Elution chromatography concentration-gradient

The acceptable separation of Am(III) and Cm(III) by countercurrent chromatography (CCC) was achieved using both isocratic elution, and a new approach to the creation of the reagent concentration gradient in the stationary phase [1]. This way allows reduce the experiment length. 

The column is eluted by gradient elution the concentration of acetone in water increases almost linearly from 0 to 95%. Gibberellins A8, Alf A6, and A5 were eluted with the following percentages of acetone in water 27 to 38, 38 to 41, 43 to 49, and 51 to 56%, respectively. The gibberellins are obtained pure after further chromatography on a column of silicic acid and Celite, followed by crystallization. 

Fig. 4. Chromatography of dog pancreatic juice on DEAE-cellulose (13, 14). The column is equilibrated with 0.005 M phosphate, pH 8.0 and eluted by a concentration gradient of phosphate indicated in the figure by a straight line. (1) Unfractionated cationic proteins (2) amylase (3) lipase (4) deoxyribonuclease (5) anionic chymotrypsinogen (6), (9), and (10) carboxypeptidase A and its precursor (7) and (8) carboxypeptidase B and its precursor. Ordinates on the left, optical density of the fractions at 280 m/ . Ordinates on the right, molarity of the phosphate. Abscissas, volume of eluate expressed in number of interstitial volumes of the column.

Ion-exchange chromatography DEAE-Sephadex elution via pH gradient. Fractions 50-60, 5 ml each, pooled, dialyzed, and concentrated 25 9 225 88 9.8 2200 44 23.6... 

Ion exchange chromatography DEAE-Sephadex elution via KC gradient. Fractions 21-31, 2 ml each, pooled and concentrated 5 1 35 364 52 1820 36.4 125... 

The observations discussed above may have implications for any system where water, dissolved salts and hydrophobic entities are present and there must be many. Currently the separation of hydrophobic proteins can be achieved using a hydrophobic chromatography column, by elution with salt solutions. There is no adequate theory for this process and present understanding is purely empirical. Suppose then, that while all salts reduce electrostatic forces, only those salts that reduce bubble coalescence also reduce the hydrophobic attraction. Further, these salts have a significant effect on the hydrophobic attraction only above their transition concentration. With this notion in mind the experimental results are explained. This then enables separations to be simplified, as the salt type and concentration gradient required are easily determined. 

Icq and m in Eq. 5 depend on the nature of the solute and on the chromatographic system, but are independent of the concentration of the strong solvent B v in the mobile phase. Assuming the validity of Eq. 5 in NP gradient chromatography with hnear concentration gradients of a polar solvent B, the elution volume Vr of a sample solute can be calculated from Eq. 

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