New Instrumentation and Applications at HPLC 2012

A marketplace is usually dynamic, with a cross-current of disparate interests meeting to find mutual satisfaction. In the sciences, traditional major meetings are so big that the trend is to hold many more vertical meetings where the size is compatible with comprehension. After all, you do not go to a produce market to buy meat. HPLC 2012—the 38th International Symposium on High Performance Liquid Phase Separations and Related Techniques—attracted 834 scientists to the Anaheim Marriott Hotel in CA from June 16 to 22, 2012. HPLC 2012 was held in one large room, where the major and smaller vendors to the liquid phase separation technology space congregated to show their stuff to an audience with similar interests.

Instruments for ultra high-performance liquid chromatography (UHPLC)

Gradient elution pumps

As with conventional (6000-psi) HPLCs, multipump gradient elution systems in which each mobile phase requires a dedicated pump are expensive. They do offer the lowest dwell volume, however. Agilent (www.agilent.com) and Thermo Fisher Scientific (www.thermoscientific.com) joined the ACQUITY H-Class from Waters (www.waters.com) by introducing UHPLC pumps with pre-pump proportioning. The pre-pump proportioning design is a less expensive and arguably more flexible approach.

Quaternary metering pumps

The most distinguishing component of the UltiMate 3000 XRS UHPLC from Thermo Fisher Scientific is a quaternary metering pump utilizing force feedback design to minimize pressure pulsations (www.thermoscientific.com/ecomm/servlet/techresource?storeId=11152&langId=-1&taxonomy= 4&resourceId=97314&contentType=Datasheets&productId=12778144). The figures of merit are impressive: Maximum flow is 2 mL/min; Pmax is 18,130 psi; pressure ripple is less than 7 psi without a pulse dampener; dwell volume is 65 μL; and four-channel vacuum degassing is standard. The pump controller continuously adjusts valve timing and pump displacement based on the compressibility of the mobile phase components to deliver an accuracy of better than ±0.5%. The UltiMate 3000 incorporates a new flow cell design and Viper connection technology to reduce peak dispersion.

Mobile phase proportioning system with quaternary pump

Agilent introduced a wide range of new products and capabilities, including the 1290 Infinity Quaternary LC mobile phase proportioning system; 1260 Hybrid supercritical fluid chromatography (SFC)/UHPLC; versatile valve-based automation, including supporting software; and 2D LC for the 1290 Infinity series.

Previously the 1290 Infinity series used two pumps for gradient elution. The 1290 Infinity Quaternary Pump offers interesting engineering and performance at a lower price. The pump is rated to 18,000 psi up to a flow of 2 mL/min. Then the Pmax declines to 12,000 psi at an Fmax of 5 mL/min. With dynamic mixing of methanol/water, retention time in reversed-phase liquid chromatography (RPLC) is shorter than a premixed mobile phase with identical nominal blend. When water is mixed with methanol, a significant volume contraction occurs. Thus, the % modifier may not be exactly the same for the premix and dynamically mixed liquids. Data from the company show that dynamic mixing is much more reproducible than preparations involving multiple operators and reservoirs. A retention time reproducibility of <0.07% and data better than 0.02% are claimed for the quaternary pump.

LC×LC system

Agilent introduced an impressive implementation of comprehensive LC (LC×LC) for the 1290 Infinity line. This is designed for chromatography of complex samples, including natural products. The unit uses a two-position/four-port valve to provide 0.6-min fractions to the second-dimension column. Also, the gradient program is tuned to the gradient position of the first dimension. Tedious programming of each 2-D run is avoided by drawing in general gradient shapes, and the software calculates each individual run. One example used two RPLC columns—C18 for the first dimension and a short phenylhexyl for the second.

The 1290 Infinity Flexible Cube is a multivalve station with a 900-psi pump. A selection of multiport valves can be mounted and actuated under software control for techniques such as heartcutting, sample cleanup, sample enrichment by trapping, column selection, and LC×LC. The software provides several features that facilitate implementation of these techniques.

