Hidrológiai Közlöny 1967 (47. évfolyam)

7. szám - A „Szervesanyag meghatározási problémák édesvizekben” című 1966. szeptember 25–28. között Tihanyben rendezett Szimpózium előadásai - Shapiro, Joseph: Különböző tavakból száramzó szervesnyagok összehasonlítása

Shapiro, J.: Különböző tavakból származó szervesanyagok Hidrológiai Közlöny 1967. 7. sz. 291 than the not heated sample is that a portion of the material forming this peak is retained in the butanol during back extraction into ammónia. The conclusion is that samples should be dried at as low a temperature as possible, but the fractions are not normally the result of this procedure. Eííect of Ion Exchange and Freeze Concentration The samples used in the above experiments were obtained from laké water whieh had been ion exchanged and freeze concentrated. Therefore an experiment was done in whieh these steps were omitted i. e. the yellow acids were extracted directly from a laké water sample whieh had been merely filtered and adjusted to pH 1. The extract was dried, dissolved in ammónia, and applied to the column. Figure 5 shows the result. The peaks are clearly not the result of ion exehange and freeze concentration. Table 1. Effects of different procedures on shape of Sephadex elution curves Sample Planimeter reading of fraction as % of the totál 1 2 3 4 1.0 mg in 1.5 mg in 3.0 mg in 100 A, 21 ml/hr 100 A, 21 ml/hr 100 A, 21 ml/hr 64.1 62.9 60.8 17.6 16.6 18.1 12.3 15.0 14.2 6.1 6.2 6.8 1.5 mg in 1.5 mg in 300 A, 21 ml/hr 100 A, 8 ml/hr 64.3 64.5 15.1 14.1 13.2 13.2 7.7 8.2 HISH Molecular Weight Fig. .5. Elution pattern of an extract of a water sample that had not been ion exchanged or concentrated Eífect oí Application and Elution Procedures As it was intended to use the data quantitatively, it was important to determine wether the shape of the curve i. e. relatíve proportions of different components, is affected by operational procedures. Figure 6 shows the effect of a threeíbld variation in weight of sample applied to the column, other factors being equal (100 X applied and elution rates of 21 ml/hr). In two other experiments the volume applied and elution rates were alsó varied three-fold. Results of all these experiments are shown in Table 1. Apparently the changes in procedure affect the proportions of the components only slightly if at all. In any event, all further experiments were done at an application volume of 100 X using about 1 mg of material and an elution rate of about 21 ml/hr. Eííect of Column Characteristics In order to further insure the validity of a particular elution pattern it was necessary to demonstrate that its shape is not a function of a particular column, but that it could be duplicated on another column of the same grade of Sephadex of the same dimensions. Figure 7 is a comparison of two elution curves of the same extract. These were done on different columns whieh were prapared several months apart. The upper curve is somewhat more sharplv defined but there is no questioning the close similarity of the two. HISH Molecular Weight LOW niun . ! LUH Molecular Weight Fig. 6. Elution patterns of different qnantities of the same extract Fig. 7. Elution patterns of the same extract run on two similar columns prepared on different days Separation oí Components Finally, to show that the peaks represented true components of defined molecular weight, fraetions were taken during elution, dried, and reapplied singly to the same column. The results in Figure 8 show that the individual fractions are remarkably diserete. It is likely that by re-cutting these fractions to eliminate traces of other components, even better defined components could be obtained. It should be pointed out that in other experiments of this type where the columns were overloaded the results were much less satisfactorv.