varian Cary 50 Uv-vis
Unique Optical Performance of the Varian Cary 50 UV-Vis Spectrophotometer Ideal for Analyzing Microvolume Samples
Daren Fyfe
Varian, Inc.
Figure 1. Varian Cary 50 spectrophotometer
Summary
• The Hellma Traycell™ fitted to a Cary 50 UV-Vis spectrophotometer is ideal for the measurement of microlitre (<4 µL) sample volumes
• Significantly higher light throughput in the Cary 50/ Traycell system compared to a competitor’s system means higher photometric range
animaltube• Removal and replacement of the Traycell for cleaning or other purpo has negligible affects on data, meaning excellent reproducibility
pointoutAim
To evaluate the reproducibility and light throughput of a Varian Cary 50 UV-Vis spectrophotometer fitted with a Hellma Traycell TM ultra-microvolume cuvette for accurate and reproducible absorbance measurements of microvolume liquid samples.Introduction
Given the increasing demand for simple, rapid and non-destructive methods to analyze microlitre volumes of chemical and biological samples, we have previously demonstrated that the Varian Cary 50 UV-Vis spectrophotometer fitted with a Hellma Traycell™ ultra-microvolume cuvette can accurately measure concentration values of 4 µL samples of DNA1. Given the occasional need to a) remove and replace the Traycell for practical purpos and b) analyze lower still concentrations of biochemical samples, in this paper we extend our initial obrvations to evaluate the reproducibility and light throughput of the Hellma Traycell™ when fitted to the Cary 50 UV-Vis
spectrophotometer.spoil
Figure 2. Hellma Traycell TM microvolume accessory
Materials and Methods
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A Traycell™ ultra-microvolume cuvette (Figure 2; Helma GmbH & Co., Germany) was fitted into a Varian Cary Eclip cell holder ba (standard with the Traycell Kit, part number 9910123500) in the sample chamber of a Cary 50 (Figure 3). The cell holder is optimized for the Traycell product on account of enhanced tilt and height adjustment capabilities compared to the standard Cary 50 cell holder. The Traycell was aligned vertically and horizontally to optimize light thoughput at 500 nm using the Align application module in the Varian Cary WinUV™ Bio software. Adjustments were
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made in the cuvette holder in order to obtain light transmission values greater than 25% at 500 nm, relative to transmission readings taken at 100%T (with air at ambient laboratory temperature in the sample beam). Results
For comparative purpos only, data prented in Figure 4 show a single transmission scan throughout the wavelength range 250–1000 nm using a competitor’s conventional UV-Vis spectrophotometer properly fitted with a Traycell. Air was initially scanned at the same wavelengths as a baline. Data in Figure 5 show the results from the same test using the Varian Cary 50 UV-Vis spectrophomometer, however in this ca the Traycell was repeatedly removed and replaced five times from the cell holder ba to asss reproducibility.Figure 5. Reproducibility of wavelength scans of 4 µL samples of DNA, scanned over the UV region using the Hellma Traycell TM with the Cary 50 UV-Vis spectrophotometer
Table 1. Statistical data derived from raw reproducibility data given in Figure 5officially
Figure 4. Reproducibility of wavelength scans of 4 µL samples of DNA, scanned over the UV region using the Traycell fitted to a conventional, competing UV-Vis spectrophotometer, not equipped with Varian’s patented2 xenon flashlamp optical design. Note noi in NIR region.
Figure 3. Varian Cary 50 Bio UV-Vis spectrophotometer sample chamber fitted with a Hellma Traycell
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Data shown in Figures 4 and 5 demonstrate that the Cary 50 fitted with the Traycell yields significantly higher %T values (>10% throughout the range 200–1000 nm)
compared to %T values recorded using the Traycell fitted into a UV-Vis spectrophotometer from a competitor. Moreover, data in Table 1 show that repeatedly removing the Traycell from the cell holder and replacing it has negligible effects on the %T values recorded, thus
demonstrating excellent reproducibility througout complete u of the Traycell. Comparing data in Figures 4 and 5 also clearly show that whilst there is excessive noi in the competitor’s instrument in the NIR range 800–1000 nm, the Cary 50/Traycell system is well suited for sample measurements within the NIR range.
Discussion and Conclusion
Quantitation of microlitre volumes of DNA by UV-Vis spectrophotometry has a variety of benefits:
1) Low volumes - save on precious samples and reagents 2) Cleaning/purcha of quartz cuvettes or other vesls is not required
3) The method is non-destructive to samples 4) Accurate and reproducible measurements can be recorded in conds
5) No chromophores or fluorophores are required to visualize the sample
6) No compromi in data quality compared to results recorded using larger volumes in a cuvette or microplate In this short review, we evaluated some performance characteristics of the Hellma Traycell TM microcell fitted to the Varian Cary 50 UV-Vis spectrophotometer to confirm that a) light throughput and therefore photometric range of the Cary 50 system is far superior to that of a competitor and b) results are extremely reproducible (maximum RSD = 1.2% across the wavelength range 200–1000 nm) even when the Traycell was removed and replaced quentially between wavelength scans.
We believe the obrvations indicate that the Traycell/Cary 50 system is ideal for rapid, reproducible and accurate measurements of a broad range of concentrations of microlitre volumes of DNA. It is the patented 2 optical design of the Cary 50 instrument that makes the measurements possible, since unique xenon flashlamp dynamics and a millimetre beam size maximizes the light flux through the optical system of the Traycell. We now intend to further our investigations to explore the dynamic range of this instrument system using a variety of biomolecules and samples.
Acknowledgements
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The author gratefully acknowledges Dr Alison Coates, Department of Physiology at the University of Adelaide, Australia for her kind collaboration during the generation of data for this document.
References
1. Keighley, RA and Fyfe, DJ. (2005) Simple and rapid quantitation of microlitre DNA samples using the Varian Cary 50 UV-Vis spectrophotometer. Varian application note #91;
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2. Hammer, MR. (1999) Spectrophotometer. United States Patent 6,002,477.
