Mcllvain’s buffer

The tetracyclines are well known for their ability to form complexes with polyvalent cations. This property changes their solubility characteristics in the mobile solvents and often results in troublesome streaking. To overcome this difficulty, Selzer and Wright used paper dipped in Mcllvaine s buffer (pH 3.5) which contains citrate ions capable of binding the metallic ions. The chromatograms were developed with a mixture of nitromethane, chloroform, and pyridine (20 10 3) on paper still damp from the treatment with the buffer solution. 

For the same purpose, Kelly and Bryske used paper impregnated with 0.1N disodium ethylenediaminetetraacetate (EDTA) and two mobile solvents the organic phase from a mixture of n-butanol, ammonia, water (4 1 5) and the organic phase from a mixture of n-butanol, acetic acid, water (4 1 5). Disodium EDTA (0.1N) works as well as Mcllvaine s buffer when it is used to treat the paper in the method of Walton et al. (49). A circular paper chromatographic method also using paper dampened with Mcllvaine s buffer (pH 4.5) was reported by Urx et al. (50). They used a mixture of chloroform and n-butanol (4 1) as the mobile solvent. 

Fig. 2. Decay of Cp and growth of both Cp and Up as a function of time at pH at 25°C in about O.OlAf Mcllvaine s buffer (Johns, LeBlanc, and Freeman62).

Mcllvain s buffer concentrated Mcllvain s buffer is prepared by dissolving 6.558 g sodium phosphate and 5.162 g sodium citrate in 100 ml demineralized water. The pH is adjusted to pH 4.5. 

KH2P04/Na2HP04) or citrate/phosphate (Mcllvaine s) buffer, pH 4.0 to 6.5... 

Hold buffer at 30°C and aerate with a stream of bubbles via a fine capi llary tube. An aquarium aerator is a convenient way to achieve this. Select the buffer and pH to suit the enzyme being investigated. Citrate/phosphate (Mcllvaine s) buffer (appendix 2A)in the pH range of 4.0 to 6.5 is usually satisfactory for the assay of most DPOs. Recipes for a wide range of buffer solutions may be found in appendix 2a or in any standard textbook of practical biochemistry (for example, Dawson et al 1986). 

Stain slides for 30 min in quinacrine mustard solution (12 mg of quinacrine mustard dissolved in SO ml of H2O and diluted 1 1 with Mcllvaine s buffer). 

Rinse twice with Mcllvaine s buffer and once in water, air-dry and mount. 

DAPI (4, 6-diamidino-2-phenylindole) Prepare a stock solution of 100 pg/mL in water. DAPI is a potential carcinogen. To avoid weighing out the powder, order small quantities and use the whole vial to make the stock solution. Aliquot and store at -20°C (it is stable for years). Prepare a working solution of 2 pg/mL by dilution in Mcllvaine s buffer, aliquot, and store at -20°C (see Note 8). 

Mixtures of two systems are most difficult to treat by any means other than the graphical, or computer, methods based upon the n function. For a final example in this section, we look at such a mixture published in handbooks as Mcllvaine s buffers. 

A. Benzene-nitromethane-pyridine (10 20 3) Whatman No. 4 satd. with Mcllvaine s buffer, pH 3.5, ascending... 

B. Impregnated with Mcllvaine s buffer, pH 5.0, containing 10% w/v urea... 

Leningrad Quicky soaked with Mcllvaine s buffer, pH 4.5, and dried Solvent... 

D. Mcllvaine s buffer soln.. pH 4.6- -butanol-chloroform-methanol (5 3 1 1)... 

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