Far UV Performance of the LAMBDA 850/950 UV/Vis and UV/Vis/NIR Research Spectrophotometers
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1 Far UV Performance of the LAMBDA 850/950 UV/Vis and UV/Vis/NIR Research Spectrophotometers UV/VIS AND UV/VIS/NIR SPECTROSCOPY A P P L I C A T I O N N O T E Summary The far UV region (generally covering the range from 190 nm to 150 nm) has great potential experimental interest. All compounds have electronic transitions in this region, including the first allowed benzenoid transition, the amide absorption bands, the first transition of the olefinic double bond, and the n-σ* absorption bands of water, alcohols, ammonia, and sulfur compounds. 1 In particular, spectra of steroids, nucleic acids, proteins, and polypeptides can be found in this region. The purpose of this application note is to investigate the far UV performance of the new PerkinElmer LAMBDA 850 and 950 UV/Vis and UV/Vis/NIR spectrophotometers. Both of these instruments are high research grade systems, incorporating double monochromators, and offer ultra high optical resolution to < 0.05 nm and a wide dynamic range to 8 A. Both units include complete far UV capability and allow scanning to 175 nm. The use of nitrogen purging, selection of appropriate solvents and cuvettes, selection of instrumental parameters, and validating experimental results will be discussed. Examples of far UV application data will also be presented. Introduction Usually, UV/Vis spectrophotometers with conventional optics using a monochromator and photomultipliers as detectors have a working range from 190 nm to 900 nm. The wavelength range below 190 nm down to 150 nm is part of the far ultraviolet range and is usually only accessible with dedicated instruments prepared for transmission measurements in a vacuum. Most of the compounds analyzed by UV/Vis spectroscopy have an absorption maximum above 200 nm. Nevertheless, there are a number of applications where there is either only a low absorbance above 200 nm, as in the analysis of sugars, or the absorbance increase below 200 nm is used for the determination of impurities, e.g. in highly pure isooctane or alkylpyridines.
2 Instrument requirements for far UV measurements The far UV range is made available in the PerkinElmer LAMBDA 850 and 950 spectrophotometers through incorporation of hardware not typically present in a conventional spectrophotometer. In particular, the LAMBDA 850 and 950 include as standard: Nitrogen purge ready, sealed optical system Special water-free Suprasil W for all windows High efficiency purge fittings with multiple lines routed to the monochromators and the source, sample, and detector compartments An exclusive far UV photomultiplier (R 6872) with fused silica optical window The latest generation high energy deuterium sources incorporating fused silica optical windows Fused silica photomultiplier and deuterium source Photomultipliers usually incorporate a quartz window for surface protection, which contributes to energy decrease below 200 nm. A photomultiplier with a fused silica window has a noise level reduced by a factor of 3 to 5 below 200 nm, when compared to a normal photomultiplier with a quartz window. Similarly, deuterium lamps with fused silica windows give two times higher radiation density at 190 nm and about five times higher radiation density at 180 nm when compared to a standard quartz window deuterium lamp. Nitrogen purging The constituents of air start to absorb below 200 nm. The UV cutoff of ozone starts at 295 nm, of oxygen at 200 nm and of nitrogen at 170 nm. 2 Thus, for working below 200 nm, it is necessary to remove the oxygencontaining compounds from the light path to increase energy and decrease noise level. The effect of nitrogen purging on the energy level between 200 nm and 180 nm is shown in Figure 1. The bottom energy curve is without purge, the top curve is with purge. Because of the relatively high diffusion coefficients of gases, it is necessary to have a permanent flush of nitrogen. The flow velocity is dependent on the size and the sealing of the optical system. An initial nitrogen flow rate of 20 l/min is recommended for 20 to 30 minutes, and then the flow rate can be dropped to about 6 to 7 l/min. Only nitrogen gas of the highest purity should be used. Instruments specifically designed to accommodate far UV measurements, such as the PerkinElmer LAMBDA 850 and 950 UV/Vis and UV/Vis/NIR spectrophotometers, have sealed optical compartments. Radiation from the source compartment enters the monochromators through a water-free fused silica window and the sample compartment area is sealed with water-free fused silica windows. Thus, the entire optical path from the source lamp to the detector can be purged largely free of water vapor and oxygen. To facilitate effective purging, the PerkinElmer LAMBDA series spectrophotometers incorporate a high efficiency purge system, with multiple purge lines routed to the monochromators, the source, the sample, and the detector compartments. Stray radiation Usually, stray radiation in an optical system increases toward shorter wavelengths because of higher scattering. The LAMBDA 850 and 950 spectrophotometers are high-performance, double monochromator systems, with a specified stray radiation level of less than % transmission Figure 1. Energy spectrum with and without nitrogen purging. Data collected at 10 nm/min with a 0.1 nm slit. 2
