HPLC 2016 Sets the Stage for the Next 50 Years

Over 1100 scientists attended HPLC 2016, held June 19–24 in San Francisco, looking for a glimpse into what the method may offer in the future, as well as to see new developments, the best of which are reported here.

Nanoflow UHPLC

Prof. Milton Lee (Brigham Young University, Provo, Utah) reported on the development of a compact nanoflow UHPLC, the components of which were displayed by VICI (Valco Instrument Co., Inc., Houston, Texas). The dual-pump gradient system is only about 4 × 8 × 12”, with a Pmax of 16,000 psi and flow maximum of 500 nL/min, which is adequate for 150-μm-i.d. capillary columns packed with sub-2-μm, and even sub-micron column packings. With this low flow rate, the nanoflow LC is compatible with MS detection. Its small size facilitates placement close to the MS inlet, which improves detection and throughput. Alternatively, if a UV detector is sufficient, a small light-emitting diode (LED) UV detector that uses on-column illumination is available. It’s surprising how well the UV works, given the small capillary column diameter.

The nanoLC may be an interesting and potentially timely development since it enables the sub-micron column packing technology emerging from the lab of Mary Wirth at Purdue University (West Lafayette, Ind.). It is not clear if nanoflow UHPLC technology will fill a significant application need. In any case, it is something to watch.

Surprising thermal effects in LC

Temperature-assisted sample focusing

Steven Groskreutz (University of Pittsburgh, Penn.) showed that temperature-assisted sample focusing can dramatically improve resolution at the low k’ region of a UHPLC chromatogram. This is significant, since other resolution-improving methods are typically useful at high k’.

Groskreutz made a low-thermal-mass, high-powered thermoelectric element, which was used to cool a short pre-column connected to a 2.1-mm-i.d. analytical column. Heat transfer was improved by milling flats to facilitate thermal contact between the cooler and pre-column. Pre-column temperature was decreased to 5 °C using the cooler. The analytical column thermostat was set at 80 °C. An alkyl parahydroxybenzoate sample was injected and trapped in the low-temperature pre-column. To inject, the pre-column was rapidly heated to 80 °C, which released the sample components to the eluent in a tight band. Early-eluting peaks were found to be narrower and taller, by as much as two times. Peak trapping could be useful when injecting a dilute sample or as an interface between columns in multidimensional LC.

Adiabatic column insulation improves efficiency up to 40% in LC and SFC

A session on high-efficiency separations drew a packed house. One lecture, by Fabrice Gritti of Waters (Milford, Mass.), described how mounting an LC column in a vacuum jacket improved column efficiency by as much as 40%. Why? It is known that mobile-phase friction in the column generates heat in LC (adiabatic expansion of mobile phase in supercritical fluid chromatography [SFC] cools the column). When the column is positioned in a water bath, column heater/cooler, or even in still air, heat exchange with the environment results in a radial thermal gradient across the column. Because the region near the column wall has a different temperature than at its center, the viscosity and flows of the mobile phase vary. This phenomenon results in an apparent loss in column efficiency, or even split peaks. However, if the column is mounted in a vacuum chamber, no heat is exchanged with the environment. A vacuum jacket coated with aluminum foil (to minimize the heat radiation) is more efficient than other means of column thermal insulation. With vacuum-based insulation, the on-column flow profile approaches a square wave (a.k.a. plug flow), resulting in narrower bands and higher column efficiency. Gritti demonstrated that optimal column performance is achieved at higher mobile-phase linear velocity than is typically observed in the literature.

Computer-aided method development and transfer

Several posters described the use of computer-aided method development and robustness evaluation. Recent upgrades to DryLab software (Molnar Institute, Berlin, Germany) include peak tracking using UV/VIS and MS during method development. Traditional methods use area counts and absorbance ratios or UV/VIS spectra. DryLab uses a combination of UV and mass spectra to track peaks. The combination of spectra is particularly useful when modeling complex mixtures in which peak order is not constant and co-elution or ion suppression is a concern. MS and UV/VIS detector chromatograms are used to construct a peak table for the sample. Several runs are made using a factorial design approach. DryLab constructs a resolution table supported by a 2-D heat map. One result indicated that a gradient from 5 to 95% MeOH at 27 °C would give a 35-minute separation. The prediction was compared to the experimental run, and agreement was excellent. Combining MS and UV/VIS spectra for peak tracking significantly improved method development speed and aided in the development of tolerance bands for robustness studies.

