Determination of Urea

The substance is employed for the determination of urea and for the characterisation of amides (compare Section 111,110). 

Martinez-Eabregas, E. Alegret, S. A Practical Approach to Ghemical Sensors through Potentiometric Transducers Determination of Urea in Serum by Means of a Biosensor, /. Chem. Educ. 1994, 71, A67-A70. 

A new kinetic enzymatic method for the routine determination of urea in semm has been evaluated. This method is based upon an enzymatic reaction and formation of a coloured complex. The method is based on a modified Berthelot reaction. The reaction was monitored specRophotomebically at 700 nm (t = 25 0.1 °C). The optimal pH value, chosen for the investigation of complex, is 7.8. 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Hydrolase enzymes catalyze the hydrolysis of a substrate and are most commonly coupled with potentiometericela trodes The pioneering work in this field focussed on de loping an enzyme electrode for the determination of urea. Urease catalyzes the hydrolysis of urea to ammonium and bicarbonate ions according to the reaction detailed below. 

Koebel, M. and Elsener, M. (1995) Determination of Urea and its Thermal Decomposition Products by High-Performance Liquid Chromatography, J. Chromatogr., 689, 164. 

As the one of the main end products of protein metabolism in living organisms, urea is a primary source of organic nitrogen in soil (from animal urine, fertilizers, etc.). Monitoring the level of urea is important for medicine, as well as for environmental protection. Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia and water. This enzyme is widely used for determination of urea in... 

Emmet RT (1969) Spectrophotometric determination of urea in natural waters with hypochlorite and phenol. NAVSHIPRANDLAB Annapolis Report 2663... 

Despite the advances made in high-performance liquid chromatography in recent years, there are still occasionally applications in which conventional column chromatography is employed. These methods lack the sensitivity, resolution and automation of HPLC. They include the determination of urea herbicides in soil, polyaromatic hydrocarbons, carbohydrates, chloroaliphatic compounds and humic and fulvic acids in non-saline sediments. The technique has also been applied in sludge analysis, e.g. aliphatic hydrocarbons and carboxylic acids. 

J.C. Oxley, J.L. Smith and S. Naik, Determination of Urea Nitrate and Guanidine Nitrate Vapour Pressures by Isothermal Thermogravimetry , submitted to Propellants, Explosives, Pyrotechnics. 

Among potentiometric enzyme sensors, the urea enzyme electrode is the oldest (and the most important). The original version consisted of an enzyme layer immobilized in a polyacrylamide hydrophilic gel and fixed in a nylon netting attached to a Beckman 39137 glass electrode, sensitive to the alkali metal and NHj ions [19, 2A Because of the poor selectivity of this glass electrode, later versions contained a nonactin electrode [20,22] (cf. p. 187) and especially an ammonia gas probe [25] (cf. p. 72). This type of urea electrode is suitable for the determination of urea in blood and serum, at concentrations from 5 to 0.05 mM. Figure 8.2 shows the dependence of the electrode response... 

Figure 5.19 — Flow-through biochemical sensor based on the twofold immobilization of the catalyst (urease) and reagent (an acid-base azo dye) in the sensing microzone for the determination of urea in kidney dialysate. (A) Sensing microzone held in a microcircuit. (B) Valveless flow injection manifold. P pumps T timer S sample W waste. For details, see text. (Reproduced from [57] with permission of Elsevier Science Publishers).

Trichosporon cutaneum [89]. The principle of this method is based on the measurement of the rapid alteration (or acceleration) of respiration after addition of ammonium ions in the presence of glucose. The physiological background of this principle is probably the uptake of ammonium ions in connection with the respiration process. Finally, the combination of nitrifying bacteria with urease on a hybrid sensor also allows an amperometric determination of urea [93]. 

Xanthydrol is employed as a reagent for the determination of urea, with which it forms an insoluble condensation product. The material obtained is entirely satisfactory for this purpose if desired, however, it may be recrystallized from alcohol, whereupon the melting point is raised by about i°. 

UREASE. Enzyme present in low-percentages in jackbean and soybean water soluble, its action is inhibited by heavy-metal ions. Its principal use is in the determination of urea in urine, blood, and other body fluids it splits urea into ammonia and carbon dioxide or ammonium carbonate. 

