Advances in Liquid Phase Separation at Pittcon® 2012

Eight years have passed since Waters (www.waters.com) introduced the ACQUITY UPLC®. This is a long life cycle for a groundbreaking instrument. Already, vendors are offering components (tubing, valves, and fittings) that will enable operation of next-generation ultra high-performance liquid chromatographs (UHPLCs) at 30,000 psi or so. The components will be expensive, since stronger alloys will be required to replace 316 stainless steel. While we wait for the next generation of liquid chromatographs, we can take a look at the noteworthy advances that have been made in instruments, columns, applications, and components for liquid phase separation.

Capillary ion chromatographs

Chromatographers at Thermo Fisher Scientific (www.thermoscientific.com) have extended the Pmax of the Dionex (www.dionex.com) ICS-5000 capillary ion chromatography system to 5000 psi. This includes the very popular RFIC™ (Reagent-Free™ Ion Chromatography) capability. More pressure facilitates faster separations using columns packed with 4-μm-diam particles. The ICS-5000 still uses the IC Cube module, which integrates sample and mobile phase handling in a small, convenient, chromatography-efficient package. With capillaries, mobile phase consumption is only 5.25 L/yr in 24/7 operation. With RFIC, the mobile phase reservoir is refilled with pure water. This facilitates walk-up-and-inject operation. In addition to conductivity, the ICS-5000 is compatible with UV-VIS, MS, and the new Thermo Scientific QD. Control is via Chromeleon chromatography software.

Not all labs need the flexibility offered by the ICS-5000, so Thermo Fisher Scientific introduced the Dionex ICS-4000 capillary ion chromatography system, which also uses the IC Cube module and offers “always on” operation, 5000-psi capability, and multiple detector options. The 4000 has a smaller footprint than the 5000. Chromeleon software manages the I/O and operation.

Nano LC platform

Nanospray electrospray ionization (ESI) detection typically provides superior detection in proteomics research. However, plumbing the components together (injector, column, ESI interface, and mass analyzer) is often frustrating. MassTech (www.apmaldi.com) introduced the “The-LCP” thermally stabilized platform for high-temperature nano LC separations. Running at an elevated temperature increases the separation efficiency. In one case, only 137 peptides were found for a protein digest with a column temperature of 20 °C. However, at 65 °C, the peak count increased to 303. Some of the improvement is attributed to efficiency and some to an increase in recovery. Another study showed that running at 60 °C greatly improved consistency of relative peak heights, enabling quantitative protein profiling.

Modular LCs

Analytical Technologies Ltd. (ATL) (www.ais-india.com) is a new name to Pittcon in HPLC and other instruments. Products include a range of modular LCs, from several analytical systems to lab-scale prep and even larger to process-scale instruments for bioprocessing. Dedicated analyzers include ion chromatography and amino acid. Pump pressure is the most differentiating feature of HPLC. ATL offers a prep pump rated from 1500 to 4000 psi. Analytical pumps start at 6000 psi and extend up to 18,000. Pre- and multipump gradient elution systems are also available. Detectors include refractive index, UV-VIS, diode array detection (DAD), digital amperometric, fluorescence, and evaporative light scattering. Columns range from analytical to axial compression preparative columns with diameters from 30 to 1000 mm. AnalChrom™ chromatography workstation software supports both preparative and analytical systems. The firm is represented in over 90 countries.

Low-pressure LC components

Occasionally, I attend a press conference where the subject matter is completely different than expected. This year, SFC Fluidics® (www.sfc-fluidics.com) hit me in the face. Premeeting promotional information led me to expect a new HPLC or perhaps an SFC system. However, after the first few minutes, I was certain there was much more. “SFC” in the company name stands for Small, Fast, Cost-effective components and systems, not supercritical fluid chromatography.

SFC is all about novel plastic components such as QuickConnect™ connectors, manifold, pump, and micromachined devices. QuickConnect fittings provide a butt-to-butt union of plastic tubing with very small dead volume. The fittings are held in place magnetically. The ePump® model 190 uses electroosmotic flow to deliver accurate, pulse-free flow over the nL to μL range. It is small (~1 in.3) and lightweight, and Pmax is 300 psi. The pump operates by applying a voltage across a porous membrane. The ions drag water of hydration across the membrane. The volume increases, but is confined, so the pressure increases. A second impermeable flexible membrane separates the osmotic section from the delivery fluid, which is expelled. A small controller operates the valves required for the pumping action.

