Column loading sequence

The Fur protein from E. coli was isolated in one step due to its high affinity for metal-chelate columns loaded with zinc. In DNase footprinting experiments, the Fur protein was shown to bind DNA in the promoter region of several iron-regulated genes. The consensus sequence, called the Fur box, is GATAATGATAATCATT ATC. In vitro binding is dependent on the divalent cations Co2+ Mn2+ /s Cd2+ Cu2+ at 150 iM, while Fe2+ seemed to be less active at this concentration, probably due to oxidation to Fe3+ (De Lorenzo et al., 1987). The unspecificity for divalent metals observed in vitro shows that the cells have to select the ions transported carefully and have to balance their active concentrations. In addition, it is a caveat for the experimenter to test a hypothesis on metal-ion specificity not only in vitro, but also in vivo. 

In a three column system, on Column One the column operational sequence will be sample, flush, equilibrate, and then on Column Two one will have to equilibrate, sample, flush and to set up Column Three in sequence, one is going to equilibrate through the first two steps and then load sample and then flush. Note that the product is continuously coming out of the system after each sequence. This is different than if one had a single column and one had to wait for longer periods of time between events. It allows optimum utilization of downstream recovery equipment with less batch operation. 

Loading Sequence. Two similar columns of heparin-PVA beads were prepared ( ). The first column was loaded with thrombin followed by antithrombin III the sequence was reversed for the second column. The thrombin load was either crude bovine (62 U, 18 nmoles), pure bovine (23 U, 0.35 nmoles), or pure human (1072 U, 9.4 nmoles). The antithrombin 111 load was either crude human (3 mg 48 nmoles, 15 mL of defibrinated plasma) or purified human (1.5 mg, 24 nmoles, 0.29 mg/mL). After loading each protein, 100 mL of PBS was passed through the column and the residual thrombin activity measured by the chromogenic substrate method. 

Fig.4.6 Schematic diagram of a FI manifold lor high efticiency on-line column preconcemra-tion for flame AAS with countercurrent elution, a. sample loading sequence b. elution sequence. P]. P peristaltic pumps E. eluent S. sample B. buffer/reagent C. conical column V injector valve W. waste and AAS. flame AA detector [16].

Fig.4.7a-c a, b, schematic diagram of a dual column FI on-line preconcentiation manifold for flame AAS with parallel sample loading and sequential elution using two pumps, a, loading sequence b, elution sequence for column Ca. Pi, Pn, peristaltic pumps Sa, Sb, samples Ea, Eb eluent (2 M HNO3) Ra, Rb, ammonium acetate buffer, T, timer for pump control V, 8-channel multifunctional valve Ca> Cb columns packed with chelating ion-exchangers W, Wa, Wb, waste flows Wa same waste line as Wa and AAS, flame AA detector [12]. 

Fig.4.9 Schematic diagram of a dual column FI on-line preconcentration manifold for vapour generation AAS with parallel column loading and sequential elution, a, elution sequence for column CA b. loading sequence. V, 8-channel multifunctional valve (missing channels in figure are blocked) Vjj, 2-way valve for controlling column elution sequence. SA. SB, samples B, buffer, E, eluent R, reductant SP, gas-liquid seperator. A, quaitz tube atomizer. Ar, argon flow W, waste [26].

To calculate the vapor load for a single column of a sequence, start by assuming a feed condition such that q can be fixed. Initially assume saturated liquid feed (i.e., q = 1). Equation (5.1) can be written for all NC components of the feed and solved for the necessary values of 0. There are (JVC - 1) real positive values of 0 which satisfy Eq. (5.1), and each lies between the a values of the... 

When the integration of sequences of simple columns was considered, it was observed that sequences with higher heat loads occurred simultaneously with more extreme levels. Heat integration always benefits from low heat loads and less extreme levels, as we shall see later in Chap. 12. Now consider the effect of thermal coupling arrangements on loads and levels. Figure 5.18 compares a... 

Establish simple sequences. Using methods described in Chap. 5, sequences of simple columns with low overall vapor load are established. Consideration should not be restricted to the single sequence with the lowest overall vapor load, since many factors need to be considered in finally arriving at the best design. 

The calculation is repeated for all columns in the sequence and the vapor loads summed to obtain the overall vapor load for the sequence. Different sequences can then be compared on the basis of total vapor load. 

Example 11.2 Using the Underwood Equations, determine the best distillation sequence, in terms of overall vapor load, to separate the mixture of alkanes in Table 11.2 into relatively pure products. The recoveries are to be assumed to be 100%. Assume the ratio of actual to minimum reflux ratio to be 1.1 and all columns are fed with a saturated liquid. Neglect pressure drop across each column. Relative volatilities can be calculated from the Peng-Robinson Equation of State with interaction parameters assumed to be zero (see Chapter 4). Determine the rank order of the distillation sequences on the basis of total vapor load for ... 

Solution Figure 11.25 shows three sequences that reduce the vapor load compared with the best sequence of simple columns. The performance of the three sequences is effectively the same, given the assumptions made for the calculations. The total vapor load is around 10% lower than that the best sequence of simple columns from Table 11.5. The differences between networks of simple and complex column in terms of the vapor load vary according to the problem and can be much larger than the result in this example. 

In addition to these issues regarding constraints for simple columns, there is also the issue of the introduction of complex columns into the sequence. Figure 21.13a illustrates the thermal characteristics of a direct sequence of two simple columns. Once the two columns are thermally coupled, as illustrated in Figure 21.13b, the overall heat load is reduced. However, all of the heat must be supplied at the highest temperature for the system. Thus there is a trade-off in which the load is reduced, but the levels required to supply the heat become more extreme. The corresponding case for the indirect sequence is shown in Figure 21.14. As the indirect sequence is thermally coupled, the heat load is reduced, but now all of the heat must be rejected at the lowest temperature. Thus, there is a benefit of reduced load but a disadvantage of heat rejection at more extreme levels. The same problem occurs with... 

Distillation sequencing. The separation of homogeneous nonazeotropic mixtures using distillation usually offers the degree of freedom to choose the distillation sequence. The choice between different sequences can be made on the basis of total vapor load, energy consumption, refrigeration shaft power for low-temperature systems, or total cost. However, there is often little to choose between the best few sequences in terms of such measures of system performance if simple distillation columns are used. 

The prepared sample may be applied in pH 2.2 buffer directly to the top of the column and the analysis sequence started, and after elution of all the amino acids and regeneration of the resin column, the next sample can be applied. However, many newer models incorporate an automatic loading device which enables several samples to be stored ready for analysis either in sample cups or in small Teflon coils. After the completion of an analysis the next stored sample is automatically applied to the resin and the buffer cycle restarted. 

The six sequencing heuristics are formulated to reduce the separation load on downstream columns, favoring easier separations early and difficult separations in the absence of nonkey components. If only two products are to be derived from a mixture and all of the components in one product are more volatile than all of the components in the other product, then the next split should divide the mixture into the two products. The presence of hazardous or corrosive materials can gready increase costs, and such components should be removed as early as possible. The most plentiful product in a mixture should be removed (if it can be) with one separation and if the relative volatility is favorable. Direct sequences, ie, removing a light product as distillate, generally are favored over indirect sequences, ie, removing a heavy product as bottoms. If no product dominates the feed composition, then separations that yield approximately equimolar splits are favored. Only if no other heuristic applies should the easiest separation be performed next. 

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