Several cleanup methods have been developed for the determination of urea pesticides, involving different basic procedures, such as liquid-liquid partition (30-32,34,36,37), steam distillation (31), and liquid-solid chromatography (9,30,32,34,36,38,56-58). Different factors, e.g., water solubility, ionic and polarity properties, thermal stability, and the molecular weight of the compounds, determine the choice of the cleanup method. Moreover, micro-cleanup procedures and online enrichment techniques have been introduced for the automated determination of phenylureas (60). Table 6 summarizes the use of the different cleanup procedures in the determination of urea pesticides. 

Table 6 Cleanup Procedures for Sample Extracts Used in the Determination of Urea Pesticides...

Table 9 HPLC Determination of Urea Pesticides in Food Samples...

Determination of urea pesticides has been performed by gas chromatography (37,56,160, 161), a technique that has highly sensitive and selective detectors. Nevertheless, some substituted ureas are thermolabile and decompose during the analysis by GC (102). Therefore, direct determination of unchanged compounds by GC is possible only for some urea pesticides under determined chromatographic conditions (161). In other cases, a degradation product is quantified instead of the parent compound (29,162-164), or these substituted ureas must be derivatized before GC determination (37,102). 

Direct determination of urea pesticides by high-performance liquid chromatography has been widely reported in the literature (10,32-36,127-130). Ultraviolet detection has often been used (32,33,35,36,60,127) with usually acceptable sensitivity, although this detector is nonspecific and the sensibility is, in general, low. To overcome this problem, several techniques have been assayed, such as precolumn enrichment (60), postcolumn derivatization (34,10), and the use of other detection techniques such as the electrochemical (129), photoconductivity (128,130), and fluorescence detectors (9,10,34). Table 9 summarizes representative papers using these techniques in HPLC analysis. 

The successful combination of mass spectrometry with gas chromatography (GC-MS) and, subsequently, with liquid chromatography (HPLC-MS) allowed not only the determination of urea pesticides in food but also the identification of their residues at trace level. Mass spectrometry is a technique that can be used as a general detector, with cyclic scanning. The selectivity and sensibility of analysis can be enhanced using characteristic ions of the molecule, with selected ion monitoring (SIM). Urea pesticides have been determined by HPLC-MS directly (175-180), without the thermal instability problems of GC analysis. 

For monitoring catalytic (enzymatic) products, various techniques, such as spectrophotometry [32], potentiometry [33,34], coulometry [35,36] and amperometry [37,38], have been proposed. An advantage of these sensors is their high selectivity. However, time and thermal instability of the enzyme, the need of a substrate use and indirect determination of urea (logarithmic dependence of a signal upon concentration while measuring pH) cause difficulties in the use and storage of sensors. 

Urea Nitrogen Macro and Micro. The flow diagram of the urea nitrogen method is shown in Fig. 18. The method originally used for citrulline (FI) has been adapted to the determination of urea (Ol). Although the method has had limited success in routine clinical chem-... 

Ml. Marsh, W. H., Fingerhut, B., and Kirsch, E., Determination of urea nitrogen with the diacetyl method and an automatic dialyzing apparatus. Am.. Clin. Pathol. 28, 681-688 (1957). 

Ol. Ormsby, A. A., Direct colorimetric method for determination of urea in blood and urine. J. Biol. Chem. 146, 595-604 (1942). 

Urastrat a complete quantitative system for the determination of urea nitrogen in serum Marketed by General Diagnostics, Warner-Lambert (UK) Ltd, Eastleigh, Hampshire, UK... 

Goeyens, L., Kindermans, N., Yusuf, M. A., and Elskens, M. (1998). A room temperature procedure for the manual determination of urea in seawater. Estuar. Coast. Shelf Sci. 47, 415—418. 

Mulvaney, R. L., and Bremer, J. M. (1979). A modified diacetyl monoxime method for colorimetric determination of urea in soU extracts. Commun. Soil Sci. Plant. Anal. 10, 1163—1170. 

Mulvenna, P. F., and Savidge, G. (1992). A modified manual method for the determination of urea in seawater using diacetylmonoxime reagent. Estuar. Coast. Shey Sci. 34, 429—438. 

Urine The slides for the determination of urea from the serum can also be used for determination from urine. However, the urine samples must be diluted 1 20 with deionised water before analysis (E59, E88, El 16). 

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