The components have been assembled to make a low-pressure LC pumping system. It appears to be suitable for short columns using plastic capillary column technology with Superdex™ 75 column packing (GE Life Sciences, www.gelifesciences.com) for separating proteins, including bovine serum albumin (BSA), ovalbumin, myoglobin, and transferrin. SFC components should also be useful in flow injection analysis.

Recycling module

From time-to-time, recycling chromatography reappears. Those who have been around long enough may recall that Waters promoted recycling intensely to improve resolution in gel permeation chromatography (GPC). The key is to make sure that the extracolumn band broadening, measured in volume, is small compared to the peak width. Japan Analytical Industry Co., Ltd. (JAI) (www.jai.co.jp/english) introduced the NEXT recycling module for all of its preparative LCs. With recycling, the effluent from the column is routed to a detector and then back to the pump inlet for another pass through the column. In the ideal case, the chromatographic resolution increases by the square root of number of passes through the column. Two passes give an improvement of 1.4, and four cycles a twofold increase, etc. JAI points out that recycling conserves solvent use and reduces the investment in columns.

DNA analyzer based on HPCE

Despite the high resolving power, high-performance capillary electrophoresis (HPCE) is not in the spotlight. Indeed, it is not even on stage, or even in the audience, for that matter. Slab gel electrophoresis is still the most common technology for assaying nucleic acids and proteins. However, polyacrylamide gel electrophoresis (PAGE) and agarose are slow, tedious, and often insensitive compared to HPCE with gel capillaries. Improved assay technology is needed, particularly for quantitative PCR of DNA. In response, BiOptic, Inc. (www.bioptic.com.tw) introduced the Qsep100 fluorescence dna-CE analyzer. The analyzer uses a pen-shaped gel cartridge, which feeds the DNA sample to a laser-induced fluorescence (LIF) HPCE. The system provides 2.5-min separations with high detection sensitivity (4 bp resolution at 50–500 bp range) from a 96-well plate. Average %RSD in migration time is less than 0.75% for a 10-run set from the same well. The unique optical design, which uses ball lenses, enables a price point that is much lower than currently available LIF-HPCE instruments. The Qsep100 received a Pittcon Editors’ Award honorable mention.

Field flow fractionation interface

PostNova Analytics (www.postnova.com) and Malvern Instruments Ltd. (www.malvern.com) announced an agreement that marries the PostNova AF2000 Series asymmetrical flow field flow fractionation system and the centrifugal field flow fractionation system with the Malvern Zetasizer Nano. The firms developed a field flow fractionation-dynamic light scattering (FFF-DLS) interface that increases the detection sensitivity by 3–4 times. Targeted applications include proteins, i.e., aggregates, and nano particles of gold, silver, and carbon.

HPLC detectors

QD detector for ion chromatography

At Pittcon 2011, a lecture by Dr. Kannan Srinivasan of Dionex described a new detector idea for ions. Compared to traditional suppressed conductivity, it was within about a factor of 10 in detection sensitivity, but for weak acids or bases, it was better. All ions with the same charge produced the same signal, that is, the detector responded to normality rather than molarity. A year later, Dionex is now part of Thermo Fisher Scientific, and the detector was introduced as the Thermo Scientific Dionex QD.

