o-Phthalaldehyde, in the presence of 2-mercaptoethanol, reacts with primary amines to form highly fluorescent products. Picomole quantities of amino acids, peptides, and proteins can be detected easily. oPhthalaldehyde is five to ten times more sensitive than fluoresenamine and is soluble and stable in aqueous buffers.o-Phthalaldehyde, in the presence of 2-mercaptoethanol, has been described by Roth (1, 2) as a reagent for fluorometric detection of a-amino acids. However, Roth found that lysine and cystine showed only about 5% of the fluorescence of other natural amino acids, and that peptides also showed greatly reduced fluorescence values. Roth's work was confirmed by Taylor and Tappel (3), who also found that peptides yielded greatly reduced fluorescence with o-phthalaldehyde. Probably because of these shortcomings, the reagent has not been widely used for routine detection of primary amines.In this report we demonstrate that a modified formulation of Roth's reagent can be used to detect a-amino acids, peptides, and proteins in the picomole range. By increasing the concentration of 2-mercaptoethanol 10-fold and adding Brij to the reagent mixture, we have overcome most of the shortcomings described. In comparison with ninhydrin and fluorescamine, o-phthalaldehyde exhibits greater sensitivity.Udenfriend et al. (4) first described fluorescamine, a fluorogenic reagent that also allows high sensitivity detection of primary amines. They compared fluorescamine with ninhydrin, attempting to show the greater sensitivity of the fluorogenic reagent. Georgiadis and Coffey (5) later published their own comparison of ninhydrin and fluorescamine, and their results also imply much greater sensitivity with the fluorometric method. As has been observed by Hare (6), these data are misleading because the amino-acid analyses displayed were not performed on the same instrument. The analysis utilizing ninhydrin was performed using 9-mm diameter resin beds, whereas the analysis utilizing fluorescamine was performed with 2.8-mm diameter resin beds. The reduction in resin bed cross-sectional area alone would account for a 10-fold increase in sensitivity; consequently, the increase in sensitivity attributed to fluorescamine is exaggerated.We compared ninhydrin with fluorescamine and o-phthalaldehyde using a single analytical system. The 0.20 X 25-cm stainless steel column was filled with DC-4A cation exchange resin (Durrum Chemical Corp., Palo Alto, Calif.). A singlecolumn procedure was used with sodium formate and sodium borate buffer solutions in stepwise addition. Thick-walled polystyrene reservoirs, pressurized with nitrogen gas, were used to 619 drive the buffers through the resin bed (7 Fig. 1A. Full-scale deflection on the recorder corresponded to an absorbance of 1.0.For fluorometric detection, the ninhydrin system was replaced by an Aminco filter fluorometer (American Instrument Co., Silver Spring, Md.) with a 70-Mil flow cell. A Corning 7-51 filter was used for excitation, and a Wratten 2A filter was used for emissi...

Urea is currently considered to be a requirement for the propagation of T-strain mycoplasmas. We report here the replication of T-strain 960 (ATCC 25023) in media prepared from dialyzed components with added putrescine and allantoin but without added urea, or in dialyzed medium containing small amounts of added urea. The least amount of urea which allowed growth in the medium without allantoin was above 10 Ag/ml. The amount of urea estimated to contaminate the added allantoin or putrescine was 5 Ag/ml or less, which is insufficient to support T-strain replication. T-strain 960 was grown in the presence of urea and the urease inhibitor acetohydroxamic acid AHA where the organisms multiplied at a slower rate in the presence of AHA than in its absence. Urea hydrolysis occurred with concomitant ammonia accumulation and pH increase in cultures with AHA added. 765 on July 16, 2020 by guest

Polymeric columns are becoming used more frequently in modern liquid chromatography applications as improvements in polymeric packing materials are realized and as more applications are developed for these materials. Modern polymers have overcome earlier problems associated with their use; higher rigidity now allows them to be used at normal eluent flow rates resulting in faster analyses, and improved synthesis techniques have resulted in efficiencies comparable to the best silica materials, sometimes exceeding 100,000 plates/m. In addition, polymers offer distinct advantages over silica packing for particular applications. Because silica packings are readily degraded by aqueous eluents, they are not always suitable for separations involving ionic species. Polymeric columns are particularly useful in determinations of amino acids, peptides, proteins, organic acids, carbohydrates, and inorganic cations and anions. Polymers are also characterized by exceptional lot-to-lot reproducibility. Reproducibility is often problematic with silica packings, particularly the bonded phases.

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