3 at 220 nm measured according to ASTM recommendation E 387. Potassium chloride solution can be used to determine stray radiation levels below 200 nm. Stray light values below 200 nm can typically increase to about 0.01% transmission. Experiments designed for the far UV should take into consideration the lower dynamic range due to the higher stray radiation present. It is generally recommended to keep solutions below 2 A when measuring in the far UV. Choice of solvent and cuvette Many solvents absorb greatly in the range of 200 and 175 nm. Most solvents are completely opaque in the far UV range in a standard 10 mm pathlength cuvette. For example, distilled water measured in a 10 mm pathlength cuvette will totally cut off at 185 nm. Therefore, narrow pathlength cuvettes are required or thin film demountable liquid cells with fused silica or CaF 2 windows. A maximum pathlength of 1 mm is recommended with the solvents that are transparent in the far UV. Common far solvents with the wavelength of their absolute cut-offs include water (170 nm), deuterium oxide (166 nm), methyl alcohol (182 nm), trifluoroethanol (165 nm), hexafluoro-2-propanol (164 nm), acetonitrile (173 nm) and pentane (167 nm). 1 A general recommendation before conducting an experiment in the far UV is to fill a 1 mm silica cuvette with the solvent of choice, and scan this against a purged air baseline. The solvent absorption observed will be a consideration in the dynamic range allowed for the analyte. If the solvent exceeds 2 A, it should probably not be used, or a narrower pathlength cell needs to be used. Choice of bandpass The quality of spectra obtained in the far UV range can be optimized by using broader bandpasses. In particular, energy is extremely low from 180 to 175 nm. The absorbance noise data presented in Table 1 shows the effect of slit width. The bandpass chosen for any particular experiment will be a compromise between the resolution required and maximizing the signal-to-noise. The general recommendation is to select the widest possible bandpass to achieve the resolution required for the compound being studied. Typical far UV corrected baselines are presented in Figure 2 using a 4 nm bandpass with nitrogen purging. Effectively purged baselines will normally be within +/ A for a 2 nm bandpass and +/ A for a 4 nm bandpass. The far UV experiment checklist Purge the instrument with nitrogen at approximately 20 l/min for at least 20 minutes prior to use. After 20 minutes, the nitrogen flow can be reduced to about 6-7 l/min. Use 1 mm pathlength silica cuvettes to start. Create a method with a 4 nm bandpass, a 1 nm data interval, and a 1.44 second integration time (40 nm/min scan speed). Select a suitable far UV solvent for the analyte. If possible, use pure distilled water. Against an air background, determine the absorbance of the solvent in a 1 mm cell. If it is over 2 A near the peak for the analyte, select a different solvent or use a demountable liquid cell with a shorter pathlength. The sample compartment will flood with oxygen when the compartment lid is opened. The Table 1. Absorbance RMS noise level at a 2 nm and a 4 nm slit as a function of wavelength. Wavelength (nm) Slit Figure 2. Expanded scale overlay of 5 corrected baselines versus air with nitrogen purge. Conditions were 40 nm/min scan speed, 1.44 second integration, and 4 nm slit (LAMBDA 950). 3
4 cover should be opened only for the shortest time possible. The sample compartment should be purged for about 5 to 10 minutes after inserting a sample. Optionally, to reduce the time between samples, an additional purge line can be routed to the sample compartment to accelerate the oxygen purge. If possible, keep the absorbance of the sample solution at 2 A or less as this will minimize any stray light effects. Validating experimental results A real problem for a researcher studying the far UV is the validation of results. Because of the difficulty in obtaining true spectra in the far UV, with demanding instrumental and technique requirements, there is a suspicion that many of the deep UV publications contain spectra