Ureas detection systems

Fig. 9. The linear range for urea detection using a miniaturized thermometric system...

The coupling of the CZE step to detection systems other than UV has required the development of separation conditions compatible to the detection system used. For instance, the presence of primary amines, such as DAB, in buffers needed to be avoided for compatibility with laser-induced fluorescence (LIE) of compounds derivatized with fluorogenic substrates through their amino groups [90]. Baseline resolution of eight peaks in approximately the same time was achieved by substituting DAB by morpholine and tricine by boric acid (to avoid potential traces of primary amines in the tricine buffer) and by adjusting the concentration of other buffer components to compensate for the increase in electrical current. In the same work, modifications were also required to achieve compatibility with MS detection where nonvolatile salts, urea, and amines should be usually avoided. A physically adsorbed polyethylenimine-coated capillary was used to overcome protein adsorption to the capillary walls in the absence of cationic additives and the use of an acetate buffer at pH 5.05 allowed the partial resolution of at least five bands of rhEPO. Other types of coated capillaries have been used for the analysis of EPO by CE-MS as detailed in Section 22.4.3.3 [30,37,42,62,96]. 

Plots of phase angle difference in the interferometer arms vs. time are related to heat-production vs. time, and this in turn is related to the concentration of the species responsible for heat production. Typical instrument output for the urea/urease system is shown in Figure 3. Calibration curves can be constructed as shown in Figure The system is quite stable, and reasonably sensitive. Minimum detectable levels of urea are 5 mM, compared to the 0.1-5 mM limits for traditional detectors. Over extended time periods (7 days) the relative standard deviation at 5 mM concentrations is better than 5 /.. The optimum FIA conditions were around 1.0 ml/min flow rate, with a sample loop of 0.1-0.25 ml. 

The performance of several column packings has been assessed and it has been stressed that low eluent flow rates are necessary for high performance separation. The effects of water contamination in eluents has been studied by Berek et a/. " highlighting the need for rigorou dried systems. Phase equilibria studies in polymer-polymer-solvent systems have proved feasible using a dual detection system and could be extended in the future. Other applications are concerned with copolymer analysis, polydispersity, oligomers, and melamine-formaldehyde and urea— and phenol-formaldehyde resins. New techniques, recycle liquid SEC, phase-distribution chromatography, and the measurement of diffusion coefficients from GPC have been described. 

Scheme 27.1 Design of CL-based detection systems for vitamin Bn analysis. The addition of sequential addition of luminol (5gM, 50 gL), vitamin Bn (lOpg/mL, 100gL) and urea-H202 (0.1 mM, 100gL) at 60-s intervals produces CL signals that were analysed using a photomultiplier tube-based luminometer (Sagaya et al. 2009).

It is worth to notice, that this cahbiation curve is obtained for SNARF-1 dextran/ urease capsule in pure water without substantial contamination of aity salt, which could bulFer the systems and spoil truly picture for urea detection. We carried out experiments to build a similar calibration curve in the presence of the 0.001 M TRIS-maleate buffer (were used solutions with the pH 6.5 and 7.5) but it resulted in overwhelming effect of pH buffering. Buffering the solution eliminates the pH change caused in a course of enzymatic reactions. Thus, it sets a hmit for detection of urea concentration using SNARF-l-dextran/urease capsules. However the calibration in conditions of particular experimental system is reasonable at salt free solution assumption. Summarizing, one can state the presented in Fig. 20.7 calibration curve as suitable for estimation of urea concentrations in-situ in water solutions. 

A simple, fast and specific color test for urea nitrate was reported recently by Almog et al. It is based on the reaction between urea nitrate and ethanolic solution ofp-dimethylaminocinnamaldehyde (p-DMAC) (9) under neutral conditions [91]. A red pigment is formed within 1 min from contact. Its structure has also been determined by the same group, by X-ray crystallography [92]. It appears to be a resonance hybrid between a protonated Schiffbase (10) and a quinoid system (10a) (Eq. (14)). The limit of detection on filter paper is 0.1 mg/cm. Urea itself, which is the starting material for urea nitrate, does not react with p-DMAC under the same conditions. Other potential sources of false-positive response such as common fertilizers, medications containing the urea moiety and various amines, do not produce the red pigment with p-DMAC. 

The sensing microzone of the flow-through sensor depicted in Fig. 5.9.B1 integrates gas-diffusion and detection with two analytical reactions [28], viz. (a) the urease-catalysed formation of ammonium ion by hydrolysis of urea (the analyte), which takes places on a hydrophilic enzyme membrane in contact with the sample-donor stream, which contains a gel where the enzyme is covalently bound and (b) an acid-b reaction that takes place at the microzone on the other side of the diffusion membrane and involves Bromothymol Blue as indicator. This is a sandwich-type sensor including a hydrophilic and a hydrophobic membrane across which the sample stream is circulated —whence it is formally similar to some enzyme electrodes. Since the enzymatic conversion of the analyte must be as efficient as possible, deteetion (based on fibre optics) is performed after the donor and acceptor streams have passed through the sensor. Unlike the previous sensor (Fig. 5.9.A), this does not rely on the wall-jet approach in addition, each stream has its own outlet and the system includes two sensing microzones... 

Male Fischer 344 rats were exposed by inhalation to 1% 2-chloro-1,1,1 -trifluoroethane for 2 h and then urine was collected for 24 h. Urinary metabolites identified by 19F nuclear magnetic resonance and gas chromatography/mass spectrometry were 2,2,2-trifluoroethyl glucuronide (16%), trifluoroacetic acid (14%), trifluoroacetaldehyde hydrate (26%), trifluoroacetaldehyde-urea adduct (40%) and inorganic fluoride (3%). A minor, unidentified metabolite was also detected. No covalent binding of fluorine-containing metabolites was observed in the liver and kidney from the exposed rats (Yin et al., 1995). In-vitro incubation of 2-chloro-1,1,1-trifluoroethane with rat liver microsomes and an NADPH-generating system has been shown to involve a dechlorination reaction (Salmon et al., 1981) that produced trifluoroacetaldehyde hydrate as the only metabolite (Yin et al., 1995). 

The results show that a number of ruthenium carbonyl complexes are effective for the catalytic carbonylation of secondary cyclic amines at mild conditions. Exclusive formation of N-formylamines occurs, and no isocyanates or coupling products such as ureas or oxamides have been detected. Noncyclic secondary and primary amines and pyridine (a tertiary amine) are not effectively carbonylated. There appears to be a general increase in the reactivity of the amines with increasing basicity (20) pyrrolidine (pKa at 25°C = 11.27 > piperidine (11.12) > hexa-methyleneimine (11.07) > morpholine (8.39). Brackman (13) has stressed the importance of high basicity and the stereochemistry of the amines showing high reactivity in copper-catalyzed systems. The latter factor manifests itself in the reluctance of the amines to occupy more than two coordination sites on the cupric ion. In some of the hydridocar-bonyl systems, low activity must also result in part from the low catalyst solubility (Table I). 

The objective is to describe a new non-enzymatic urea sensor based on catalytic chemical reaction. The sensor consists of screen-printed transducer (IVA, Ekaterinburg, Russia) and catalytic system which is immobilized on the transducer surface as a mixture with carbon ink. The sensor is used for measuring concentration of urea in blood serum, dialysis liquid. Detection limit is 0.007 mM, while the correlation coefficient is 0.99. Some analysis data of serum samples using the proposed sensor and urease-containing sensor (Vitros BUN/UREA Slide, Johnson Johnson Clinical Diagnostics, Inc.) are presented. 

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