Pittcon 2013®: Developments in Liquid Chromatography

The first commercial liquid chromatograph was probably an amino acid analyzer. These ion exchange chromatographs were so specialized that they had almost no impact on chemical analysis outside of amino acids. In contrast, in the late 1950s, Mr. John C. Moore working at Dow Chemical Co. (www.dow.com) developed gel permeation chromatography (GPC), which Dow licensed to Waters Associates (www.waters.com) in 1961. In 1963, Waters introduced the GPC-100, which included a refractive index detector. Jim Waters is credited with designing the original refractive index detector, solving the major technical hurdle at the time.

Gel permeation chromatographs

Over the last 50 years, Waters and others have developed a series of GPC instruments. However, prior to Pittcon® 2013, most would characterize GPC as mature. The installed base is about 6400 instruments. Sales are mostly replacements of instruments that are obsolete due to lack of spare parts. New GPC technology consisted of an occasional new column and instrument introduction.

About 15 years later, Hamish Small working at Dow invented ion chromatography. This was offered to several major vendors, but only tiny Durrum Instrument Company, an amino acid analyzer developer, pursued the opportunity, and ultimately became Dionex, for “Dow Ion Exchange.”

In 2004, Waters introduced the ACQUITY UPLC®. It reduced run time by 90% and improved detection limits by 3×. It was a discontinuous change in technology that has been copied by all major vendors. The key change was that the columns had very narrow peak widths. This required low-dispersion instrumentation, including new detectors. UV and MS got the attention, then fluorescence, but refractive index (RI) was a project perpetually relegated to the back burner. No one saw a need for a better detector for GPC.

Then, in 2010, another phone call came from Dow…Do you know that GPC benefits from low-dispersion detection? The examples were compelling: GPC separations in less than 3 min. Opportunity had knocked; priorities were revised. Waters reacquainted themselves with GPC. But the critical path on the plan was developing a low-dispersion refractive index detector. Even Jim Waters dropped by for a review meeting.

In addition to the RI, Waters needed to develop columns compatible with the high pressure and faster linear velocity. The BEH chemistry had already given a 200-Å particle that was strong and rigid. It did not shrink or swell in different mobile phases used to dissolve polymers such as THF, toluene, DMF, etc., so Waters added columns with 125-Å and 450-Å porosity.

With the new RI and some columns, the Waters® ACQUITY® Advanced Polymer Chromatography™ System (APC™) was introduced at a packed press conference early Monday morning at Pittcon. A smiling Jim Waters was in the audience. While industrial polymers are the initial focus of the APC, I expect that rapid measurement of molecular size of biopolymers will become a much larger application segment.

Refractive index detector for UHPLC

Detectors for ultrahigh-performance liquid chromatography (UHPLC) need to provide low chromatographic dispersion as well as fast response. UV and MS were quickly optimized, but refractive index detectors have not been upgraded. This is a particular problem for large polymers, since the column performance for size exclusion chromatography (SEC) is far ahead of the detectors. Wyatt Technology Corp. (www.wyatt.com) addressed the problem by improving the detection speed and liquid flow path to create the Optilab UT-rEX. Detection involves using a 512-element diode array with a sample rate of 80 Hz. These are averaged to produce an output rate of 25 Hz. The flow path was also redesigned to reduce dispersion. As a result, the overall dispersion was reduced by 90% to 4 μL. Yet the noise level remains constant, with an industry-leading noise of only ±7.5 × 10–10 RI units.

The results are useful. Detection of unlabeled sugars is one obvious application. The UT-rEX gives much higher resolution and speed. In another example of a monoclonal antibody, the new RI detector showed a well-formed rider peak following the main peak. With the legacy RI, the rider peak was an indistinct mound. Peak deconvolution showed the ratio of the RI signal to UV was different for the main peak and shoulder, indicating that the structures are probably not similar compounds.