Catalytic degradation of antibodies traced to injector needle

Shishan Zhao and colleagues at Charles River Labs (Senneville, Canada) demonstrated that metal ions from corrosion of injection needles can catalytically degrade monoclonal antibodies by oxidation or hydrolysis. Degradants generally appear shortly before the main peak in the chromatogram.

SFE and SFC of natural products

Supercritical fluid extraction (SFE) and SFC of natural products, primarily cannabis, was a timely topic. Care By Design (Santa Rosa, Calif.) uses SFE with CO2 to extract nonpolar fractions from cannabis plants. The process is much less dangerous than extraction using liquefied petroleum gases (LPGs) such as propane, butane and pentane.

SFE and SFC instruments

PIC Solution Inc. (Media, Penn.) exhibited a range of instruments for SFE and SFC. The SFEPICLab Ex-20 is based on the SFC-PICLab Hybrid 20, which is designed for liquid flow up to 20 mL/min, ideal for 30-mL extractor vessels. This size is convenient for development of larger-scale SFE processes. The SFE-PICLab Ex-20 can sequentially process up to 10 extraction vessels and deliver fractionated material to collection bottles. Chromatographers can program the pressure, flow and co-solvent as a function of time. The controller is identical to those used in larger systems such as the SFEPICLab 100 and 400, facilitating scaleup.

SFC-PICLab Analytic, for analytical applications including method development, includes a column compartment that accommodates a maximum of 10 analytical columns (4.6–10 mm) to scout for usable selectivity. Column compartment temperature can be programmed from 15 °C to 60 °C. Maximum flow rate is 10 mL/min at a Pmax of 350 bar, and detection is diode array UV/VIS with up to four simultaneous wavelengths. PIC supports the instruments with flow rates up to 600 mL/min with columns, column packer and software.

2-D and higher-dimension LC separations

For about the last decade or two, I’d relegated LC × LC to the back burner. It seemed to be a technology looking for an application. However, at HPLC 2016, at least 42 posters and lectures referenced 2-D LC, “comprehensive” LC or LC × LC. The applications below show that 2-D LC has crossed the chasm from technology push to applications pull.

Examples of 2-D LC

Xiyu Ouyang and colleagues at the University of Amsterdam, The Netherlands, presented a poster, “Unraveling the composition of environmental samples from complex matrices using LC × LC/TOF/MS.” Ouyang’s lab uses effect-directed analysis (EDA), which involves repeated fractionation and bioanalysis, with qualitative analysis the particular focus. LC × LC significantly increased throughput of the EDA cycle. The 2-D part used C18 reversed-phase liquid chromatography (RPLC) in the first dimension and a perfluoropropane phase in the second. Twenty percent of the column effluent was routed to the electrospray ionization/time-of-flight/mass spectrometer (ESI/TOF/MS) for chemical identification; 80% was passed through a UV detector and onto a fraction collector (4 × 96 wells) for drying and toxicology assay.

A team at Novartis (Basel, Switzerland), led by Imad Ahmad, presented a poster illustrating the usefulness of 2-D LC for purposes other than increasing peak capacity. They used 2-D LC: 1) to facilitate the correlation of MS data to impurity peaks collected with a mobile phase with poor compatibility with MS due to phosphate buffer; peaks of interest were collected from the first dimension by heart-cutting and were reinjected into the second dimension, where a volatile LC/MS-compatible buffer was used; 2) for complex samples, such as formulation and degradation assays, to confirm the purity of a peak after method development using an orthogonal column in the second dimension; 3) to characterize degradants or aggregates in small- and large-molecule combinations; and 4) to aid in column selection for resolving critical peaks.

A team at the Merck Bioprocess Development unit in Kenilworth, N.J. used a combination of ion exchange (IEX) and hydrophobic interaction chromatography (HIC) to fractionate oxidation products from an antibody-derived therapeutic protein. IEX chromatography with a weak cation exchange column provided separation of acidic and basic fractions from the main peak. HIC using a butyl phase delivered resolution sufficient for quantitative analysis of the oxidation products.

RP18-amide for orthogonal selectivity to C18 for 2-D LC

Fortis Technologies, Ltd. (Neston, U.K.) introduced RP18-amide as a stationary phase with different selectivity, targeted for 2-D RPLC applications. Embedding the amide phase among the C18 changes the selectivity and preserves column efficiency.

Sonja Schneider of Agilent (Waldbronn, Germany) presented a poster describing 2-D LC/MS for characterization of charge variants of antibodies and fragments using IEX chromatography followed by RPLC. The order of the modes is important, since RPLC reduces the salt content, improving MS performance and reducing maintenance.