Instrument systems

High-pressure ion chromatograph

There is no better example of the close coupling of columns and instruments than ion chromatography. Ion chromatography columns were usually among the largest in particle diameter, which reduced efficiency, but PEEK or similar high-strength plastics were needed for an inert flow path. Thermo Scientific Dionex (www.dionex.com) introduced new capillary columns packed with 4 μm diameter for ion chromatography. The new instrument (Thermo Scientific Dionex ICS-5000 Reagent-Free™ HPLC™ system with Eluent Generation) is capable of operation at 5000 psi. The pressure is limited by the limited strength of plastic tubing and parts. Volumetric flow rates are 1/100 of conventional 4-mm-i.d. columns. This enables the instrument to run on a 24/7 basis on 5 L of mobile phase per year, which enables “always on” walk-up-and-inject injection.

SFC/UHPLC chromatograph

The Agilent Hybrid SFC/UHPLC instrument provides SFC with UV detection as good as conventional LC. A CO2 pumping module provides the CO2 to the 10-port control valve and onto the mixer, injector, column, and detector. Effluent from the detector travels back to the SFC module through the backpressure regulator, which keeps the mobile phase homogeneous. The maximum pressure is 9000 psi. In addition to using CO2 as a mobile phase moderator, the hybrid system can be used as a 2D SFC-UHPLC hybrid, which provides more orthogonal technologies. Applications reported include food dyes and packaging.

Triple-quad liquid chromatography-mass spectrometry (LC-MS)

The short duration peaks that come from “sub-2” LC columns really challenged MS detection, especially triple quadrupoles. Shimadzu (www.shimadzu.com) responded with the LCMS-8040, which is designed for fast LC-MS (www.ssi.shimadzu.com/news/news_info.cfm?newspgsID=4&press_ id=311&press_group=Mass%20Spectrometer%20Systems&date_story=2012). Polarity can be switched in 15 msec. Scan rate is 15,000 u/sec. The New UFsweeper™ II technology enables up to 550 multiple reaction monitorings (MRMs)/sec. Detection sensitivity is improved five times.

Ion source for LC/MS

Bruker (www.bdal.com) introduced a refined electrospray design called ionBooster™ that improves ionization efficiency and hence minimum detection limit (LOD) by 5–100 times (www.bdal.de/products/lc-ms/ion-sources/ionbooster.html). Detection limit is the most commonly used figure of merit in LC detectors. The ionBooster is recommended for Bruker amaZon, maXis, maXis impact, or solariX MS detectors, where it provides LODs competitive with triple-stage quadrupole analyzers. The ionBooster maintains the vaporizing temperature in the electrospray ionization (ESI) source, which improves desolvation. It is compatible with UHPLC separations.

UHPLC-TOF for assay of designer steroids

A poster from LECO (www.leco.com) (“Screening Designer Steroids in Urine Using Ultra-High Resolution Time-of-Flight Mass Spectrometry and Liquid Chromatography Time-of-Flight Mass Spectrometry with Comprehensive Fragmentation”) described the use of time-of-flight/mass spectrometry (TOF-MS) with and without chromatography for assay of designer steroids in urine. Infusion of samples with TOF-MS is time-competitive with immunoassays for screening of samples. Chromatography is used when speciation or confirmation is required.

LC-NMR options for the chromatographer

Nuclear magnetic resonance (NMR) can provide information to define molecular structure. This is very attractive in HPLC as is LC-MS. However, NMR lacks detection sensitivity, which requires long acquisition times or high analyte concentration. Bruker presented a workshop discussing various options for the chromatographer for LC-NMR. The ideal case is using a flow-through probe that monitors the column effluent. This requires high concentration and use of deuterated mobile phases. The second option is to simply stop the flow when the analyte band is in the coil of the probe. Many spectra can be acquired and averaged to improve signal-to-noise. The third option is to trap the analyte and concentrate it by making replicate injections onto a trap column. The trapped material can be released into a suitable NMR-friendly solvent. The spectrum is obtained off-line. Applications reports focused on hemoglobin variants, peptides, and small proteins, including glyco forms.

Mobile phases

The cost of preparing mobile phases for liquid chromatography are probably hidden in the overhead of most labs. It is just another routine task. This is not to say the cost is zero—zero is not even close. Honeywell Burdick & Jackson® (www51.honeywell.com/sm/rlss/bandj/index.html) LabReady® Blends of mobile phases reduce cost and improve assay reliability. A few mobile phases are frequently used across many labs. These include mixed eluents of water and acetonitrile. For LC-MS, formulations containing trifluoroacetic acid (TFA) or formic acid are available.