The QD is the world’s first charge detector for ion chromatography. It detects all ionizable species. It is recommended for detection of phosphates in environmental samples, organic acids in food and beverages, and amines. In some cases the relative response of the conductivity and QD detector can confirm identity. The detector received a Pittcon Editors’ Award honorable mention. Also, Prof. Sandy Dasgupta (University of Texas at Arlington), who is named as inventor on the patent that is the basis of the QD, was honored with the Dal Nogare Award

850 IC Amperometric Detector

Many of the analytes that are most suited for separation by ion chromatography lack strong chromophores or fluorophores for detection. Carbohydrates, amines, and acids are typical problem analytes. Metrohm (www.metrohm.com) introduced the 850 IC Amperometric Detector as an option to the Model 850 Professional IC, 881 Compact IC pro, and 882 Compact IC plus. The detector can operate in series or in parallel with other detectors such as conductivity and UV-VIS absorbance. Detection sensitivity is low ng/L for electroactive analytes. Metrohm offers gold, silver, platinum, and glassy carbon working electrodes as required by the analytes. The reference and auxiliary electrodes are maintenance free. A standalone version of the detector is offered as the Model 896 Professional Detector.

Quadrupole mass spectrometers for LC/MS/MS

IONICS Mass Spectrometry Group (www.ionics.ca) introduced two new lines of triple quads for LC/MS. Both the Series 100 and 200 have two probe options. One option can be switched between ESI and atmospheric pressure chemical ionization (APCI) detection in less than 20 msec, and the other is dedicated to one. Mass range is 5–1500 m/z. Dynamic range is better than 106. Scan rate for the Series 200 is fast, at 27,000 data points per sec. With the Series 100, the data rate increases further to 50,000 pts/sec.

Ion mobility detector for HPLC and HPLC/MS

Ion mobility spectrometry (IMS) is particularly useful in separating isomers. Excellims Corp. (www.excellims.com) introduced the AIMS2100 ESI high-performance ion mobility spectrometer as a detector module for HPLC and HPLC/MS. Adding the AIMS2100 to an LC/MS provides an orthogonal method for compound identification since IMS resolves isomers based on ion mobility rather than mass. Typically, the column effluent is split between the AIMS2100 and other detectors. The orthogonal nature of the detection reduces the need for LC×LC and SFC×LC, even for the most complex samples. Ionization with ESI improves detection sensitivity to the subnanogram range, especially of low-volatility analytes. Linear dynamic range is 102 to 103. Typical applications include separation of skeletal peptide isomers, diastereomers, cis–trans isomers, and carbohydrates.

The most significant figure of merit for HPLC detectors is usually the noise level. SCINCO (www.scinco.com/en) introduced the DAD-208 diode array detector with a noise level of ±2.5 × 10–6 AU. The diode has 1024 elements covering the range from 190 to 950 nm. Chromatographers can select up to eight wavelengths to monitor at 50 Hz. Three flow cell options have a volume of 2–15.5 μL. The company is looking for OEM partners.

Flow splitter for detectors

Splitting column flow between detectors is a technique that sounds good in concept but is frustrating in practice. Analytical Scientific Instruments, Inc. (ASI) (www.hplc-asi.com) introduced the QuickSplit™ AS650 to automate flow splitting. The AS650 should open up many new applications that would benefit from multiple detectors. The company points out that the QuickSplit is the first fully automated flow splitter for HPLC. It can split the column flow to deliver microliter flow rates to an optical detector and nanoliter flows to an MS. Inlet and outlet flows are entered with a keypad. A four-line light-emitting diode (LED) display provides instantaneous status reports to the operator and to a PC for recording. The dynamic control keeps the split ratio constant during gradient elution, where viscosity excursions can be large. Volume input ranges in mL/min are 1–5, 5–25, and 25–90. Flow ranges of the side stream (in μL/min) are 0.1– 0.5, 0.5–2.5, 2.5–12.5, and 12.5–62.5. Key applications are expected to include proteomics and environmental, and even preparative chromatography, where a side stream is sent to the analyzer. ASI points out that the AS650 takes the guesswork out of flow splitting.

UHPLC module

Now with UHPLC capable of giving subminute runs, high-throughput labs need to load and process more samples for overnight operation. To meet this need, Shimadzu Scientific Instruments (www.ssi.shimadzu.com) introduced the SIL- 30ACMP multiplate autosampler as an add-on module for the Nexera MP UHPLC. The autosampler triples capacity to six 384-well plates. The specifications are impressive: Carryover is <0.0015%; %CV is <1% for 0.5-μL injections and <0.2% for injection volumes greater than 5 μL. Minimum cycle time is 14 sec.

