Stirring semi-micro

By analogy, the anion exchangers like Amberlite IR-4 should bind preferentially the dicarboxylic acids. This is also the case. A mixture of amino acids and hydrochloric acid, which is stirred for 1 to 3 hours with sufficient resin IR-4 to give a final pH of 6 to 7, will adsorb completely the acidic amino acids and leave the neutral and basic amino acids unaffected (Cannan 1944, Cleaver et al., 1945). On the pure fraction eluted with HCl from the resin, Cannan determined the aspartic and glutamic acids separately by a combination of electrometric titrations in water and formaldehyde solutions and a gasometric ninhydrin estimation—a method well suited for work on a semi-micro scale. 

The calcein release from the vesicles was measured as follows the vesicle suspensions of HCO-10 were prepared in 100 mM calcein/20mM Tris buffer (pH 7.4, 388 mOsm), in which the fluorescence intensity of calcein is self-quenched. The vesicles were separated from un-trapped calcein by gel-permeation chromatograph using a Sephadex G-75 gel (0.5 x10 cm column) equilibrated with an isotonic buffer, glucose/20mM Tris buffer (pH 7.4). The osmolarity of buffers was monitored with an Osmometer (Semi-micro Osmometer, Knauer). The separated vesicles, suspended in a cold isotonic buffer, were rapidly diluted (1 200) into the well-stirred isotonic buffer equilibrated to the experimental temperature. The fluorescence emission intensity of a sample was recorded continuously subsequent to this dilution (zero time) on a RF-5000 fluorescence spectrophotometer, Shimadzu (excitation A = 490 nm, emission X = 520 nm) equipped with temperature control accessories and a magnetic stirrer [11]. 

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