellulose. ChromegaChiral CCO-F2 columns have been used successfully with chiral SFC and HPLC modes. ChromegaChiral CCO-F4T3 surface chemistry—4-fluoro-3-(trifluoromethyl) phenyl cellulose bonded to 5-μm silica particles—appears to have preferential affinity for analytes containing trifluoromethyl groups. GreenSep Naphthyl is a unique phase for SFC of natural products such as quinines. The naphthyl phase is planar with a strong pi-electron cloud. This seems to provide differential interaction with other aromatics, including heteroaromatics found in natural products. Recently, Supelco and ES announced a cooperation agreement whereby Supelco will distribute ES SFC columns on a global basis.
Field-flow fractionation (FFF)
Postnova Analytics (Salt Lake City, Utah) introduced AF2000 MultiFlow asymmetric-flow field-flow fractionation (AF4) instruments for the separation and characterization of polymers, proteins and nanoparticles. Measurement range is 1 nm to 100 μm, and particles can be separated in liquids with pH of 2 to 11. Channel flow is 0–10 mL/min with a cross-flow of 0–8 mL/min; Pmax is 35 bar. A metal-free flow chamber with a ceramic frit and a range of membranes offer broad sample compatibility. Instrument setup and runs are controlled with NovaFFF expert software, which includes a method wizard.
The PN3621 MALS detector is part of Postnova’s FFF suite. It scatters light from samples as small as 7.8 nL, despite a sample cell volume of 63 μL. Scattered light is detected by 21 photosensors placed in a ring surrounding the sample cell. The molar mass range is 103–109 Da.
Chromatographs of the future
Micro-GCs
Qmicro B.V. (Enschede, The Netherlands) introduced the Qmicro gas chromatography module, which has a unique foreflush and backflush injector and a range of micro-detectors for gas analysis. Submodules provide flexibility. The thermal conductivity detector is chip-mounted internally, but the FID and MS are external.
Columns are in a second module, along with the heater. A wide range of wall-coated open-tubular and porous-layer open-tubular columns are available with fused-silica or metal tubes. The column+ heater module clicks into place on the base chassis. Limit of detection is about 500 ppb, depending upon the analyte and columns. Communication and control are via industry-standard protocols. The base unit is 20 cm × 20 cm with a depth of about 8 cm, making it small enough to fit almost anywhere, such as on top of an MS or inside a rack mount.
Super-fast nano-LC
In LC, commercial instruments have a hard time keeping up with advances in column technology. The next column advance may be capillaries featuring internal diameters under 10 μm and submicron packings or wall-coated phases. Mary Wirth’s lab at Purdue University (Lafayette, Ind.) appears to be leading in the development of this technology, which offers tremendous column efficiency through generation of an effect often called slip flow. The flow profile obtained when using larger-diameter packing materials resembles a blunt parabola with trailing edges. However, in slip flow, the small interstitial spaces between the nanoparticles facilitate “slipping” of the polar mobile phase along the “greasy” reversed-phase particle surfaces, essentially eliminating the trailing edges of the blunt parabola flow profile and significantly narrowing the chromatographic band widths. Column efficiencies of 2 million plates per meter have been reported.
A nano-flow LC plumbed with a very narrow capillary column was prominently displayed in the VICI booth. Two nano-syringe pumps feed a mixing T just before a valve that contains the injection loop and column inlet. The nano pumps deliver 2.5 nL per step and 10 pL per microstep. Smooth delivery of mobile phase at <10 nL/min is now realistic. Nano-flow LC operates with small-diameter columns, including slip-flow columns, so this LC is clearly designed for analytical applications.
Submicrometer fused-silica capillaries
With the introduction of nano-capillary fused-silica tubing by Polymicro Technologies (Phoenix, Ariz.), chromatographers and engineers will be able to conveniently explore nano-scale instrumentation ultimately leading to single-molecule analysis. Tubing internal diameters range from 200 to 1000 nm and interface with current fitting technologies. Fittings (unions and splitters) and cleaving stones are also available.
Pittcon 2017 is scheduled for March 5–9 in Chicago, Illinois. Please monitor pittcon.org for more information.
Reference
- www.americanlaboratory.com/914-Application-Notes/182544-Calibration-Free-Catalytic-Microreactor-for-Analysis-of-Pesticidesin-Food/
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]