Typical specifications for LC-MS-grade TFA in water are: TFA 0.1% ± 0.01%, no UV-absorbing peaks greater than 0.10 AU at 254 nm, and less than 0.02 AU and 215 nm. Maximum trace metals are 50 ppb. Background in LC-MS is less than 50 ppb as reserpine.

pH effects and ionic strength

A team from Waters presented a poster (“Systematic Control of Mobile Phase pH and Ionic Strength in Reversed-Phase Method Development”—poster 460) demonstrating the utility of AutoBlend Plus™ technology for systematic scouting of pH effects on separations. The ability to dial in the pH facilitates comparing runs with a resolution of 0.1 pH to search for optimum space or define the operating space. AutoBlend Plus is a combination of instrument characteristics and software algorithms resident in the ACQUITY UPLC® H-Class LC with a unique user interface that facilitates programming runs in the units of pH and molarity. AutoBlend can run a pH gradient, keeping ionic strength of the buffer constant; run a salt gradient, keeping the pH constant; or run a simultaneous pH and salt gradient.

A poster from Linda Lloyd of Agilent (“Increasing Throughput: The Advantages of pH Gradients in High Speed Ion Exchange Analysis of Proteins”—poster 74) presented a similar approach implemented on the Agilent 1260 Infinity Bio-inert Quaternary LC. Agilent Buffer Advisor software provides an intuitive graphical user interface that facilitates independent control of ionic strength and pH.

HPLC columns

Advances in core-shell particles took the spotlight, followed by sub-2-μm porous particles, and then columns for hydrophilic interaction liquid chromatography (HILIC).

Core/shell columns

Core/shell columns are developing a following since they offer higher efficiency and peak capacity and often lower backpressure than columns packed with porous beads. Monoliths still offer the lowest flow resistance, however. Please see www.americanlaboratory.com/913-Technical-Articles/118862-HPLC-2012-New-Mysteries-in-Column-Efficiency/.

Aeris™ core/shell stationary phases for peptides and proteins

Phenomenex (www.phenomenex.com) introduced new core/shell stationary phases for proteins (Aeris Widepore [300 Å] pore) and another optimized for peptides (Aeris Peptide [90 Å]) for protein digests (www.phenomenex.com/Aeris/AerisWidePore). The dividing line is 10,000 Da. Aeris Widepore has a thin, 20-nm active shell that encases the 3.6-μm core. The thin shell promotes rapid mass transfer of molecules >10 kDa, which diffuse slowly. The capacity is sufficient for top-down protein analytics. Surface chemistry is C4, C8, and C18, which provides useful retention for hydrophobic to hydrophilic proteins. Endcapping and isobutyl side chains reduce nonspecific adsorption compared to analogous porous phases. Chromatographic resolution is usually much greater than columns packed with 3- or 5-μm porous particles.

Aeris Peptide is optimized for smaller molecules (<10 kDa) that have higher diffusion coefficients. This allows a thicker and hence higher-capacity shell. Shell thickness for the 3.6-μm packing is 0.5 μm and 0.2 μm for the 1.7-μm phase. Surface chemistry is C18.

NUCLEOSHELL® core/shell column packings

NUCLEOSHELL core/shell column packings from Macherey-Nagel (www.mn-net.com) now include phases for HILIC as well as pentafluororopyl (PFP) (www.mn-net.com/tabid/11635/Default.aspx). These are bonded to 2.7-μm silica beads with a 1.7-μm dense core. The shell is 0.5-μm-thick porous silica (90 Å), with a S0 of 130 m2/g. The plate height is about 4 μm at a linear velocity of 2–6 mm/sec. The Pmax is 9000 psi.

Accucore HPLC columns

Thermo Fisher Scientific extended the Accucore line to include the Accucore 150-C18 with C18 surface chemistry, and Accucore 150-C4 with C4 surface chemistry (www.thermoscientific.com/ecomm/servlet/newsdetail?storeId=11152&contentId=54908&ca=accucore). Packed columns are available in conventional and nanospray diameters. Target applications are peptides and small proteins.

