According to our HeNe laser providing an excitation wavelength of 632.8nm, we had to choose an adequate label for the use dye. The CY5 dye, possessing an absorption maximum at 650nm and an emission maximum at 670nm, match in this configuration. This improved detection device allows for the simultaneous monitoring of the surface plasmon reflectivity and fluorescence emission.³⁰

Total Organic Carbon Analysis

The TOC analyzer is so elemental an instrument that it defies radical change. TOC of water is measured by oxidizing organic carbon to produce CO₂, which is measured. New developments are incremental and depend on the specifications for the water. A search of listings in http://www.globalspec.com/ showed 32 companies competing. Attacking it differently as a word search pulled up 1,455 entries; but only 14 were directly related.

An analytical centrifuge is essentially s preparative centrifuge with an optical system for measuring the distribution of molecules inside while the rotor is spinning. Computer programs that interpret the data are the newest development. Peter Schuck of the National Institutes of Health is the webmaster of a homepage for SEDFIT, software for the analysis of analytical ultracentrifugation and other hydrodynamic data, which was written at NIH and is distributed without charge for research use. The program can be downloaded from the home page, http://www.analyticalultracentrifugation.com/default.htm. (It is also available directly from NIH.) SEDFIT is closely related to SEDPHAT, which provides global modeling capability for both sedimentation equilibrium and sedimentation velocity data. It also can serve as a platform for isotherm analyses.

A general introduction to the study of protein interactions by analytical ultracentrifugation can be found at the website of the PBR/DBEPS at NIH. This includes an introduction to the general principles of AUC and detailed experimental protocols. A general introduction of analytical ultracentrifugation can be found in Protein Science 2002; 11:2067-2079. A workshop on hydrodynamic and thermodynamic sedimentation analysis with SEDFIT and SEDPHAT at NIH in Bethesda is scheduled to take place September 20-22, 2005.

UV-VIS Spectroscopy

A research-grade scanning spectrometer measures the intensity of transmitted light in a narrow bandpass, and scans the wavelength over time in order to collect a spectrum. Because absorption is a ratiometric measurement, these instruments generally require the user to measure two spectra, one sample and one blank. The blank should be identical to the sample in every way except that the absorbing species of interest is not present. This can be done either consecutively with a single-beam instrument followed by a ratio calculation, or simultaneously with a dual-beam instrument. The dual-beam method is faster and has the added advantage that lamp-drift and other slow-intensity fluctuations are properly accounted for in the ratio calculation. Collecting spectra with scanning spectrometers is slow, but the instruments often have very high resolving power owing to the use of photo multiplier tube detectors, which can be used with very narrow slit widths.³¹

A photodiode spectrometer houses a one- or two-dimensional stack of individual photodiode detectors, each of which makes an independent measurement of the incident light intensity at its particular location. Typically, 2-D arrays are used for electronic imaging applications while the one-dimensional arrays are used in spectroscopic instrumentation. If the array is placed at the focal plane of a monochromator, then the position of each photodiode will be associated with a specific bandwidth of light. ³¹

A popular model (Agilent 8453) has 1,024 diodes in the linear array associated with a 1 nm band of light spanning from 900 nm to 1,000 nm. The individual diodes are separated by about 25 microns and the physical slit width matches this spacing. Thus the maximum resolution of the instrument is about 1 nm. This is not as good as the typical resolution of a scanning instrument (resolutions of about 0.2 nm are not uncommon with scanning instruments), but for many applications (especially molecular solution spectroscopy where adsorption bands are very broad) 1 nm is adequate. The real advantage of he instrument is its speed. A single spectrum can be taken in about 0.1 sec.³¹

Peter Silverberg is an associate editor with Biopharm International.