Diode array detector for LC

This year, Shimadzu Scientific Instruments (www.ssi.shimadzu.com) introduced several product extensions for the Nexera LC, called the Nexera X2 series for UHPLC and HPLC. Extensions include the new SPD-M30A photodiode array detector, which features a new total reflection capillary flow cell that improves light throughput. More light reduces noise to an impressive 0.2 × 10–5 AU. Improved temperature control also aids in noise reduction.

The detector is smart—when absorbance is strong, which means that the detector is in the range of saturation, it will select and then ratio with other wavelengths with weaker absorbance to extend the linear dynamic range. This aids in running impurity profiles for detecting peaks as small as 0.005 area% of the main peak. Shimadzu calls this the Intelligent Dynamic Range Extension Calculator (i-DReC). Previously, two runs often had to be made to capture the signal from the major component, and then a subsequent run determined the percentages in the impurity profile.

Too often a small peak nearly coelutes with another. The experienced eye is able to discern its presence, but estimates of magnitude are notoriously inaccurate. Shimadzu has developed a new on-board method for deconvolution analysis called i-PDeA (Intelligent Peak Deconvolution Analysis). It uses spectral differences to tease the signals of the individual components from each other and presents the signal for each peak.

Chromatography system for protein purification

Bio-Rad (www.bio-rad.com) has a long history of supplying products for biochemical research including chromatography, electrophoresis, and recently PCR. The company recognizes that the midmarket of biochemistry still wants to purify and analyze rather large amounts of proteins, especially compared to biomarker research with UHPLC/MS/MS. Thus, Bio-Rad introduced the NGC™ (Next Generation Chromatograph) family of medium-pressure, modular chromatography systems for protein purification.

The industrial design of the NGCs is unique. The instrument is designed in layers like a wedding cake. The layers have bays that accommodate a range of modules for pumps, detectors, and valves. More complicated systems need more layers, which are easily added. To configure the NGS for a particular application, one selects the flow path from the library in the computer, and then starts plumbing according to the novel point-to-plumb GUI design. For example, if one is connecting the pump outlet to the injector valve, blue light-emitting diodes appear at the pump outlet fitting and another appears at the injector inlet. The blue dots are connected with PEEK spaghetti tubing. All liquid connections are easily accessible on the front of the instrument.

Redesigned HPLC system

First introduced almost 20 years ago, the Alliance HPLC® from Waters has had a design update. Key parts for the system were no longer available. However, with so many validated methods running on Alliance, it was recognized that the users did not want any change that would require revalidation.

Waters also introduced the XP (eXtended Performance) line of columns with 2.5-μm particle diameter specifically to improve efficiency over 3- and 5-μm porous column packings.

Electrochemical detector

Electrochemical detection (ECD) is useful for quantitative detection of analytes that can be oxidized or reduced. This includes almost all drugs, metabolites, carbohydrates, and even general biologicals. Thermo Fisher Scientific (www.thermoscientific.com) updated Dionex’s design of the UltiMate® 3000 ECD to meet the advanced requirements of UHPLC. The new UltiMate 3000 electrochemical UHPLC/LC detector includes reduction of peak dispersion through redesign of the flow cell and increasing the sampling rate to 200 Hz. While the engineers were looking at the basics, they also took the opportunity to add new features such as advanced digital processing to reduce noise and improve dynamic range (10 pA to 100 μA) with autoranging. The thermostated detector base houses up to four coulometric or amperometric detection modules, which include the cells and column. A range of electrodes are available, including boron-doped diamond. ECD is not as popular as MS, but for many applications, it is probably the most economical choice.

Advances in liquid chromatography applications

AB SCIEX (www.absciex.com), Phenomenex (www.phenomenex.com), and Eurofins (www.eurofins.com) announced a cooperative program to develop and support methods for assay of animal feeds for antibiotics, and residues of fungicides and pesticides. Also, feeds may be processed by adding vitamins, fats, binders, preservatives, carbohydrates, and occasionally adulterants such as melamine, which increases the nitrogen and hence the apparent protein content.