SunShell RPLC columns

Historically, the concept of core shell was to provide near sub-2-μm column efficiency to users of legacy (~6000 psi) HPLCs. This is potentially relevant to a global installed base of 300,000 to perhaps 500,000 active LCs. ChromaNik Technologies (www.chromanik.co.jp) has earned a reputation for developing HPLC packings using novel chemistry. This year, the company introduced SunShell RPLC columns (“Development of Superficially Porous Silica with Polyfunctional C18 Bonding Technique for Reversed Phase HPLC”—poster 298). These have a 0.5-nm shell encasing a 1.6-nm solid silica core. The surface chemistry bonds hexamethyloctadecyltetrasiloxane (HMODTS), which is subsequently endcapped with trimethylsilane. Packed columns show plate count efficiencies similar to totally porous sub-2-μm C18 phases but with less than half the pressure drop. Acid and base stability were better than competitive products, and capacity was higher. Plus, the SunShell columns showed less bleeding in LC-MS.

Core/shell particles with diamond cores

Diamond Analytics (www.diamond-analytics.com) presented a poster (“New Diamond-Based Coatings on Carbon Spheres for Use in High Temperature and Extreme pH Reversed Phase HPLC”—poster 311) describing synthesis and use of core/shell particles with diamond cores. Diamond is expected to be very inert and offers the best possible heat conduction through the core. The particles were prepared by coating 3.6-μm carbon core with a 0.2-μm shell of alternating layers of polyallylamine and nanodiamond for a total diameter of 4 μm. The RPLC surface chemistry consisted of a web of 1,2-eposxyoctadecane linked with 1,2,7,8-diepoxyoctane. These were packed into a 4.6 × 30 mm stainless steel column. The columns are used for a variety of applications, and demonstrate stability to pH 13 at 35 °C. Another poster (“Comparison of Efficiencies of Diamond-Based Core-Shell Materials for HPLC Made with Different Sizes of Nanodiamonds and Core Carbon Particles”—poster 291) showed good stability at 120 °C.

Larger core/shell particles

For over at least a half century, Dr. Jack Kirkland of Advanced Materials Technology (www.advancedmaterialstech.com) has developed a series of core/shell particles. The first was Zipax, which used 35-μm particles, but was very easy to dry-pack. There were several intermediate products, but today, the interest is on the Halo particles, particularly the 5 μm diameter. Surprisingly, the 5-μm Halo particles nearly double the plate count/column of totally porous stationary phase. Plus, the pressure drop is reduced by 50%. This makes Halo particles attractive for very long columns that could be required for high peak capacity. The shell has 90-Å pores with S0 of 90 m2/g. Pmax is 6000 psi.

Sub-2-μm porous columns

While columns packed with particles smaller than 2 μm have been around for two decades, most vendors now offer instrumentation that can use the columns effectively to routinely provide subminute separations. This has motivated many vendors to develop columns for the “sub-2” segment.

Titan columns

Supelco (www.sigma-aldrich.com) introduced Titan columns packed with 1.9-μm spherical silica particles (“Development of a New Monodisperse Porous Silica for UHPLC”—poster 301). Supelco believes that the low polydispersity of the particles (d90/d10 <1.2) leads to a more homogeneous bed structure, with less eddy diffusion and fingering. The particle has 80-Å pores with S0 of 400 m2/g. Performance testing shows that the columns have reduced plate heights as small as 1.69. This is comparable to columns packed with core/shell particles.

Triart column packings

Triart from YMC America (www.ymcamerica.com) is another new column packing for the “sub-2” class. The Triart particle consists of multiple layers of organic and inorganic silages for RPLC. The particles are available in 1.9, 3, and 5 μm, and larger for prep. The particles have a Pmax of 14,500 psi. With a 120-Å pore diameter, Triart is suitable for small molecules. Stability to pH 12 is a unique feature.