LC: past, present and future

Over the last 50 years, separation science has led the growth in analytical chemistry. Prof. Barry Karger (Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Mass.) compared HPLC in the 1960s to today. At that time, academic research garnered most of the prestige and attention, with an emphasis on new instruments. Prof. Karger was highly instrumental in helping HPLC develop into a reliable analytical technology. Today, the focus is on applications, particularly ’omics. Life scientists have adopted tools such as HPLC and MS, and many do not have formal training in analytical chemistry. Karger reported that reproducibility of results has become a major concern. However, he is optimistic that the major trends, such as increasingly smaller samples, improved detection limits and automation, will open up new applications, which will then drive the development of new technology. He advised, “It is important to understand the problem first and then creatively find the successful strategy to innovate and apply separations.”

Chromatographers and mass spectrometrists have long argued about which technology is the most important, the fastest, the most information-rich, the most useful. For 50 years, chromatographers have sought to improve column efficiency (plate count) as a way to improve chromatographic speed and, thus, throughput.

In a thought-provoking lecture, Prof. Fred Regnier (recently retired from Purdue University) argued that, if chromatographers focus on selectivity, completely new paradigms open up. These could offer much higher throughput and be more useful than traditional hyphenated techniques such as LC/MS. He used proteoforms, which include all of the protein forms and derivatives arising from a single gene, as an example. A gene codes for the primary protein sequences, but post-translational modification (PTM) can give several thousand potential derivatives, each with a potentially unique biofunction. With the growing success of clinical diagnostics, he predicted that the annual sample load of blood assays will soon reach 100 million per year.

Regnier proposed a paradigm change. In clinical diagnostic tests, he noted, clinicians want to focus on a small set of disease markers, rather than on a high-resolution chromatogram showing thousands of peaks in an hours-long run. Using natural bioselectivity as a guide, he suggested developing bioaffinity capture reagents such as antibodies bound to engineered nanoparticles to fish out the analytes (biologically active proteins) of interest. Once loaded with analytes, the particles are transported to the inlet of an MS for analysis.

Regnier expanded on the design of his analyte sequestering transport particle (ASTP). The particles are 10–100 nm in major dimension. They have affinity footprints for specific families of compounds. Regnier also engineered a transport interface with 10-nm side pores to retard matrix components of the sample while the analyte bearing ASTPs races toward the MS inlet. The beauty of this approach is that bioaffinity interactions can be highly specific. Regnier envisions that one can develop affinity ligands, each selective for a particular family of analytes, or markers of specific diseases. Each can be bound to ASTP units. A disease assay can have a mixture of beads, some up-regulated and some down. With multivariant markers, the pattern is the key diagnostically relevant result.

The affinity-footprint ASTP can be selective to PTMs—in particular, positions along the peptide backbone, which is diagnostically important but not available from MS alone. Top-down and bottom-up MS protocols do not provide this information unambiguously.

New chromatographs

Agilent Technologies

Two years ago, Agilent (Palo Alto, Calif.) upgraded the 1290 Infinity I UHPLC, calling the new instruments the 1290 Infinity II. The dual-pump low-dispersion instrument offered premium performance and price. Now, Agilent has upgraded its 35-module LC line, including column compartments and detectors, to Infinity II standards and appearance. The 1260 Infinity II, with a Pmax of 600 bar, is compatible with columns with particle diameters larger than about 2.5 μm. The fast detectors can follow the narrow peaks achieved with core/shell particles, including Poroshell 120 series core/shell columns with 2.7-μm-size particles. These columns offer similar efficiency to sub-2-μm totally porous particles with half the backpressure.

The Infinity II Multisampler automates an interior and exterior wash protocol that reduces sample carryover to below 10 ppm. Dual-injectors rinse one syringe while the other makes an injection. For high-throughput studies, the Multisampler has a maximum capacity of 6144 samples (16 384-well plates). The race for the shortest cycle time between LC and MS is very competitive, especially in quantitative analysis.

SIELC Technologies

The Alltesta HPLC analyzer from SIELC (Wheeling, Ill.) targets routine isocratic analysis of pesticides, dyes and drugs. The small, lightweight instrument can be placed almost anywhere, including in a bank of analyzers, each dedicated to a particular assay. Service is by rapid module exchange, for pump, detector or autosampler, using express shipping.

Waters Corp.

Waters (Milford, Mass.) introduced the ACQUITY Arc LC system, which allows users to change from HPLC to UPLC with the flip of a switch. This is particularly useful in bridging studies in updating validated methods.