Chromatography simulation and method development software

DryLab® 4 from Molnár-Institute (www.molnar-institut.com) is the latest extension of the popular series of chromatography simulation and method development software. The new release extends DryLab into 3-D separations and also supports quality by quality-by-design (QbD) principles to LC method development. This can be extended to multifactorial modeling needed to delineate the design space and ensure that the operating space falls within it.

For method development using QbD, one must start with the best column. Using DryLab’s built-in Design of Experiment (DoE) module, the user selects the input parameters for optimization including mobile phase components, pH, temperature, dwell volume, and run time. This will provide a list of experiments to run. After the results have been imported, DryLab tracks the peaks and constructs a map of the response space in a heat map. This facilitates describing the design space. A local optimum is selected for validation experimentally. Usually the agreement between predicted and actual results is within 5% for retention time and critical pair resolution. Anecdotal reports show a reduction in method development validation time by 50%. Plus, the heat map is useful in showing regulators that the assay is understood and under control. Another advantage is that method transfer between different instruments is predictable and hence easier.

Solvent gradient optimization system

In the booth of Bischoff Chromatography (www.bischoff-chrom.de), I was introduced to a program that optimizes solvent gradients with the mixed-mode columns of Phase OPtimized LC, aka POPLC®. With POPLC, the stationary phase in the column is optimized for the sample by stacking cartridges filled with different stationary phases such as C18, C8, NH2, CN, etc. Previously, Bischoff offered software to assist in the optimization of isocratic runs. Today, the company has gradient elution optimization.

Sample prep

Dried blood spots sampling systems

Despite decades of experience with dried blood spot (DBS) sampling for phenylketonuria (PKU), the FDA has not approved sampling of other analytes on dried blood spots. Without a guidance document, DBS sampling awaits approval, at least in the States, although this is not the case for some European companies. For example, CAMAG Scientific Inc. (www.camag.com) introduced the DBS-MS 500 for sample prep for MS. For DBS, the dried cards from Whatman (www.whatman.com) or ID Biological Systems (www.id-biological.com) are picked up with a new gripping tool and moved to the optical card recognition (OCR) module, which records the bar-code identification and spot position. The card then passes to the spotting station where the internal standard is applied. CAMAG was able to apply its decades of experience with thin layer chromatography (TLC) spotting to give precise standard addition. Next, the card travels to the extraction station, where it is pressed between the solvent inlet and exit ports. The card is removed and replaced in the sample rack for archiving. One study showed that the %CV for 560 replicate DBS samples was 2.8% with the CAMAG systems. Manual extraction had a %CV of 4.57%.

Protein digestion platform

Digesting proteins with enzymes is one of the common techniques in proteomics. Shimadzu has teamed with Perfinity Biosciences (www.perfinity.com) to design a new Perfinity Integrated Digestion Platform (IDP). This automates the digestion work flow to reduce time and improve reproducibility over manual protocols. These involve a three-day cycle time including an 18-hr digestion. With IDP, the cycle time is reduced to 30 min maximum. Perfinity explains this is accomplished through optimum automated integration of buffer exchange, digestion, desalting, and reversed-phase separation and simplification of the work flow.

TLC/MS interface

TLC is still popular in many organic synthesis labs. However, these labs also want to characterize the spots with MS. Until now, the best available technology involved scraping the spot from the TLC plate and eluting the material in a tube, then transferring the liquid into the MS, which is cumbersome by today’s standards. CAMAG introduced the TLC/MS interface for on-line transfer of compounds from TLC spots to APCI/MS, ESI/MS, or APPI/MS (atmospheric pressure photoionization-MS) interfaces. The spot is located with cross-hairs. An arm lowers and presses the solvent inlet/outlet probe over the target spot. The solvent is metered in and passes up the outlet directly to the MS. The on-line nature of the operation gives a throughput of a spot per minute.