Epic C18 1.8-μm columns

ES Industries (www.esind.com) introduced several high-performance sub-2-μm columns for HPLC and SFC (www.esind.com/pdf/AppNewsEpic%20C18_1_8u.pdf). The columns have been engineered to meet the demands of UHPLC. As with other sub-2s, they can be used over an exceptionally wide range of linear velocity (i.e., flow rate) while maintaining high-resolution separations. Phases include 17 surface chemistries covering both normal and RPLC.

FortisBIO™ C18 column

FortisBIO C18 (Fortis Technologies, www.fortis-technologies.com) is now available with a 300-Å pore that scales in particle diameter from 1.7 to 5 μm. The smaller silica particle improves detection sensitivity by more than 50% compared to conventional 5-μm column packings. These packings are optimized for separation of peptides and smaller proteins.

Porous columns with particles larger than sub-2s

eXtended Performance (XP) 2.5 μm columns

Waters (www.waters.com) introduced eXtended Performance (XP) 2.5-μm columns, which appear to be a response to the sub-3-μm core shell particles that were offered as a lower-pressure response to sub-2s, but preserving chromatographic selectivity. The sub-3s offer improved efficiency, but are compatible with legacy (Pmax 6000-psi) instruments. Particle chemistries include silica, ethylene bridged hybrid, and charged surface hybrid (www.waters.com/XP).

InertSustain™ columns

For over 50 years, chemists in Japan have earned a deserved reputation for innovative column packings. InertSustain C18 columns from GL Sciences (www.glsciencesinc.com) are one more example (www.glsciencesinc.com/ProductInfo/HPLCColumns/InertSustain-ODS.aspx). The column packing starts with a “radically new type of silica” that provides a pH-stable surface while maintaining the traditional chromatographic performance advantages of silica. The company claims that the new surface enables tight control of the silanol content. Next, the C18 groups are added, followed by “complete end-capping." The columns are recommended for operation from pH 1 to 10. Comparative evaluation against modern wide-pH-range stationary phases shows greatly improved stability at pH 10. The particle size is 5 μm, but for some unexplained reason, the pressure drop is 20–50% lower than competitive columns with the same nominal dimensions. In addition to C18, InertSustain is available with C8, phenyl, and amino surface chemistries. The latter make particular sense since aminos on silica phases are vulnerable to base attack.

Bio-Bond™ RPLC columns

Chemists at Dikma Technologies (www.dikmatech.com) must have been very busy over the last few years, since four new LC column lines have been developed. The Bio-Bond series of RPLC columns is designed to provide RPLC separation of proteins and peptides. Surface chemistry is C4, C8, and C18. The silica has 300-Å pore. Particle diameters are 3, 5, to 10 μm. Spursil™ features a C18 surface chemistry, which also contains polar modifiers to reduce silanol interactions. Particles are 3-, 5-, or 10-μm silica spheres with 100-Å pore. The phase also provides unique steric selectivity. Leapsil™ is Dikma’s first entry into the sub-3-μm segment. The columns are packed with 2.7-μm spherical silica with a 100-Å pore. Carbon loading for C18 is 27%, which is extremely high. For the sub-2-μm market, Dikma introduced Endeavorsil™ 1.8 μm. This packing is C18 bonded to 120-Å porous silica.

Hydrocell surface chemistries

Biochrom Labs (www.biochrom.com) is an established developer of column packings based on polystyrene/divinylbenzene (PS/DVB) beads. These come in a variety of pore sizes that are selected to optimize capacity and resolution. PS/DVB has one major problem: high nonspecific adsorption of many biomolecules to the aromatic part of the bead. Biochrom has developed a series of surface chemistries that mitigate this interference in chromatography (www.biochrom.com/reversedphase/index.html). At HPLC 2012, Hydrocell RP 5D and Hydrocell 10D were introduced. Bead size is 5 μm and 10 μm, respectively. The columns have similar selectivity to silica-based C18 particles. Since the pore size is 1500 Å, the operating range is from small peptides to thyroglobulin (670 kDa). Pmax is 6000 psi. The packing is available in bulk as well as packed columns.

HILIC and related columns

Triart Diol-HILIC columns

YMC’s Triart particles provide excellent chemical and mechanical stability, and thus are attractive in HILIC mode. Triart Diol-HILIC is one example. A dihyroxypropyl group is bonded to the Triart particle. As expected, the neutral diol has low nonspecific adsorption. Columns are packed with 1.9-, 3.0-, or 5-μm-diameter spheres with 120-Å pore.

