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HPLC in the Nanoscale: Tipping point of a new analytical technology
Nick Roelofs, Ph.D.
Technology breakthroughs come in a variety of forms. Many follow a path in which the perceived usefulness of an invention at its introduction is increased many times by further innovations.
In the field of analytical chemistry, this pattern is clearly evident in the nearly complete replacement of packed GC columns by capillary GC columns during the late '70s and early '80s. The availability of a "standard," off-the-shelf capillary GC column with an integral stationary phase enabled shorter and consistently reproducible retention times, higher sensitivity and the ability to process far smaller samples. These improvements soon rendered the "do-it-yourself" packed GC column, and the hands-on labor it required became unnecessary for most applications.
HPLC-Chip
Figure 1. Integrated components of the HPLC-Chip. The chip integrates traditional nanocolumn/nanospray LC-MS components on a precisely replicated microfluidic chip. Click to enlarge. |
The recent development of the HPLC-Chip seems to be following a similar but potentially more dramatic path with respect to HPLC. This microfluidic nanoscale device, currently configured for LC-MS applications, integrates all of the traditional LC-system "plumbing" - packed enrichment and analytical columns, frits, filters, connections, and nanospray MS emitter - on a credit-card-sized biocompatible, solvent- and pH-resistant polyimide substrate (Figure 1).
Just as capillary GC column performance greatly exceeded that of the packed GC column, so too does the performance of the nanoscale HPLC-Chip surpass that of a standard bore LC column, with an estimated 3700-fold improvement in chromatographic efficiency. Additional enhancements in sensitivity gained from increased nanospray MS ionization (compared with a conventional column) can bring detection limits down to the attamole range.
As with the capillary GC column, the integrated on-chip microbore LC columns and fittings provide reproducible LC-MS analyses with notably shortened retention times and the ability to work with extremely small samples. This is a pressing analytical requirement in challenging fields from genomics to proteomics, in disease-targeted medical and pharmaceutical research, and in screening for low-level biologically active and/or toxic environmental residues such as metals, pesticides and herbicides, endocrine and other hormonal disrupters, and more recently, environmentally pervasive pharmaceuticals.
The HPLC-Chip executes these analyses with few of the operational difficulties encountered with traditional nanocolumn assemblies. The latter systems can be sensitive to small variations in configuration and require significant operator expertise and time to achieve reproducible performance. Here too, new labor-saving robotics designed to interface with the HPLC-Chip plays an important part in the accurate and reproducible deployment of this device without the need for manual intervention.
A multiplicity of MS configurations and applications
Today the mass spectrometer is morphing into a variety of ultrasensitive multisector instruments such as the Triple Quadrupole MS and the Quadrupole TOF MS. These new MS technologies are empowered with higher levels of mass accuracy and sophisticated informatics to extract, process, and integrate data to yield more meaningful results than ever before. These are the kinds of instruments that will increasingly be required to execute cutting-edge techniques such as metabolomic profiling or the detection of post-translationally modified (PTM) biomarker proteins, in which the HPLC-Chip is also playing a part.
Chips are being manufactured with different column packings and more are being added as the community of users makes their needs known. These same users now have the option of providing their own packing for the manufacture of custom chips. Already, the chip has acquitted itself consistently well in a variety of documented applications. And the very real potential for more sophisticated chip configurations supporting new analytical methodologies is generating a great deal of excitement about this new technology.
Nick Roelofs, Ph.D. is the Vice President and General Manager of the Life Sciences Solutions Unit at Agilent Technologies Inc. He may be contacted at ChromatographyTechniques@advantagemedia.com
Agilent Technologies, Inc.
5301 Stevens Creek Blvd.
Santa Clara, CA, 95051-7201
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