not totally free of instrumental artifacts, and often the spectra are difficult to reproduce. Most importantly, insufficient or inefficient nitrogen purging creates high oxygen backgrounds, greatly reducing the dynamic range in the far UV, and causing spectra to be affected by stray light. Spectra affected by stray light are invalid, often yielding irreproducible spectra with shifted peak maximums. Therefore, validating experimental results in the far UV is critical. The following procedures are recommended for validating experimental results: Run replicates on the same sample to see if the peaks overlay consistently. If time permits, repeat the experiment with a new corrected baseline, running the same sample. Run the sample at different times during the purge process. The spectra should overlay with acceptable precision. Acquire fresh corrected baselines before each sample measurement for the best precision. The oxygen backgrounds can fluctuate slightly with time. Prepare and run different concentrations of the sample, and keep the absorbance at the peak at < 2 A. The spectra and peak maximums should be the same for the different concentration. To reduce the possibility that a stray light induced peak is being observed, validate the experimental peaks by scanning a number of neutral density screens bracketing the absorbance range of the results (Figure 3). The screens should give a flat spectral response to 175 nm against a fresh corrected purged baseline. If peaks are observed in the corrected baseline, or in the neutral density screen spectra, then most likely the purging was not adequate, and oxygen is still present. Applications Among the possible applications in the far UV range with conventional UV/Vis spectrophotometers are the study of gases with large absorption coefficients in this range, 3 and the high sensitivity of organic compounds in solution such as sugars, substituted benzenes and pyridines. A spectrum of a 10 ppm solution of benzene in water is shown in Figure 4. Method parameters were a 40 nm/min scan speed, a 1.44 second integration time, and a 4 nm slit. Figure 5 shows two overlaid spectra of deoxyribonucleic (calf thymus, Sigma D 1501) acquired at different concentrations. The nearly identical spectra help validate the experiment by assuring stray light artifacts are not a factor. The transmission characteristics and purity of deep UV transmitting materials can also be studied. Figure 6 shows two overlaid spectra of different calcium fluoride lens materials. Figure 3. Overlay spectra of four neutral density screens scanned into the deep UV with nitrogen purging. A flat spectral response can help validate the experimental results by assuring stray light artifacts are not present. Figure 4. Spectrum of 10 ppm benzene in water scanned in a 1 mm cell. Conditions were 40 nm/min scan speed, 1.44 second integration, and 4 nm slit. 4
5 Conclusion High quality far ultraviolet data can be obtained with a correctly configured UV/Vis spectrophotometer, using appropriate experimental materials and procedures. The PerkinElmer LAMBDA 850 and 950 UV/Vis and UV/Vis/NIR double monochromator spectrophotometers are ideal systems for conducting far UV experiments. These research grade units include the hardware required for these measurements, as standard. This includes sealed optical systems, high efficiency multiple purge lines, water-free Suprasil windows, and a special extended range photomultiplier. Also, the ultra-low stray light characteristics of the LAMBDA 850 and 950 allows publication quality spectra, free of artifacts, to be collected to 175 nm. References 1 Malcolm F. Fox, Applied Spectroscopy, Volume 27, Number 3, 1973, pg W. Boehme, G. Naundorf, Applied UV Spectroscopy 13E, Bodenseewerk Perkin-Elmer + Co. GmbH, Uberlingen, West Germany B.A. Thompson et al., Journal of Geophysical Research, Vol. 68, No. 24, December Figure 5. Overlaid spectra of DNA in distilled water (calf thymus sodium salt) scanned in the deep UV in a 1 mm pathlength cuvette at approximate concentrations of 15 (lower) and 35 (upper) µg/ml. Figure 6. Overlaid spectra of two calcium fluoride crystals scanned into the deep UV with nitrogen purge. The differences observed are caused by impurities. PerkinElmer Life and Analytical Sciences 710 Bridgeport Avenue Shelton, CT USA Phone: (800) or (+1) For a complete listing of our global offices, visit /lasoffices 2004 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. LAMBDA is a trademark and PerkinElmer is a registered trademark of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. Suprasil is a registered trademark of Heraeus Holding GmbH. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors _05 Printed in USA
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