LC columns

Aeris™ Core-Shell HPLC/UHPLC columns

Aeris™ Core-Shell HPLC/UHPLC columns (www.phenomenex.com/Aeris/Index) from Phenomenex (www.phenomenex.com) are designed to provide rapid separation of proteins and peptides. They are offered in two pore sizes: Aeris Protein has the large pore; Aeris Peptide has smaller pores and is suitable for peptides and small (<10 kDa) proteins. The other key consideration is the pressure rating of the instrument. One who has a legacy HPLC (i.e., Pmax of about 6000 psi) should select columns packed with 3.6-μm particles. One running UHPLC should take advantage of the 1.7-μm particles. These columns provide unexpectedly high column efficiency and resolution compared to even porous columns with similar particle diameter. One counterintuitive point is that if the highest resolution is needed, then a long column can be constructed by connecting several columns packed with 3.6-μm particles. In HPLC, maximized peak capacity (peak count) is achieved with long columns packed with larger-diameter particles. The tradeoff is much, much longer run time.

Kinetex® phenyl–hexyl ligands

Bonded to Phenomenex Kinetex® particles, phenyl–hexyl ligands (www.phenomenex.com/Products/HPLCDetail/Kinetex/Phenyl-Hexyl) are useful in separating moderately polar analytes such as chlorophenols, steroids, phthalates, and geometric isomers. An animation of the phase showed a benzene ring connected to the Kinetex core shell particle via an n-hexane tether. As with the Aeris Core-Shell, columns packed with 2.6-μm particles provide good separation efficiency for HPLC (~6000 psi) instruments. Columns packed with 1.7-μm particles are designed for UHPLC instruments. Users can expect outstanding speed and detection limits if their system can keep up.

SecurityGuard™ ULTRA guard columns for UHPLC

Since particulates are more of a problem with narrow columns packed with very small particles, and the columns are also expensive, guard columns are especially recommended. These must also be designed to minimize extracolumn band broadening. Phenomenex extended the SecurityGuard™ product line to SecurityGuard ULTRA (www.phenomenex.com/Products/HPLCDetail/SecurityGuard%20ULTRA), which is specifically designed for UHPLC columns from any vendor (www.phenomenex.com/Products/HPLCDetail/Kinetex/Phenyl-Hexyl). The columns are rated to 20,000 psi. They are small (<0.3 μL) and not a significant source of extracolumn band broadening.

Ultra HPLC columns

Restek (www.restek.com) extended its Ultra HPLC column line with three new surface chemistries. The Ultra line is based on 3- or 5-μm spherical silica with 100-Å pore diameter. After the ligand is bonded, the particles are endcapped to remove the H-bonding effects of residual silanols. Compared to conventional alkyl and phenyl phases, the biphenyl phase exhibits both increased retention and selectivity for aromatic and/or unsaturated compounds (www.restek.com/catalog/view/11086). The pentafluorophenylpropyl (PFP) phase has a pentafluorobenzyl group attached to the particle by a propyl tether. The PFP is particularly recommended for nitrogen heterocyclics and halogenated analytes (www.restek.com/catalog/view/11087). The Ultra Aromax column sports a proprietary surface chemistry that shows stronger retention of aromatic analytes than conventional C18 or even phenyl phases. It is particularly effective in separating steroids, tetracyclines, and drug metabolites, especially if they have some unsaturation (www.restek.com/catalog/view/32171).

USLC™ columns

The section describing DryLab’s QbD approach to method development and validation starts with selecting the best column. “Best” can include many attributes (availability, price, fitting compatibility, etc.), but selectivity for the analytes is probably the most important. Restek introduced the USLC™ four-column set with diversity in selectivity to quickly scout for favorable selectivity (www.restek.com/USLCarticle). The phases include Ultra Aqueous C18 with an embedded polar group; Ultra IBD, also with an embedded polar group that improves separation of acids; Ultra PFP Propyl, which shows selectivity for bases; and Ultra Biphenyl, which demonstrates selectivity for aromatics. These all offer diverse selectivity patterns compared to Ultra C18, which is used as a benchmark. It is selective for hydrophobic analytes.

Hydrophilic interaction chromatography (HILIC) columns

COSMOSIL Triazole phase

Nacalai USA, Inc. (www.nacalaiusa.com) introduced the COSMOSIL Triazole stationary phase for HILIC. It demonstrates useful selectivity for anionic analytes such as oxamic acid and oxalic acid. In another example, the separation of ascorbic acid and isoascorbic acid on the triazole column was run under ion exchange conditions (dilute salt), and a partial separation (R = 0.7) was obtained. If the salt concentration increases to 100 mm, even this poor separation is lost entirely. However, under HILIC conditions, achieved by increasing the acetonitrile to 80%, the R improves to over 4.

