Pressurized Capillary Electrochromatography: A New Tool for Quantitative Analysis

The common buzz at some meetings is that separation science is a mature technology with only small, fine-tuned improvements expected, since the big advances are behind us.

In 2004, Waters introduced the ACQUITY platform. This gave chromatographers a new high bar in performance, with column efficiencies of 250,000 plates per meter. Run times were reduced by about 90% compared to legacy chromatographs. Precision and accuracy were sufficient qualitative analysis based upon retention time. Peak areas gave RSDs in the 2–5% range.

If qualitative analysis is the target, HPLC/MS is hard to beat. Mass spectra can help the analyst focus on the few possible ambiguities provided by the retention time and accurate mass, including molecular formula and fragmentation patterns.

However, for complex samples such as proteomics, lipidomics, and toxicology, the problem involves both qualitative and quantitative. Chromatographers recently responded by introducing new technology that is even faster and much easier to use.

A case in point is the performance available from the pillar columns for capillary and nanoflow LC/MS designed and developed by PharmaFluidics (Ghent, Belgium). These microfluidic column chips will make nanoflow LC/MS practical. This feeds the opportunity provided by the fact that mass spectrometers work better when the solvent flow is reduced.

Then, there is the cyclic IMS (cIMS) instrument introduced by Waters in June at ASMS 2019. This novel instrument has a circular flight path that increases the flight length by one meter as the ion makes each revolution. Small differences in the structure can be conveniently explored for very large molecules. Sure, this is not chromatography, but it is gas phase electrophoresis.

But, back to liquid chromatography. Recall that about 15–20 years ago, separation scientists sought to combine electrophoresis with packed columns with a technique called capillary electrochromatography (CEC). The technique was slow to develop convincing applications. Plus, controlling the injection volume was difficult. So, quantitative analysis had to rely on relative measurements with internal standards. This is not a comfortable scenario for achieving precise measurements.

A paper from the laboratory of Professor Chao Yan at Shanghai Jiao Tong University shows that his perseverance of over 20 years with pressurized capillary electrochromatography (pCEC) has paid off. His team recognized that pCEC combines hydraulic and electroosmotic flow (EOF) to force the mobile phase through the capillary. With longer capillaries and higher voltage, EOF is responsible for most of the mobile phase velocity.1 EOF also has the advantage that it produces a plug flow profile, in contrast to the bullet profile associated with pumped flow.

The TriSep-2010 pCEC instrument from Unimicro Technologies (Pleasanton, CA) uses an injection valve (1-µL loop) fed by an autosampler and UV absorbance detector. With no applied voltage, the pump flow rate was 50 µL/min, but only 1/5600 of the volume was directed to the column. When the voltage was tuned on to get the acceleration of EOF, the split ratio ranged from 5600:1 at low voltage to 500:1 at 11 kV applied.

The fused-silica capillary (100 µm i.d., 375 µm o.d. × 100 mm l) was packed with monodisperse SiO2 spheres. The particles were synthesized by a modified Stöber process by adding tetraethyl orthosilicate (TEOS) to ethanol + water + ammonia solution. For research purposes, the silica particles had a diameter of 320 nm and 420 nm. Mixed C18 +C1 surface chemistry was added to the particles by reaction with n-octadecyltrichlorosilane and methyltrichlorosilane. The blue color of the packed columns is produced by Bragg diffraction of a silica colloidal crystal and confirms the face-centered cubic structure of the column packing. With this capillary column packed with submicron particles, the column efficiency for the three peptides was 1,752,000 plates per meter, 460,000 and 230,000 plates per meter for LY-6, DE-11, and DE–11p, respectively, at an applied voltage of 6 kV. For reference, the bar for efficiency in UPLC columns is about 250,000 plates per meter.

Separation performance was measured with a six-component mixture where thiourea was the T0 marker. With no applied voltage, the run required 38 minutes. The peaks were wide. When pressurized flow was assisted with EOF, run time decreased to less than 5 minutes. The relative retention time for six consecutive runs had a %CV ranging from 0.039% to 0.076%. The range of CVs for peak areas was 1.12–1.96%.

These values demonstrate that pCEC has come a long way and should be useful in quantitative analysis, especially of complex mixtures such as tryptic digests, metabolomics, disinfection by-products, and pesticide residues.

Reference

  1. Liu, Q.; Yan, K. et al. Preparation of silica colloidal crystal column and its application in pressurized capillary electrochromatography. J. Chromatogr. A Feb. 2019, 1587, 172–9.

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

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