A Microfluidics-based LC CHIP for Mass Spectrometry

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BY KEVIN KILLEEN, PHD

Today, proteomics is centred on analysis by mass spectrometry — once the domain of physicists and chemists. As biologists turn to liquid chromatography/mass spectrometry (LC/MS) to separate and identify proteins, their progress is constrained by bottlenecks, including difficulties with sample preparation, data analysis and the challenge of using equipment designed for experts. Thanks to a new application of microfluidics technology called HPLC-Chip/MS, scientists can now handle the separation and detection of even complex samples confidently, reliably and much more easily.

Unveiled by Agilent Technologies Inc. (Palo Alto, CA) this spring, the HPLC-Chip/MS technology takes traditional nanoflow LC/MS and mounts much of it into an integrated polymer chip. Designed for use with the Agilent 1100 Series Nanoflow LC System and Agilent ion trap mass spectrometers, the HPLC-Chip/MS system is expected to be available for purchase before year’s end. The first chips will be designed for proteomics applications.

Plug and Play
The technology is centred on the high performance liquid chromatography (HPLC) chip, a microfluidic device that combines the separation column and sample enrichment of HPLC with an electrospray MS tip. The enrichment, separation and spray-tip components form a single unit, with a channel that joins the LC column to the spray tip and eliminates fittings and connections.

When inserted into the HPLC-Chip/MS interface (a module that mounts on the Agilent mass spectrometer), the chip is automatically positioned with its electrospray tip in the optimal position for mass analysis. A chip can be snapped in or out in a matter of seconds, without the tweezers and fuss required by traditional LC column/electrospray needle connections.

By integrating separation and detection, HPLC-Chip/MS technology also promises better MS signal-to-noise measurements. Compared to traditional HPLC, separations performed on the chip-based platform pass through up to 50% fewer connections. This dramatically reduces the possibility of leaks, and virtually eliminates the possibility of dead volumes. Dead volume leads to band widening, resulting in less certainty that a peak is a true signal.

Optimizing LC for MS with a narrow column and low flow rate can complicate the HPLC setup, and puts a premium on detecting dead volume and leaks. At 200 nanolitres per minute, a leak could be undetectable due to evaporation. Another low-flow LC problem arises from dead volumes created by connections, which can cause peak dispersion and adversely affect measurement of true signal. HPLC-Chip/MS minimized post-column dispersion with a fluidically integrated electrospray tip.

How Liquid Enters the Chip
A challenge in using a microfluidic device for LC is getting the liquid into the chip. Generally, a rotary valve provides the fluid connection to the chip. While some are working on miniaturized, integrated valves, this approach typically limits devices to operating pressures of one or two bar. High pressure gives more efficient chromatography, so Agilent used available rotary valves that act as a high-pressure interface. The HPLC-Chip/MS chip fits onto the valve and is automatically clamped in place — a key technical achievement. This enables leak-free, high-pressure connections and fluid switching, so the chip can accommodate pressures of up to 100 bar.

Sample is loaded into the HPLC chip via an autosampler connected to the valve, and passes through the valve to an enrichment column on the chip that concentrates the sample. This column is packed with the same type of media as the chip’s analytical separation column. This sample pre-concentration allows researchers to load high volumes of sample quickly at relatively high flow rates of several microlitres per minute, then switch to nanolitres-per-minute gradients to do separations through the analytical column.

How the Chip is Made
The same type of process that produces ink-jet printer cartridges allows the incorporation of HPLC in a polymer chip. A high-power laser creates the channels, columns and fluid-access ports by ablating tiny sections of polymer film. Designers can control the laser’s movement to a tenth of a micron. Once etched, the films are laminated to form the internal three-dimensional structures, and then trimmed to form the electrospray tip and final shape of the chip. Direct-write laser ablation allows tremendous flexibility in chip design, and even customization.

Validation Experiment
Agilent scientists tested the HPLC-Chip/MS’s ability to separate protein digests, evaluating sensitivity and reproducibility. Analysis of a digest of bovine serum albumin produced good protein coverage and high-confidence protein scores with less than one femtomole of sample.

Media used included the Agilent Zorbax and StableBond C18. The gradients ranged from 2% to 85% acetonitrile. The Agilent nanoflow pump delivered the gradients at flow rates between 200 and 300 nanolitres per minute.

Samples were loaded with the Agilent 1100 Series microwell plate autosampler, with a capillary pump set at four microlitres per minute. Injection volumes typically were of one to five microlitres. The samples were preconcentrated on the chip, before separation, in under three minutes.

MS data were generated by an Agilent 1100 Series LC/MSD Ion Trap mass spectrometer with a prototype of the HPLC-Chip/MS interface. Nitrogen was the drying gas (at 350 C) and flow was two litres per minute. The researchers searched protein databases for matches to the MS/MS spectra using Agilent’s SpectrumMill software.

The HPLC-Chip/MS technology improves sensitivity for better protein coverage. More protein will go through the run and be seen by MS. Higher sensitivity makes it easier for databases to match the resulting spectra, so researchers will be more certain of the protein’s identity.

Agilent plans to develop specific chips for different applications, beginning with proteomics. Two-dimensional LC is expected to be incorporated into single chips, and custom chips may also be offered. HPLC-Chip/MS technology combines the separation of HPLC with the advantages of microfluidics, providing improvements in productivity, sensitivity, speed and efficiency.

Kevin Killeen, PhD is project manager for the Microfluidics and Biosensors Group in the Molecular Technology Laboratory within Agilent Laboratories.