Epic HILIC-PI phase

ES Industries (www.esind.com) introduced the Epic HILIC-PI phase for the separation of polar amines. The aromatic character of the stationary phase provides selective retention based upon π–π interactions. The stationary phase is a proprietary ligand bonded to 1.8-μm porous silica. This provides excellent column efficiency and speed. The new phase offers unusual but stable selectivity, which provides robust methods.

Ascentis® Express

Supelco (www.sigmaaldrich.com) introduced three surface chemistries to the Ascentis® line of Fused-Core columns for HILIC (www.sigmaaldrich.com/analyticalchromatography/analytical-products.htmlTablePage=104698603). For Ascentis Express F5, a PFP group is bonded to 2.7-μm Fused-Core particles via a propyl tether. The PFP phase can function as a reversed-phase packing with π–π selectivity. It also functions in the HILIC mode when the acetonitrile concentration is very high, where it shows general selectivity.

Ascentis Express ES-Cyano consists of diisopropyl-cyanopropylsilane bonded and endcapped to 2.7-μm Fused-Core particles. The CN provides a stable, reversed-phase packing suitable for basic, acidic, or neutral analytes (www.sigmaaldrich.com/analytical-chromatography/analyticalproducts.htmlTablePage=108679944).

Ascentis Express OH5 (Pentol) is a proprietary phase developed by Supelco that contains a polyol surface chemistry. This probably interacts with analytes with potential hydrogen bonding functional groups.

Fortis HILIC column for assay of polar analytes

The sub-2-μm market offering is expanding as more vendors add new stationary phases. Fortis HILIC from Fortis™ Technologies (www.fortis-technologies.com) is now available in columns packed with 1.7-μm particles. These are optimized for the assay of polar analytes with HILIC conditions. The silanol content is controlled to attract a water-rich layer on the beads. This produces retention of polar analytes that are typically not retained on more hydrophobic stationary phases.

Deamidation with RPLC and ERLIC

Deamidation of asparagine and glutamine residues in proteins occurs naturally. In vivo deamidation is believed to play a role in cataracts and Alzheimer’s disease. It can also be promoted by the conditions of bioprocessing, storage, and proteomics digestion. Deamidation reduces the shelf-life of protein therapeutics. In addition to naturally occurring deamidation, deamidation sites are used in proteomics as markers for sites of N-glycosylation. Thus there is a need for an assay to differentiate between native and ex vivo or artifactual deamidation.

Dr. Andrew Alpert, President and Founder of PolyLC Inc. (www.polylc.com), explained that deamidation of asparagine occurs when the amide side chain of asparagine attacks the adjacent residue on the C-terminal side, forming an asymmetric succinimide intermediate. The ring opens to form aspartic acid or isoaspartic acid in a 1:2 ratio. The presence and ratio of the latter two are markers of deamidation. All three isoforms can be separated by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) (PolyLC) and identified on-line by mass spectrometry. One study showed that much deamidation was artifactual and could be identified incorrectly as glycosylation sites. Dr. Alpert went on to propose an improved digestion protocol that suppresses the artifactual deamidation.

Ion exchange columns

IonPac columns for ICS-4000/5000

To take advantage of the extended pressure and flow rate capability of the new ICS-4000 and upgraded 5000, Dionex introduced new columns packed with 4-μm-diam particles packed into 0.4-mm-i.d. tubes. Prior columns utilized 5.5- or 9-μm-diam particles. The new columns are much more efficient, and since they are also narrower, they have greater detection sensitivity for the same sample load. The IonPac AS18-4μm anion exchange column is the hydroxide-selective column of choice for compliance monitoring of inorganic anions in drinking water and wastewater samples in accordance with U.S. EPA Methods 300.0(A) and 300.1. However, the cycle time is reduced to 3 min. Other applications run on the IonPac AS18-Fast column can be easily transferred to the IonPac AS18-4μm column with the benefit of reduced run time with no loss in resolution. The IonPac AS18-4μm column is available in capillary format, 0.4 × 150 mm (www.dionex.com/en-us/products/columns/ic-rfic/hydroxide-selective-packed/ionpacas18-4um/lp-111535.html).

