Separation and determination of cobalamins on an SP-Sephadex column

✍ Scribed by G. Tortolani; P. Bianchini; V. Mantovani

• Publisher: Elsevier Science
• Year: 1970
• Tongue: English
• Weight: 282 KB
• Volume: 53
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(01)98517-6

✦ Synopsis

Separation of isopro~yl~zoradr~tzaline and the corres+on&zg

O-methyl derivative a&g n tadem chromatografih'ic jwocedwe on celluloso~ltos~Jzate and carboxymetlgvlcellzclose. For the separation of both catecholamines from epinephrine and norepinephrine two columns 0.6 x 40 cm combined in series were used. These columns were filled up to 35 cm with cellulosophosphate Whatman CP II (Column No I) and carboxymethyl cellulose Whatman CM 32 (Column No II). Columns were eluted by a linear gradient of ammonium acetate buffer with a concentration change from 0.05 to 0.25 nd. The pH of the buffer was adjusted previously to 6.1; flow rate on both columns was less than 0.8 ml/min., Fractions of 2 ml were collected. Columns were loaded with 0.5 ml of a sample obtained after either purification procedure. The elution of the column was stopped after IOO ml of the eluant had passed through the system. Under these conditions 0-methylisopropylnoradrenaline is eluted in fractions No. 3-7, isopropylnoradrenaline in fractions No. 10-15 (see Fig.

Oxl'dation of isojwo;fiylnoradrenaline and its@metlzyl derivative to the corresponding luti~zes. Combined fractions 3-7 and 10-15 were evaporated to a final volume of 0.2 ml, diluted to I ml with borate buffer (0.66 M, pH 7) and the following reagents were added subsequently: 0.05 ml of 0.02 y0 CuCl,*zH,O and 0.05 ml of 0.25 y0 potassium ferricyanide. The reaction was stopped after 3 min by adding 0.05 ml of IO y0 BAL in 25 O/~ formaldehyde. After 10-20 set 0.2 ml of IO N NaOH was added, and after another 5 min 0.15 ml of glacial acetic acid was pipetted into the sample. The reaction mixture was vigorously mixed and subjected to fluorimctric evaluation.