Development of a Colorimetric Test for Uranium

This paper discusses the development of and proposed enhancements to a colorimetric test for the detection of uranium in biological samples such as urine. The goal of this work is to develop a technique for the detection of uranium that could: 1) be conducted rapidly and accurately; 2) would not require extensive sample preparation; 3) would not require expensive or complicated instrumentation; 4) would require little or no technical training to conduct; and 5) could be used in a field situation if needed. The technique described in this paper involves the following steps. A buffer is added to the sample to maintain the pH of the mixture within an experimentally acceptable range and a quaternary ammonium salt is added to aid in solubilization of the reaction components. A pyridylazo stain, 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, capable of binding a variety of metals, is used to complex the uranium. This interaction has been made specific for uranium through the use of “masking agents.” Color development indicating the presence of uranium is monitored and can be quantitated by determining the absorbance of the reaction mixture at 578 nm, using a spectrophotometer or colorimeter. At present, the limit of sensitivity of the procedure is approximately 30 μg of uranium/L. However, through the incorporation of a sample concentration step in the procedure, we believe we can greatly increase the sensitivity of the technique. The two areas we believe are amenable to our concentration efforts are prior to the addition of the assay components (pre-complexation concentration step) or after the formation of the stain/uranium complex (post-complexation concentration step). Our goal is to make the procedure more applicable while still maintaining technical simplicity and ease of use. We believe this research will provide the capability to rapidly and accurately screen biological samples for uranium. 1.0 INTRODUCTION Since the terrorist attacks of September 11, 2001, concern about the potential use of radiological weapons directed against civilians or military personnel has risen dramatically. Concern is no longer limited to nuclear fission devices delivered by rogue states or terrorist groups. Other means of spreading radioactivity, especially use of a radiological dispersion device (RDD)—the so-called dirty bomb—are now thought to be urgent risks. An RDD uses conventional explosives to disperse radioactive material over a wide area. Although a relatively small number of people may be killed or injured by blast effects from such a weapon, a much larger number Paper presented at the NATO Human Factors and Medicine Panel Research Task Group 099 “Radiation Bioeffects and Countermeasures” meeting, held in Bethesda, Maryland, USA, June 21-23, 2005, and published in AFRRI CD 05-2. Development of a Colorimetric Test for Uranium 22–2 NATO RTG-099 2005 potentially could be exposed to dispersed radionuclides. Exposures could occur by any of several routes, including inhalation, ingestion, embedded fragments, or wound contamination. Uranium has been thought by some to be a potential component of an RDD. It is a naturally occurring, radioactive, metallic element found in trace amounts in soil and rocks, water, and air. Uranium, as found in nature, consists primarily of three isotopes in the following percentages (by weight): U (99.283%); U (0.711%); and U (0.005%). As produced for power generation and nuclear weapons, uranium contains greater than 0.711% U and is considered “enriched” uranium. Uranium containing less than 0.711% U is considered “depleted” uranium. Depleted uranium (DU), obtained as a by-product of the enrichment process for nuclear reactorand weapons-grade uranium, usually contains less than 0.3% U and is therefore less than half as radioactive as natural uranium. Because it is extremely dense (1.7 times the density of lead), DU has several applications including the armor plating of military vehicles. Its mass and pyrophoric properties under conditions of extreme temperature and pressure also make it useful for penetrator munitions designed to defeat enemy armor. After internalization, uranium, no matter what the form, is eventually excreted from the body in the urine. As such, urine uranium levels are excellent indicators of exposure. There are a variety of techniques available to measure uranium levels in fluids, including neutron activation analysis [Zouridakis 2002], kinetic phosphorescence analysis (KPA) [Brina 1995, Ejnik 2000], alpha spectrometry [Ethington 2000], liquid scintillation spectrometry [Salonen 1993], and inductively coupled-plasma mass spectrometry (ICP-MS) [Karpas 1996]. The unifying feature of these techniques is that they either require extensive sample preparation times (sometimes requiring days to complete) or use expensive instrumentation with which to conduct the analysis. Because many individuals in the vicinity of an RDD detonation would not be exposed but would, as “worried well,” seek assessment and treatment, a method to rapidly assess and differentiate exposed from unexposed individuals would be useful. Colorimetric or spectrophotometric detection methods are preferable for use in the field. The pyridylazo dye, 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP), was previously used to detect uranium in organic leach liquors from nuclear fuel reprocessing plants [Johnson 1971, Pakalns 1972] as well as used histochemically to detect a variety of metals in rat liver samples [Sumi 1983]. Its use in aqueous solutions is greatly hindered by its insolubility and nonspecificity. However, recent procedural advancements have enabled its use to specifically bind to uranium in such diverse aqueous environments as water and urine [Kalinich 2000], cells [Kalinich 2001, Kalinich 2002], and buffered extracts of metallic shrapnel [Kalinich 2000]. 2.0 PROGRESS TO DATE Br-PADAP (Figure 1) has been used to bind to a variety of metal ions including cobalt, nickel, zinc, and copper [Sumi 1983], as well as uranium in organic leach liquors from nuclear reprocessing facilities [Johnson 1971, Pakalns 1972]. Development of a Colorimetric Test for Uranium NATO RTG-099 2005 22–3 O H N CH2CH3 CH2CH3 N N N Br Figure 1: Structure of Br-PADAP Figure 2 shows our procedure for detecting uranium in fluids. Figure 2: Procedure for the colorimetric detection of uranium Because Br-PADAP is capable of binding a variety of metals, a procedure was needed to eliminate binding of Br-PADAP to metals other than uranium. We have accomplished that goal through the use of “masking agents,” compounds that prevent the binding of the stain to metals not of interest. The addition of a mixture of EDTA and sodium citrate allows the stain to bind to uranium, but not to other metals. Table 1 shows the metals that do not bind Br-PADAP, those that can bind Br-PADAP but can be masked by EDTA/citrate, and those that bind Br-PADAP but are not masked. Table 1: Metals tested for the ability to bind Br-PADAP Do not bind Bind but can be masked Bind, not masked Lithium Potassium Cesium Calcium Tantalum Molybdenum Sodium Rubidium Magnesium Barium Cerium Silver Chromium Gadolinium Lanthanum Aluminum Tungsten Cobalt Zinc Cadmium Iron Nickel Copper Lead Uranium The binding of Br-PADAP to uranium is pH dependent, with an optimal pH range of 8–12. Several common laboratory buffers, including phosphate, borate, and CAPS, are capable of maintaining the pH within the acSample Add 1. Masking agents 2. Buffer, pH 8-12 3. Solubilizing agent 4. Br-PADAP Determine Absorbance (578 nm) Development of a Colorimetric Test for Uranium 22–4 NATO RTG-099 2005 ceptable range. Because of the limited water solubility of Br-PADAP, we have prepared the stain as a stock solution in ethanol or dimethyl sulfoxide (DMSO) prior to addition to the reaction mixture. Also, we have discovered that the addition of a quaternary ammonium salt acts as a “solubilizing agent” to keep the reaction mixture components in solution. After addition of the Br-PADAP, color development is rapid if uranium is present in sufficient quantity. This results in a bathochromic shift of the absorption maximum from 444 nm to 578 nm (Figure 3). This wavelength shift easily can be detected with a visible-light spectrophotometer or colorimeter. 700 600 500 400 W avelength (nanom eters ) 1.2