Thermo Fisher Scientific

Q Exactive BioPharma (Thermo Fisher Scientific, Waltham, Mass.), for bottom-up and top-down (native) proteomics, utilizes a hybrid quadrupole-Orbitrap design to extend mass range to handle antibody–drug conjugates and intact monoclonal antibodies. It also performs well with peptide mapping. The platform is supported by Thermo BioPharma Finder software, which integrates the instrument and data for full characterization in proteomics.

Knauer Wissenschaftliche Gerate GmbH

AZURA HPLC instruments from Knauer (Berlin, Germany) span HPLC and UHPLC. The AZURA Compact HPLC features an isocratic flow path with an Fmax of 10 mL/min and Pmax of 6000 psi, while the AZURA HPLC Plus offers gradient elution and a Pmax of 10,500 psi. At the top of the line is the AZURA UHPLC version with a Pmax of 15,000 psi. Options include pre- and post-pump gradient. Mixer volume is 50 or 100 μL, and degassers are optional. A tablet interface for remote monitoring and control of the chromatograph is suitable for transmitting data to a chromatography data system or LIMS.

Knauer is known for its modular designs. Simply by changing flow-path components allows a user to convert a Knauer pump from analytical to preparative flow rates (50 mL/min to 1000 mL/min; similarly, detectors scale, often by changing the flow cells. AZURA Prep LCs are supported by a range of autosamplers, fraction collectors and automated valves that facilitate plumbing the most sophisticated flow path, including simulated moving-bed chromatography and peak recycling.

Mass-directed purification

Interchim’s (Los Angeles, Calif.) PuriFlash LC-MS features a fast, single-quadrupole MS for mass-directed purification. The PuriFlash couples the output of MS detection with other detection modes to fine-tune the cut points, which can improve recovery, to typically higher than 95%. Key is a dynamic splitter that keeps flow rate to each detector constant. The MS detector features ESI or atmospheric-pressure chemical ionization (APCI). Maintenance does not require breaking vacuum. A UV/VIS detector with a 200–600 nm wavelength range is the general column monitor. One unique feature is that flow to the multiple detectors (UV/VIS, MS and electron light-scattering detector, ELSD) are parallel rather than serial. Thus, detector signals are synchronized in time, which facilitates inspection of the outputs for unexpected results.

Fittings and related hardware

EXP fitting systems from Optimize Technologies (Oregon City, Ore.) are designed for PEEKsil tubing. When fitted with a Ti-LOK ferrule (Optimize Technologies), PEEKsil tubing can be hand-tightened to over 20,000 psi. Connections made with the fittings qualify as zero dead volume.

The EXP Nano Trap Kit provides all the ultralow-dead-volume hardware required to add a trapping column to multiport automated valves. Supported thread formats include 10-31 for 1/16” PEEKsil and 6-32 and 6-40 for 1/32” tubing. The standard trapping bed is 0.125 μL, but other volumes are offered. Capacities up to 100 μL are available with the EXP Trap Column. Trapping columns are often used to remove detergents and salts for improving MS compatibility.

New LC columns and packings

YMC America

YMC America (Allentown, Penn.) introduced Chiral Art columns for the separation of chiral compounds using amylose or cellulose chiral selectors; -SA, -SB and -SC are bonded, which improves durability. Two of the phases are coated, designated by appending “-C” after the chiral selector designation (SAC, for example). All have very low background due to column bleeding. According to a company brochure, the columns show remarkable stability in terms of plate count, pH stability and mobile-phase compatibility, particularly with the bonded phases. Chiral Art columns are a direct outgrowth of YMC’s custom column business.

Chiral Technologies, Inc.

The CHIRALPAK IG column from Chiral Technologies, Inc. (West Chester, Penn.) is a 5-μm amylose p-bead derivatized with tris(3 chloro-5-methylphenylcarbamate). This unusual chiral selector was chosen after comparing chiral separations with an extensive library of more than 150 candidate chiral stationary phases. As with the other CHIRALPAK chiral stationary phases CSPs)—1A, 1B and 1C—the meta-substituents seem to give the highest selectivity, suggesting that they may have superior accessibility.

Daicel Chiral Technologies

Several million HPLC columns are replaced each year, yet initiatives to recycle the hardware have not been adopted. Daicel (Shanghai, China) offers a service to recondition used chiral columns. Common problems are plugged frits and damaged stationary phases. If a column can be resurrected, it is packed, inspected and returned to the customer. The fee is a small fraction of the replacement price for a new column. For large-diameter preparative columns, the service also includes a matching analytical column.