For metabolomics, AB SCIEX and LECO (www.leco.com) have partnered to meet the needs of customers for LC/MS and GC/MS assays. LECO will provide GCs with both nominal and Accurate Mass detection, while AB SCIEX will supply LCs with both triple quadrupole and TOF/MS systems.

AB SCIEX developed an LC/MS/MS assay for dicyandiamide (DCD) in dairy products. DCD is used to control nitrogen runoff from pastures as well as stimulate plant growth. The method requires only a quick extraction followed by LC/MS/MS. Run time is just a few minutes.

The 3200 QTRAP® LC-MS/MS for clinical diagnostics from AB SCIEX will be a common element in a series of anticipated products for clinical diagnostics. Certification of ISO 13485:2003 standards is a key step for the company to enter the clinical market segment.

Method robustness: Evaluation of key parameters in HPLC

Mólnar Institute for Applied Chromatography (www.molnar-institut.com) has expanded DryLab® 4 software, which was introduced last year, to improve the robustness of validated methods. In a typical LC run, some parameters are much more influential, i.e., “risky” than others. Previously, DryLab modeled the complex interaction of the chromatographic separation to aid in method development. It now displays the regression coefficients that were used in modeling to help the chromatographer focus on the key parameters defining the design space. Parameters include column temperature, %B start, %B end, gradient start time, gradient slope, and flow rate. The software displays a bar graph showing the impact of each parameter. This helps chromatographers and regulators focus on potential problem parameters.

Online SPE for HPLC

Solid-phase extraction (SPE) is usually performed off-line in plastic syringe tubes. The new SPExos from GERSTEL (www.gerstel.com) substitutes a small metal cartridge containing about 50 mg of sorbent. The sample plus internal standard is processed with GERSTEL’s MPS (MultiPurpose Sampler) under control of its MAESTRO software. The SPExos extracts the sample plus internal standard (if added), and then turns a valve to inject by eluting the sample onto the LC column. The quantitative transfer of analyte usually improves the method detection limit. Plus, with judicious selection of the sample solvent and mobile phase, one may further concentrate the sample prior to injection.

Sample filtration module

Sample filtration can significantly improve column lifetime, especially with the sub-2 column packings in UHPLC. The column bed traps almost any particulate larger than about 25% of the particle diameter. GERSTEL added a filtration module to its MPS, and it can process 98 samples in a batch. The filtration module is simply inserted into the automated work flow orchestrated by MAESTRO software. In a similar manner, GERSTEL also added a shaker station and a multiposition evaporation station called mVAP.

Business-to-business in HPLC

When I started attending Pittcon about 45 years ago, the focus of the exhibition was exclusively business-to-end user. However, over the last 20 years, the focus has slowly broadened to include business-to-business, particularly with firms seeking to improve their distribution in new geographic markets. My estimate is that about 15% of the booths were primarily B2B. For example, ECOM spol. (www.ecomsro.com) and Sykam (www.sykam.com) both showed HPLC instruments, and were openly looking for distribution opportunities. ECOM featured a diode array detector optimized for flash chromatography. The pathlength can be as short as 0.1 mm, which is useful in prep chromatography of highly colored compounds. The detector can be ordered with a variety of options, including wavelength number and range, displays, and power.

UHPLC fittings

UHPLC is all about reducing peak dispersion. As tubing gets smaller, it is more difficult to make and maintain good connections. With very high pressure, ferrule creep is common. This permits the tubing to creep out of the fitting, giving a mixing volume that can destroy the chromatographic resolution. Even worse, the tubing can pull out of the fitting entirely and spray liquid under very high pressure.

To meet this problem, Optimize Technologies (www.optimizetech.com) developed the Ti-lok fitting, which features a titanium/PEEK hybrid ferrule to form the primary seal. The second stage is a collet locking mechanism that grabs the exterior of the fused silica and prevents ferrule creep. The Ti-lok provides a reusable, fingertight seal rated at 15,000 psi. Threads are 6-40, 6-32, or 10-32.