Similarly, the AS11-HC-4μm column and the CS19-4μm capillaries are also packed with 4-μm particles in capillary format. The improved efficiency obtained with smaller particles allows use of 2–3 times higher flow rate with constant resolution, but reducing run time by the reciprocal factor. When operating at the standard flow rate of 0.010 mL/min, an IC system capable of operating up to 3000 psi is sufficient. However, to take advantage of the fast runs, using a flow rate of 0.030 mL/min, a high-pressure capillary IC system such as the ICS-5000 or ICS-4000 is required.

MAbPac™ SCX-10 columns

Screening monoclonal antibodies just got faster by reducing the diameter of the column particle to 3 or 5 μm diam from 10 μm. MAbPac™ SCX-10 columns from Dionex are effective for separating antibody variants differing by only one charge. Unique surface chemistry nearly eliminates nonspecific hydrophobic interaction, giving sharp peaks. The columns are suitable for platform assays for identification and stability assays. A range of column diameters and lengths are offered.

Scherzo SW-C18 column

Adenosine mono-, di-, and tri-phosphates are very polar anions with broad interest due to their involvement in biological energy processes. While separation by RPLC is not successful, Imtakt USA (www.imtaktusa.com) finds that a mixed-mode anion exchange/ C18 column is quite effective. Run times are less than 10 min with gradient elution.

Acclaim™ Surfactant Plus columns

Surfactant by HPLC seems to be a natural idea until one tries it by RPLC and the frustration starts. C18 can irreversibly sorb some surfactants such as sodium dodecyl sulfate. This was the basis of “soap chromatography mode” in the early 1980s. A few years ago, Dionex introduced the Acclaim™ Surfactant columns, which had a proprietary surface chemistry on 3- or 5-μm silica that was useful for a variety of common products, including formulations. This year, Thermo Fisher Scientific introduced an improved version called the Acclaim Surfactant Plus. The stationary phase is a mixed-mode surface chemistry with improved bonding to 3- or 5-μm-spherical silica. Compared to its predecessor, the new columns maintain the same selectivity with low column bleed, improved stability, and shorter analysis time.

LC column packings

A decade ago, I visited China several times. The entrepreneurial column vendors all struggled with the expense of procuring quality silica for HPLC columns. They had to import the column packings from Western sources. They quickly mastered bonding and especially packing. I wondered how long it would take for a firm to start producing chromatographic silica in China. Nanomicro Technology Co. had a booth that featured a wide range of column packings, including UniSil™ spherical silica with a nominal diameter of 2, 3, 4, 5…50 μm). Six pore diameters are available from 50 to 500 Å. Nanomicro also makes bonded phases including C18, C8, C4, NH2, and HILIC.

Nanomicro UniPS™ beads are made of polystyrene/divinylbenzene copolymer. Particle diameters start at 2 μm and extend to 60 μm. Photomicrographs support the claim of monodisperse particle distribution. The company also offers polyacrylic particles in three size cuts from 20 to 60 μm. A huge selection of pore diameters is available, including nonporous. Ionogens have been added to the polymeric beads to make the corresponding ion exchange resins with the trade name Uni-IEC.

Solid-phase extraction phases

SiliaPrepX™

SiliCycle (www.silicycle.com) is known for producing a wide range of products, sorbents, scavengers, and catalyst supports, based on silica. Thus, I was surprised with the introduction of the SiliaPrepX™ line of polymer-filled tubes for solid-phase extraction (SPE). A range of stationary phase chemistries is offered, including cation and anion exchange and two neutral sorbents. The polystyrene/divinylbenzene phase retains nonpolar analytes. The HLB phase presents a wettable copolymer with a balanced hydrophilic and hydrophobic surface that retains acids, bases, and neutrals. The columns are available in plastic tubes with volumes of 1, 3, to 6 mL and 96-well plates.