Scherzo mixed-mode columns

HILIC is now widely accepted for separation of polar and even moderately polar analytes that do not separate well in RPLC. Imtakt Corp. (www.imtaktusa.com) introduced Scherzo mixed-mode columns as an alternative to HILIC. Both modes work better with LC-MS than ion pair chromatography. The stationary phase starts with C18; however, three levels of anion and cation exchange ionophores are added for retention by a mixture of ion exchange and RPLC. This is effective for zwitterions, histamine analogs, and some neurotransmitters. Separations are optimized by varying the ionic strength, pH, and % organic. In contrast, the retention of neutral analytes is nearly unaffected by these changes. The animation shown on the mixed-mode series displayed the ratio of three ion exchange groups per C18 for the Scherzo SS-C18. The ratio is two ionophores per C18 for the Scherzo SM-C18, which reduces the ion exchange capacity. Similarly, the Scherzo SW-C18 has a ratio of one, for the lowest ion exchange influence (www.imtaktusa.com/hplc-columns/13/scherzo-sm-c18/).

HILIC stationary phases

Restek Corp. (www.restek.com) focused on HILIC columns and applications. HILIC is usually classed as normal mode chromatography. Yet the columns usually have some reversed-phase character. Restek has developed several stationary phases for HILIC, including an embedded-polar RPLC and pentafluorophenyl propyl. The question often arises as to which one should be used. Restek developed a hydrophobic-subtraction model that is useful in selecting stationary phases with orthogonal selectivity.

New SRT columns for SEC

SEPAX Technologies, Inc. (www.sepax-tech.com) extended the SRT line of columns for aqueous steric exclusion chromatography (SEC) by adding 150- and 300-Å porosity phases. The columns are packed with 5-μm spherical silica that is treated with a proprietary material to form a “neutral, hydrophilic film.” The backpressure of the columns is only about 700 psi, so several can be connected in series to improve resolution. The SRT columns are recommended for water-soluble polymers in bio and industrial applications.

Immobilized polysaccharide-based chiral stationary phases (CSPs) and columns

CHIRALPAK® IE™ and IF™ CSPs

Chiral Technologies Inc. (CTI) (www.chiraltech.com) showcased its R3—Robust, Reliable, Reproducible—portfolio of CSPs. The CHIRALPAK IE and IF CSPs are the newest additions to the R3 portfolio.

CTI’s polysaccharide-based CSPs are manufactured using a two-step process. First, polysaccharide derivatives are coated on silica particles. Second, the CSPs are bonded to the surface using a proprietary chemical process. The advent of the immobilized polysaccharide chiral selectors allows chemists to choose from a large assortment of organic mobile phases, including additives to improve enantioselective selectivity. The new immobilized chiral phases can be effectively employed for analytical method development under reversed-phase chromatographic conditions. Many RP and LC-MS-compatible separation methods have been identified using these CSPs.

Special-purpose columns

Inertsil® Acrolein C18 column

Acrolein is a difficult analyte because of its reactivity. It is often derivatized with diphenylhydrazine for analysis. GL Sciences has developed a C18 stationary phase on 5-μm spherical silica (Inertsil Acrolein C18) that can separate the DNPH derivatives of formaldehyde, acetaldehyde, acetone, acrolein, and propionaldehyde using a conventional water/acetonitrile mobile phase (www.glsciencesinc.com/ProductInfo/HPLCColumns/Inertsil-Acrolein.aspx). GL has also developed a sampling cartridge for acrolein.

ProteCol™ C8 capillary columns

Top-down proteomics with LC-MS is attractive since one can see occupancy and distributions of post-translational modifications. Columns are an issue. SGE (www.sge.com) introduced the ProteCol C8 capillary column with a 3-μm diameter and 1000-Å pore for proteins. First, the pore size was selected based on an analysis of restricted diffusion predicted by the Renkin equation. SGE wanted to have pores that were large enough to provide no more than 50% loss in diffusion mobility. This requires that the ratio of the analyte diameter be no more than about 10% of the pore diameter. For a protein, this is about 100 kDa and a 1000-Å pore. Next, the column needs to be inert and strong with a metal-free flow path. SGE designed a column starting with 1/16”-o.d. PEEKsil™ capillary, with 50-μm ID PEEKsil connection capillaries permanently attached. Bed supports are 100-μm-thick woven mesh with 0.5-μm pore size. For strength, the column is encased in a stainless steel sleeve. The column was able to separate single amino acid mutations in recombinant proteins (“Analysis of Single Amino Acid Mutations in Intact Proteins”—poster 289).

