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The HPLC-Chip, Part II: Applications and Future Directions
H5>Applications and Future Directions
by Nick Roelofs, Ph.D., Vice President and General Manager, Life Sciences Solutions Unit, Agilent Technologies, Inc.
Part I of this article described the HPLC-Chip and its potential for advancing nanoscale LC analysis. Part II provides some examples of HPLC-Chip/MS applications and then explains how the method of microfluidic device fabrication foreshadows a revolutionary change in the way liquid chromatographic systems are likely to be conceived and implemented in the future.
The HPLC-Chip gives users, for the first time, a nanoscale LC-MS system without the complexities of a traditional nanocolumn LC. The integrated on-chip device eliminates many of the potential problems users can have with nanocolumns, such as leaks, clogging or contamination. Even more to the point, there is no complex system to physically assemble or adjust. The chip is inserted into chip interface of the LC-MS instrument and built-in robotics does the rest. On-chip component formation and integration minimize both sample path length and post-column dead volume, thereby reducing peak dispersion. By contrast, post column band broadening due to dispersion on a typical nanocolumn can cause as much as 45% loss of resolution. Additional gains in sensitivity with the chip are realized through optimal positioning of the nanospray emitter at the MS entrance cone for more effective analyte capture and selectivity. Chip-enabled LC-MS instrumentation is increasingly attractive to users who need robust, precise and easy-to-manage nanoscale LC analytical tools.
These are the applications for which samples are often vanishingly small, not only because they are difficult to come by, but because the analytes are present at such low concentrations: Molecules such as low-abundance protein biomarkers, sometimes structurally complicated by post-translational modification; picomole or femtomole concentrations of drug metabolites or environmental residues; and systems of interacting proteins in which the nature and transient concentration of each molecular actor is critical to understanding the biological system they comprise. Typical examples of such investigations include:
• Protein profiling (1). This technique seeks to overcome the deficiencies of classical shotgun proteomics that generally lead to identification of only a subset of the proteins actually present in a sample. Utilizing a HPLC-Chip/MS-enabled ESI-TOF MS with sufficient mass accuracy and resolution, the authors explore a protein profiling approach combined with differential analysis to highlight and identify potential low-abundance biomarkers in the presence of much higher-abundance proteins.
• Characterizing glycoproteins (2). Using a HPLC-Chip/ion trap MS system, this study details the development of a quality control methodology able to detect and quantify femtomole levels of glycoprotein and glycan products including their minor isoforms, the presence of which may be significant in the stability or effectiveness of a glycoprotein drug.
• Noncovalent Protein Interaction (3). An infusion HPLC-Chip coupled to an ESI-TOF MS is used to analyze a myoglobin noncovalently associated complex. The instrumentation provides the exact mass information needed to confirm the identity of the complex. This approach is proven to be more effective and sensitive than traditional methods for the study of protein complexes.
These are only a few of the investigations being undertaken utilizing chip-enabled LC-MS.
Designs for Tomorrow's Analyses
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HPLC-Chip
Manufacturing: a Flexible, Scalable Process (4). 1. Laser ablation of the
biocompatible polyimide film is used to create surface structures required
for the HPLC-Chip layout. This makes it possible to create chips with a
wide variety of features such as analytical columns, filters and fluid access
holes (Bottom inset). 2. The surface is cleaned. 3. Lamination of several
polyimide layers together allows the creation of multifunction chips. 4.
The chip is trimmed and the electrospray tip is directly laser ablated on
the chip. 5. The surface is metallized for high voltage electrospray contact.]
The flexibility of the design and fabrication process allows for facile
modification and enhancement to generate a host of LC devices designed to
meet user needs. Retooling now means making changes to the fabrication program
in the computer that specifies the action of the "generic" toolset. Already,
an experimental 2-layer chip with separate cation exchange and reverse phase
columns has been created enabling on-chip 2DLC (Figure 2). Currently, the
development of a variety of multicolumn, multilayer chips is being explored. |
Early on in this article, the conceptual departure in LC component design represented
by the HPLC-chip was likened to the transition between packed and capillary GC
columns, but it is much more than that. Here we see a transition from a system
of individual components linked together post-manufacture to systems of components
created in place as a single integrated entity. This is a paradigm shift in the
way LC devices are conceived and implemented. In this new regime, computer assisted
manufacturing is the toolset and the device concept is transferred from the mind
of the engineer to the computer program, which implements it directly onto the
chip substrate as architecture (Figure 1, above).
The flexibility of the design and fabrication process allows for facile modification
and enhancement to generate a host of LC devices designed to meet user needs.
Retooling now means making changes to the fabrication program in the computer
that specifies the action of the "generic" toolset. Already, an experimental 2-layer
chip with separate cation exchange and reverse phase columns has been created
enabling on-chip 2DLC (Figure 2,
below). Currently, the development of a variety of multicolumn, multilayer
chips is being explored.
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Figure
2. Experimental 2-Layer Chip HPLC-Chip Enabling a Complete On-Chip 2D LC-MS
Analysis.
Sequence of Operations: Run position: a) Autosampler delivers sample to
layer one SCX column: 1 > 6, b) Breakthrough analyte fraction (now in layer
2) is transported to and captured by enrichment column: 6 > 1 > 4. c) Excess
to waste 4 > 5.
Load position: d) Sample is flushed from enrichment column to analytical
column.
Run position: e) RPLC. f) First salt step elution of SCX to enrichment column.
Load position: g) Sample is flushed from enrichment column to analytical
column.
Run position: h) RPLC.
Repeat: Steps f)-h) with increasing salt concentration elution of SCX column
followed by enrichment column capture, flushing onto analytical column followed
by RPLC-MS. |
Nick Roelofs may be contacted at nick_roelofs@agilent.com
or by phone at 877-424-4536.
AT A GLANCE
• The integrated on-chip device eliminates problems users have with nanocolumns
• The development of a variety of multicolumn, multilayer chips is being explored
• The chip design process allows for modification to generate a host of LC devices
• The transformation in the nature and production of the chip will greatly impact LC instrumentation
ONLINE
For additional information on the technologies discussed in this article, see
Laboratory Equipment magazine online at www.LaboratoryEquipment.com
or the following Web site:
• www.chem.agilent.com
Laboratory Equipment
Advantage Business Media
Rockaway, NJ, 07866
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