Elution lower

Figure 18.27 Comparison of the performance of displacement and elution chromatography. Maximum production rate in the separation of a certain mixture on a given column 25-cm- long column packed with 20 im particles. 1 3 mixture, a. = 1.20 k2 = 9.78. Top plot of maximum production rate of second component versus reduced velocity of the mobile phase. Figures by the symbols on the lines (upper line, elution, lower line, displacement) are the concentration of the collected fractions. Bottom recovery yield of the second component. Reproduced with permission from Katti et ah, J. Chrorrmtogr., 540 (1991) 1 (Fig. 10).

The chromatographic separation should whenever possible be completed in one operation. If, however, shortage of time necessitates an interruption, this can most conveniently be made immediately after the first band has been completely eluted, whereupon the lower end of the tube is closed by a short piece of rubber tubing carrying a screw-clip. Great care should be taken however not to allow even the top of the column to run dry. 

When the solvent has nearly reached the top of the adsorbent layer, the components should be well separated. The relative distance travelled by the components can be increased by using a solvent of higher or lower polarity in the order of increasing eluting power ... 

Purification of anthracene. Dissolve 0-3 g. of crude anthracene (usually yellowish in colour) in 160-200 ml. of hexane, and pass the solution through a column of activated alumina (1 5-2 X 8-10 cm.). Develop the chromatogram with 100 ml. of hexane. Examine the column in the hght of an ultra-violet lamp. A narrow, deep blue fluorescent zone (due to carbazole, m.p. 238°) will be seen near the top of the column. Immediately below this there is a yellow, non-fluorescent zone, due to naphthacene (m.p. 337°). The anthracene forms a broad, blue-violet fluorescent zone in the lower part of the column. Continue the development with hexane until fluorescent material commences to pass into the filtrate. Reject the first runnings which contain soluble impurities and yield a paraffin-hke substance upon evaporation. Now elute the column with hexane-benzene (1 1) until the yellow zone reaches the bottom region of the column. Upon concentration of the filtrate, pure anthracene, m.p. 215-216°, which is fluorescent in dayhght, is obtained. The experiment may be repeated several times in order to obtain a moderate quantity of material. 

The elution volume, F/, and therefore the partition coefficient, is a function of the size of solute molecule, ie, hydrodynamic radius, and the porosity characteristics of the size-exclusion media. A protein of higher molecular weight is not necessarily larger than one of lower molecular weight. The hydrodynamic radii can be similar, as shown in Table 4 for ovalbumin and a-lactalbumin. The molecular weights of these proteins differ by 317% their radii differ by only 121% (53). 

The behavior predicted by this equation is illustrated in Fig. 16-33 with N = 80. Xp = (Evtp/L)/[il — )(p K -i- )] is the dimensionless duration of the feed step and is equal to the amount of solute fed to the column divided by tne sorption capacity. Thus, at Xp = 1, the column has been supplied with an amount of solute equal to the station-aiy phase capacity. The graph shows the transition from a case where complete saturation of the bed occurs before elution Xp= 1) to incomplete saturation as Xp is progressively reduced. The lower cui ves with Xp < 0.4 are seen to be neany Gaussian and centered at a dimensionless time - (1 — Xp/2). Thus, as Xp 0, the response cui ve approaches a Gaussian centered at Ti = 1. 

It has been seen that this resin has also some important advantages over the other resins in the literature like high total ion exchange capacity, easy synthesis, lower cost, simple regeneration. Furthermore, very good sepai ations were obtained using a concentration gradient of elution. In these elutions, very low concentrations of sodium trimetaphosphate were used. As a result, the resin synthesized can be used as an adsorbent for the effective removal of Pb, Cd, Co, Cu, Fe, Ni, Zn and Cr from aqueous solutions. 

This, as is shown by the theory, is due to the evolution of the heat of absorption, during solute adsorption at the front part of the peak. Conversely, the back of the peak is eluted at a lower temperature than the surroundings throughout the length of the column due to the absorption of the heat of solute desorption. As a result, the distribution coefficient of the solute at the front of the peak, and at a higher temperature, will be less than the distribution coefficient at the back of the peak, at the... 

The main product, benzene, is represented by solute (B), and the high boiling aromatics are represented by solute (C) (toluene and xylenes). The analysis of the products they obtained are shown in Figure 12. The material stripped form the top section (section (1)) is seen to contain the alkanes, alkenes and naphthenes and very little benzene. The product stripped from the center section appears to be virtually pure benzene. The product from section (3) contained toluene, the xylenes and thiophen which elutes close to benzene. The thiophen, however, was only eliminated at the expense of some loss of benzene to the lower stripping section. Although the system works well it proved experimentally difficult to set up and maintain under constant operating conditions. The problems arose largely from the need to adjust the pressures that must prevent cross-flow. The system as described would be virtually impossible to operate with a liquid mobile phase. 

The temperature must be raised when there is no solvent that can dissolve samples at ambient temperature. For example, polyolefines such as polyethylene and polypropylene are usually analyzed at 130-140°C because no solvent can dissolve these polyolefines at lower temperatures. It is also preferable to perform analyses at elevated temperatures when the viscosity of the elution solvent is considerably higher at ambient temperature. However, a temperature around 25-40°C is recommended when good solvents having low viscosity are available at such a temperature. It is much more convenient to operate a GPC instrument at 25-40°C than to operate at higher temperatures. 

A SEC material should be hydrophilic if it is to be used for biological applications. One such material, introduced by PolyLC in 1990 (8), is silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) the trade name is PolyHYDROXYETHYL Aspartamide (PolyHEA). This material was evaluated for SEC of polypeptides by P.C. Andrews (University of Michigan) and worked well for the purpose (Fig. 8.1). Because formic acid is a good solvent for polypeptides, Dr. Andrews tried a mobile phase of 50 mM formic acid. The result was a dramatic shift to a lower fractionation range for both Vq and V, (Fig. 8.2) to the point that V, was defined by the elution position of water. 

Eluent. The solubility of the sample determines the elution solvent for the GPC experiment the better the solubility the lower the danger of undesirable... 

FIGURE 19.1 Theta condition effects on pMMA elution. Upper trace acetonitrile 65°C Lower trace THF 35°C... 

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