UHPLC and HPLC tubing and columns

Biocompatibility is a key issue to bioscientists. Any surface is a concern, but some (stainless steel) are of more concern than others (PEEK). The NGC from Bio-Rad uses PEEK throughout the liquid flow path. PEEK has a Pmax of only 4000–5000 psi, which is about one-third the Pmax of UHPLC instruments.

IDEX (www.idexcorp.com) is noted for developing innovative solutions to difficult engineering problems, especially in chromatography. IDEX engineers worked out a process to encase PEEK tubing, including narrow capillaries, in stainless steel tubes. This year the company introduced PEEK-lined stainless steel capillaries with 25 μm i.d. for UHPLC connections rated to 15,000 psi. Tubing is offered in precut lengths. IDEX used Upchurch Scientific (www.upchurch.com) VHP fittings and developed a “tipping” process to make sure that the liquid goes down the through-bore and not between the PEEK and stainless steel. For larger-diameter tubes such as columns, PEEK lining also provides a smoother surface compared to drawn stainless steel, which shows up as superior column efficiency. In one test, the PEEK column provided 20% higher peak area (i.e., recovery) than the corresponding stainless steel column.

Preparative LC system

The Prep 150 LC from Waters, designed for lab-scale preparative separations, has an Fmax of 150 mL/min, which is suitable for Waters OBD™ prep columns with 10–50 mm i.d.

Evaporative light scattering detector

Evaporative light scattering detection (ELSD) for analytical applications is well established. Previously, Sedere (www.sedere.com) extended its range with the SEDEX LT series for low-temperature detection. This year, the company introduced the SEDEX LT Model FP for flash and preparative applications, including LC and SFC. The compact detector appears to be at a new price point for ELSDs.

HPCE system

Some may recall the PrinCE HPCE (high-performance capillary electrophoresis) system. It was the brand name of an elegantly simple CE designed and built in The Netherlands under the guidance of Dr. Henk Lauer. At Pittcon, Prince Technologies (www.princetechnologies.eu) introduced the PrinCE NEXT 800, described as the “New platform from the CE technology leaders.” Unique features include: 1) Pressure injection from 96-well plates for improved accuracy; 2) temperature control of the capillary, sample, and buffer; 3) open design in anticipation of adding a flow cell from an external detector such as fluorescence; 4) flexible capillary length, including ultrashort for high speed; and 5) easy integration with automated liquid and plate handling robots.

Handheld CE analyzer

Analysis of glycans usually is run on an HPLC, often with MS detection. Run times for Dp1 to about Dp25 ladders are almost an hour. BiOptic Inc. (www.bioptic.com) introduced a small handheld CE analyzer, called the GL1000, that provides similar resolution to HPLC but with a run time of only 5 min. This improves productivity by a factor of 10, to say nothing of the cost. Detection is with fluorescence.

Capillary electrophoresis platform

In the Beckman Coulter (www.beckmancoulter.com) booth, emphasis was on applications of the PA 800 plus capillary electrophoresis platform. One application note was entitled, “Analysis of Monoclonal Antibody Charge Variants with Capillary Zone Electrophoresis.” Charge variants affect the pI of the antibody. These can arise from post-translational modification deamidation, lysine modification, and errors in transcription, etc. Beckman developed a method that provides quick separation of acidic and basic charge variants from the parent monoclonal antibody in 12 min with a 20-cm capillary. If higher resolution of the individual peaks is of interest, doubling the capillary length increases resolution dramatically, at the expense of longer migration time (~38 min).

Another application note from Beckman discusses improving quantitative analysis of erythropoietin (EPO) by replacing a slab gel protocol with HPCE. This is documented in the European Pharmacopoeia. Still another application note showed interesting fingerprints of white and red wines. I’m sure that the CE profiles could be correlated with taste, but this was was not included in the report.

Conclusion

Some Pittcons are remembered for their surprises. For Pittcon 2013, this was especially true since I’d lived the early days. Many of the people involved are still active and respected professional friends. However, as surprising as the ACQUITY APC was, I expect even more in the next few years. So, I plan to see you next year from March 2 to 6 in Chicago.

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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