28 CHROMATOGRAPHY/SPECTROSCOPY
The power to choose expands possibilities for measurement
The future of molecular analysis lies in speed, accuracy, and ease-of-use of microsample technologies. Philippe Desjardins reports.
capabilities of a novel microsample retention system as well as those of traditional cuvettes. This dual functionality is ideal as microsample analyses continue to push the limits of detection and molecular techniques continue to evolve that require the flexibility to perform conventional and unconventional quantitation methods. Devices are being developed that integrate
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microsample technologies with simple ‘sample-in answer-out’ capability. As sequencing, PCR (polymerase chain reaction), microarrays and other molecular techniques continue to use ever smaller amounts of sample, the quality control of these samples is essential. In order to keep pace with the miniaturisation of
sample volume throughout all of molecular biology, new quantification methods had to be developed. To meet this challenge, Dr Charles Robertson, then a physicist at the E.I DuPont Experimental Station, developed an elegant solution – remove the containment device altogether (ie cuvettes). The central idea is based on the concept that the containment device itself was a limiting factor in reducing volume. Removing the containment device in effect removes the limitation of having to fill a certain minimum volume in order to take a measurement. But how is this done? The answer lies in using the physical
new system configured for both microvolume pedestal measurements as well as cuvette measurements gives scientists the flexibility to use the unique
property of the sample itself, namely surface tension, to hold itself in place during the measurement cycle. Combining fiber optic technology with the inherent surface tension of liquids resulted in the development of a unique sample retention system that is the basis of NanoDrop technology.
The Thermo Scientific NanoDrop 2000c
Spectrophotometer employs this sample retention system by using a pair of optical pedestals to hold the sample during measurement. The user places a droplet of sample (usually 1ul of sample for aqueous solutions of nucleic acids) onto the lower optical pedestal (Fig. 1) and lowers the lever arm. The sample makes contacts with both optical surfaces, forming a vertical liquid bridge (Fig. 2). Light from a xenon flash lamp fires through an optical fibre embedded in the upper pedestal, then passes through the sample, and is collected by another optical fiber embedded in the lower optical pedestal. The light then continues to an internal CCD detector to provide the requisite data. The software displays a full UV-Vis spectrum as well as a calculated concentration of the sample being measured. The absence of a solid containment barrier allows
the distance between the optical surfaces to change in real time. The system finds the correct transmittance of light through the sample column by rapidly changing the distance or path length from 1 mm to 0.2mm, 0.1mm, and 0.05mm (Fig. 3). According to the basic laws of spectroscopy, shorter path lengths allow for higher concentrations to be measured. By having four incrementally shorter path lengths, the new system has the broadest dynamic measurement range of any spectrophotometer, essentially eliminating the need to
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Fig. 1. A microvolume (1uL) droplet of sample is pipetted directly onto the lower optical pedestal.
Fig. 2. The sample is held in place by surface tension between the upper and lower optical pedestals during measurement.
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