Alternatives to sole-sourced columns

Chromatographers are loath to change column brands since the selectivity and details of construction, particularly end fittings, are not well understood. However, there is a need to have alternate sources in validated methods to protect against possible disruptions in the supply chain.

To meet this need, ES Industries introduced Commercial Equivalents™ for popular columns from Phenomenex, Waters, and Thermo. The columns are designed to provide an economical alternative source of otherwise unique columns.

Partisep™ is one example. Partisil was very popular in the early days of HPLC when it was manufactured by Whatman. USP and other legacy methods still specify Partisil columns, despite evolution of spherical silica. Recently the commercial rights to Partisil columns were acquired by HiChrom (www.hichrom.co.uk). In response to requests for a column with similar performance, particularly selectivity, ES Industries developed Partisep columns. The columns are available in 10-μm diameter, which matches the original size, and 5 μm, which offers improved resolution or speed, since a shorter column can be used. The company reports that most methods, including USP, referencing Partisil can be transferred to Partisep without modification of the method. The following surface chemistries are available: Partisep ODS, Partisep ODS2, Partisep ODS3, Partisep C8, Partisep Silica, Partisep PAC, Partisep SAX, and Partisep SCX.

Sample prep

cHiPLC®-nanoflex chip-based platform

The proteome and related peptome are linked in many interesting ways. Eksigent (www.eksigent.com), which is part of AB Sciex and hence Danaher, presented poster 208,“Automated Microfluidic Sample Preparation Platform for Proteomics.” Eksigent developed the cHiPLC-nanoflex, a chip-based off-line sample processor that uses strong cation exchangers on a chip for trapping, reducing, and digesting proteins to peptides. The chip completely digested a 1-μg load of bovine serum albumin (BSA) to peptides in 15 min at 37 °C. Loading capacity, sample carryover (< 5%), and chip-to-chip reproducibility (5–15%, chip-to-chip) were measured quantitatively. The cHiPLC-nanoflex is predicted to become an important tool in proteomics.

GlykoPrep™ rapid glycoprotein sample prep platform

Post-translational modifications, especially glycosylation, were a frequent topic of discussion in the lectures. Prozyme, Inc. (www.prozyme.com) showed the GlykoPrep rapid glycoprotein sample preparation platform for cleavage and LC-MS assay of N-glycans. Prozyme recognizes that the type and distribution of N-linked glycans affects immunogenicity, efficacy, and pharmacokinetics of glycoprotein therapeutics. With the GlycoPrep sample preparation station, including reagents, the glycans are released from the protein and prepared for LC-MS assay in 3 hr, compared to three days with prior protocols.

EVOLUTE® and ISOLUTE® for pain management drugs

Inappropriate use of pain management drugs was a topic on the national news during the meeting. Pain management drugs may replace the imports from South America as the top concern of enforcers. Two posters from Biotage presented protocols for detection of metabolites and native drug in body fluids (“Extraction of Pain Management Drugs from Urine using EVOLUTE® CX with the RapidTrace+ Automated SPE Workstation Prior to LC-MS-MS” and “Extraction of 22 Pain Management Drugs from Urine using ISOLUTE® SLE+ in 96-Fixed Well Plate Format”). Specifically, urine from patients suspected of taking pain medications is treated with beta-glucuronidase, which releases the drugs as cations suitable for retention on an SCX ion exchange cartridge. The protocol can be used for screening urine for the presence of 22 pain management drugs. Throughput of the sample prep stage is 96 wells in less than 1 hr.