Dikma Technologies

Endeavorsil columns from Dikma Technologies (Lake Forest, Calif.) are suitable for UHPLC applications with internal diameters of 2.1 mm and lengths from 30 to 100 mm. The columns are packed with 1.8-μm spherical silica particles with 120-Å pore diameter. Surface area is 300 m2/g, coverage is 3.5 μm/m2, carbon load is 20% and pH is 1.5–10, with endcapping. Endeavorsil’s selectivity differs slightly from that of the Waters ACQUITY HSS T3 C18. However, selectivity depends upon the sample. If one is evaluating a panel of sub-2 columns, Endeavorsil should be in the library.

Dikma also introduced Leapsil columns for ultrafast LC separation using sub-2 technology. These are packed with 1.8-μm spherical silica with a 100-Å pore; the C18 phase has a carbon loading of 27%, which is quite high. The synthesis protocol is designed to deliver high-purity (99.999%) silica for strength and stability. Endcapping extends the pH range from 1.5 to 10. Other surface chemistries are planned.

GL Sciences

GL Sciences (Tokyo, Japan) exhibited InertSustain AQ-C18 phases with C18 groups bonded at equal distance to the silica gel. This, they say, offers significant retention for highly polar compounds, even under water-rich mobile phases. Steel-coated-PEEK hardware is available upon request for analyzing samples such as nucleotides having several phosphate groups, which are sensitive to stainless-steel hardware. A spider chart showed high retention of acidic, basic and neutral test compounds. The AQ-C18 column is strong enough to retain polar analytes, even with 20% acetonitrile in the eluent. Solvent peaks usually precede analytes. Chromatograms of watersoluble vitamins, organic acids, catecholamines, nucleotides, nucleosides and free bases are depicted in a product brochure.

AkzoNobel

Kromasil EternityXT is the newest column offered by AkzoNobel (Amsterdam, The Netherlands). The column packing consists of a dense silica core with a hybrid organic/inorganic gradient, with ligands such as C18 on the exterior. High-stability coupling chemistry provided by the organic layer extends the useful pH range from 1 to 12, even at elevated temperatures. This wide pH stability range facilitates searching for optimum pH for separation and column regeneration. Nominal particle size starts at 1.8 μm and scales to 10 μm for rapid prep.

SIELC Technologies

SHARC (Specific Hydrogen-Bond Adsorption Resolution Chromatography) columns from SIELC Technologies show separation based almost exclusively on hydrogen bonding. Typical eluents are nonaqueous, with acetonitrile as a weak solvent, since it has no hydrogen-bonding capability, and methanol, which is a strong solvent due to its propensity to bond to hydrogen. Methanol/acetonitrile is also much less viscous than either solvent mixed with water. Lower viscosity improves mass transfer kinetics, which can reduce run time by 80%. Typical analytes include small-molecule drugs with amine, alcohol or acid groups.

Shodex

Shodex (Tokyo, Japan) introduced columns packed with two new stationary phases for hydrophilic interaction chromatography (HILIC). Both packings start with 5-μm-diameter, 100-Å porosity spherical hydrophilic polymeric beads. For anionic analytes, the beads are bonded to proprietary quaternary ammonium groups (HILICpak VT-50); for neutral analytes, the surface chemistry is proprietary amino (HILICpak VG-50). Importantly, VG-50 columns demonstrate amino selectivity, but avoid the problem of amines relentlessly dissolving the silica when bonded to conventional silica beads.

In LB-800 OHpak columns, “LB” designates “low bleed,” which improves compatibility with light-scattering detectors. The key is that bleed of microparticles from the column has been eliminated. Previously, the SEC columns needed to be washed for days or weeks to reduce the background noise resulting from microparticle bleed from the column packing. Light-scattering detectors respond preferentially to the largest particle in the detector cell. OHpak 803 has a 800-Å pore diameter, and OHpak LB 806 has a pore diameter of 15,000 Å. Both are compatible with water and dimethylformamide (DMF) eluents, and correspond to USP L38 and L39 column classes.

Sigma-Aldrich (A Part of MilliporeSigma)

Sigma-Aldrich’s Supelco Division (Bellefonte, Penn.) displayed the Supel Genie solid-phase extraction cartridge for online removal of phospholipids from serum, plasma and whole blood. Supel Genie is designed as a disposable inline cartridge with a proven useful lifetime of more than 100 injections. Typical analytes include risperidone, clomipramine, digoxin and metabolites of Vitamins D2 and D3.

Acknowledgment

Prof. Robert Kennedy of the University of Michigan at Ann Arbor deserves credit for organizing a coherent and stimulating technical program, as does Barr Enterprises for handling the logistics and creature comforts.

Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].