Fritless SOLA SPE cartridges

Early cartridges and multitip plates for SPE used screens or frits to hold the sorbent in place. While the frits were usually selected to be inert under anticipated use conditions, they were often suspected when the mass balance was short. Chromatographers at Thermo Fisher Scientific worked to blend the frit with the plastic of the tube wall (polyethylene) and column packing. The result is the SOLA series of cartridges and plates. Separation modes include reversed phase, cation, and anion exchange in 1- or 2-mL tubes (www.thermoscientific.com/ecomm/servlet/newsdetail?storeId=11152&contentId=54068&ca=sola-spe).

LC fittings

EXP® fittings from Optimize Technologies (www.optimizetech.com) and column hardware allow indefinite make-and-break cycles of fingertight connections rated to 20,000 psi. This year, the line was expanded to include very low-dead-volume stem filters, pre-column filters, trap columns, and packed-bed guard columns. Plus, some fittings are available for 1/32-in. tubing, which is widely used in small-bore capillary applications.

However, the item that really caught my eye was a spring-loaded auto-adjusting zero-dead-volume connection. The bleeding edge of current column technology produces peaks with a volume of 1–3 μL. With conventional designs, the position of the fixed ferrule can vary due to design variances, machining tolerances, and creep. Creep is ferrule movement as the tubing starts to pull out. It is difficult to detect. Whatever the source, the hidden dead volume is 2 μL per mm of mispositioning. One millimeter could contribute significantly to extracolumn band broadening. With the Optimize zero-dead-volume connection, one pushes the tubing into the fitting until it bottoms out, then the nut is tightened by hand. Now you have made a zero-dead-volume connection rated to 20,000 psi. And, it took you longer to read it here than to do it, at least the second time (www.news-medical.net/news/20120312/Thermo-Fisher-Scientificlaunches-Acclaim-Surfactant-Plus-and-MAbPac-SCX-10-chromatography-columns.aspx?page=2).

Check valves

Decades ago, I worked on exploring the pressure limits of HPLC. We found that stainless steel was not suitable at pressures at about 30,000 psi and higher. We decided to back off to a Pmax of 8500 psi to have a very large safety margin. This year IDEX (www.idexcorp.com) introduced check valves rated to 30,000 psi. The key is to use a different alloy that can take the pressure. The company also introduced a complete range of fittings for 1/16- and 1/32-in. tubing rated up to 30,000 psi. However, the lifetime measured in make-and-break cycles reduces to 5 as the pressure increases to 30,000 psi. Tightening fittings with a torque wrench is recommended.

TitanHT™ multiport valves

IDEX introduced three new valves with 6, 7, and 10 ports for next-generation UHPLC applications. Rheodyne TitanHT™ valves use patent-pending metal-on-metal seal technology, which enables ultrahigh-pressure injection, selection, and switching. This sealing technology doubles the expected lifetime compared to the older valves that were rated to only 15,000 psi.

Analytical standards

In some respects, clinical diagnostics is a leading market model for analytical chemistry. Clinical labs run assays on dedicated, purpose-built analyzers. Vendors supply all reagents, standards, training, software, and postsales support, usually globally.

Clinical LC/MS is the fastest-growing market segment of chromatography today. Vendors of traditional MS instruments (Waters and Thermo Fisher Scientific in particular) recognized the need to develop a new business model to be compatible with the established practices of diagnostics labs. Surprisingly, Waters discovered significant interest from its traditional base in pharma. These labs do not want to spend time preparing reagents, etc. Plus, assured supply and traceable certificates of analysis were also valuable. With global R&D programs and outsourcing, the availability of common reagents and standards to all laboratories is particularly desirable since it should improve comparability and facilitate method transfer.

Pittcon 2013

Next year, Pittcon moves to Philadelphia, PA, a new location for the meeting, and one that will be attractive since so many chemical and pharmaceutical companies are located within driving distance of the convention center. The dates are March 17–21, 2013.

Click here to read Dr. Stevenson's reviews on gas chromatography instrumentation and columns  and supercritical fluid chromatography systems.

Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: rlsteven@comcast.net.

Comments