EXP® Nano Trap columns

Optimize Technologies (www.optimizetech.com) extended the EXP line of capillary column accessories with two EXP Nano Trap columns (www.optimizetech.com/product/EXP%C2%AE-Nano-Trap). Free Turn® architecture allows zero dead volume connections to be made, even at 20,000 psi. The EXP Nano Trap column is designed to remove detergents and salts, which are problematic for LC-MS.

The EXP Nano Trap HR functions as a vented trap, which provides repeatable, high-resolution nano LC. Bed volumes range from 0.25 to 4 μL. Stationary phases include 2.7-μm core/shell and 3.0 totally porous particles in a range of surface chemistries. Optimize also featured the EXP Analytical column hardware, which provides fingertight fittings rated to 20,000 psi.

Supercritical fluid chromatography

Supercritical fluid chromatography was a major topic. Agilent promoted it as part of its new technologies, but CO2 as a mobile phase was a major topic with Waters, which calls it Ultra Performance Convergence Chromatography (UPC2) (www.waters.com/waters/keywordSearch.htm?cid=505548&q=Ultra%2520Performance%2520Convergence%2520Chromatography). Waters points out that UPC2 eliminates the gap between LC and GC. It is implemented on the Waters ACQUITY UPC2 system, including columns. The low viscosity of CO2 improves mass transfer, giving narrower peaks, which improves resolution while also reducing pressure drop across the column. For separation of volatiles, CO2 as a mobile phase enables separations at much lower temperature than conventional GC. This could be important with temperature-sensitive analytes, as evidenced by some of the titles on Waters’ Web site (www.waters.com/waters/libraryList.htm?cid=511436&filter=documenttype|APNT&locale=en_US).

SFC columns

When the SFC market was in the doldrums, chromatographers used HPLC columns with some success. There was little innovation. But during the last two years, chromatographers have come to recognize that SFC columns are really different. First, the phases are normal mode. But the variety of surface chemistries is expanding rapidly. For example, YMC offers 11 stationary phases optimized for SFC. The latest is PVA-Sil SFC, which has polyvinyl alcohol bonded to 5-μm silica (120-Å pore). This phase has different selectivity than other phases, including the SFC diol.

Protein heterogeneity

While Tosoh (www.tosoh.com) has had world-leading columns for characterization of proteins for decades, the lecture focused on using them in complex work flows to characterize protein heterogeneity. Heterogeneity characterization is essential in QC of the formulated product. Tosoh described work flows for 1) single peptide variants in therapeutic antibodies, 2) deamidating an enzyme that alters activity, 3) antibody aggregation by SEC, and 4) distribution of pegylation of a protein adduct.

Standards and reagents

Recently, LC-MS has led several traditional instrument vendors to the clinical marketplace. The clinical technology segment favors complete solutions for high-volume assay that have proven diagnostic utility. The business models for the segments are also different. This creates a need and hence opportunity for instrument vendors to offer analytical standards and reagents. Waters has found that this model is also relevant to the traditional analytical laboratory. Accordingly, Waters is the first analytical instrument vendor to market a comprehensive portfolio of reference materials, controls, calibrators, and mobile phases (www.waters.com/standards).

High-performance capillary electrophoresis

Traditionally, the HPLC umbrella has been extended to cover capillary electrophoresis; after all, both are high-resolution separations in liquid phase, often with orthogonal selectivity. Beckman (www.beckmancoulter.com) is one of two firms promoting CE in the Western World. Beckman has developed a CE-MS interface that takes advantage of the nano flow rate from CE capillaries to improve ESI-MS detection sensitivity by reducing ion suppression. The company also presented a poster describing assay of recombinant human erythropoietin according to a European Pharmacopoeia method using a Beckman PA 800 + HPCE. The assay is required to ensure safety and efficacy of the product in treating anemia. Finally, a lecture by John Hudson described a rapid CE assay of the isomers of methamphetamine. In forensics, it is necessary to show that the active isomer is present in the sample (www.beckmancoulter.com/wsrportal/bibliography?docname=B2011.pd-12537f).

The future of liquid phase separation

The examples above illustrate how the entrepreneurial spirit is alive and well in the field of liquid phase separation. New research leads to advances in technology that are quickly transferred into improved results for society. Speaking personally, I can hardly wait to see what is introduced in 2013.

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

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