SEPARATION - THE COLUMN

The chromatograph is built around the column, in which the actual separation takes place. The column accommodates the two chromatographic phases the stationary phase, which remains in the column, and the mobile phase, which is transported through it. Separation is achieved because different sample components (solutes) show different distributions over the two phases. A solute, having such a high affinity towards the stationary phase that it resides in this phase exclusively, will stay in the column indefinitely. A solute, that does not enter the stationary phase at all, will be transported through the column at the same speed at which the mobile phase is transported. In chromatographic terms, the latter is called an unretained solute. 

If a column is packed with porous particles, then an unretained solute is assumed to be swept through the entire volume of the column that is occupied by the mobile phase, either outside the particles or in the pores. A solute that does not enter any of the pores is called a (completely) excluded solute. Throughout the remainder of this book we will assume that the solutes will not be (partially or completely) excluded from the pores. 

---

As noted earlier, control of the column s temperature is critical to attaining a good separation in gas chromatography. For this reason the column is located inside a thermostated oven. In an isothermal separation the column is maintained at a constant temperature, the choice of which is dictated by the solutes. Normally, the tern-... 

At the end of the separation, the column is ready for the next batch. The resolution may well exceed the one typical in the preparative-scale SEC. 

Design a column for this separation. The column will operate at essentially atmospheric pressure. Use a reflux ratio 1.5 times the minimum. 

Purify the derivative by gel filtration using a PBS buffer or another suitable buffer for the particular protein being modified. The use of a desalting resin with low exclusion limits work well. To obtain complete separation, the column size should be 15-20 times the size of the applied sample. Fluorescent molecules often nonspecifically stick to the gel filtration support, so reuse of the column is not recommended. 

The idea is to compute the elements of T in such a way that the product UT results in zeros for the shaded part of C. In order to achieve this, we can separate the columns of C and treat them individually. The i-th column c j of C is the product of Uxt , , where tis the i-th column of T. 

In Figure 26-3, an [H+l gradient was used for a cation-exchange separation. The column for this separation has nitrilotriacetic acid groups that bind lanthanide cations in the order... 

Another way to improve the single-column process is to use a membrane to split the feed into two different feed streams (see Figure 10.15). Since the membrane does part of the separation, the column has to do less work to... 

HPLC Separations. The columns chosen for the FM0CC1 and OPA-MERC experiments were a result of several considerations and observations. Although all four columns used operated in the anion exchange mode only the silica particle columns (y Carbohydrate and UNH2) were successful in chromatographing GLYPH and AMPA, ostensibly due to the interaction of the fluorenyl moiety of the... 

The worksheet can be made easier to look at by adding lines that separate the column labels and numbers. To create a line under the labels one moves the cell pointer to cell C7, type then =. In LOTUS the backslash ( ) serves as a repeating label prefix. Whatever is typed after the backslash is repeated until it fills the cell. After pressing RETURN cell C7 now contains a row of equal signs ( = ). To continue the double line across the worksheet from cell C7 to cell 07 one can use the / Copy Command. 

We want to make the worksheet easier to look at by separating the columns for the variables by vertical lines. Therefore we have first to insert columns. Move the cell pointer to appear in cell J6 select / Worksheet Insert Column. 

However, for difficult separation (q > 0.60) this is reversed. Figures 3.18a and 3.18b clearly show that for q > 0.60 the column performance, in terms of minimum batch time, is improved significantly with decreasing plate holdup and suggests that for difficult separations the column holdup should be kept as minimum as possible. This is also clear from the results presented in Table 3.3 which show that for difficult separations optimum column holdup is very close to the minimum (Mujtaba and Macchietto, 1998 used 2% as the minimum column holdup). The minimum batch times for both cases (using minimum and optimum holdup) are almost alike and no time saving could be realized when compared to one another (last column of Table 3.3). The results discussed so far clearly show that holdup may have a dramatic effect on the operation. 

Reduction in hatch time For a given fresh feed and a given separation, the column performance is measured in terms of minimum batch time required to achieve a desired separation (specified top product purity (x D]) and bottom product purity (x B2) for binary mixture). Then an optimal amount and composition of recycle, subject to physical bounds (maximum reboiler capacity, maximum allowable purity of the off-cut) are obtained in an overall minimum time to produce the same separation (identical top and bottom products as in the... 

The productivity of a typical reversed phase purification of a peptide is summarized in Table 4.3. For this particular separation the column loading is relatively low and a large number of separations are carried out in order to deliver the annual production target. On the face of it, the productivity of this separation is poor in practice the column size was chosen to balance the output with the rate of synthesis of crude peptide. The actual combined costs of synthesis and purification for this particular peptide was only a tiny fraction of the value of the formulated drug. On occasions it may be better to purify a valuable product in smaller portions rather than risk a proportionally high loss from failed separation. 

In the case of temperature-sensitive separations the column temperature profile is constrained. Appropriate methods are stripping, liquid-liquid extraction, adsorption and crystallization, as well as vacuum distillation. 

A new set of columns was used for each separation. The columns were contaminated with at least three higher-molecular-weight components, which appeared as two well-resolved purple bands and a yellow band. These compounds were held tenaciously by the packing material and could not be removed by extended CH2Q2 extraction in a Soxhiet extractor. 

Selecting an appropriate column for capillary GC is a difficult task and one which is usually left to the technician. However, it is important to be aware of some general issues and what influence they can have on the separation. The column internal diameter can affect both resolution and speed of analysis. Smaller internal diameters columns (0.25 mm i.d.) can provide good resolution of early eluting peaks (Fig. 32.5a). However, the problem is that the analysis times of the eluting components may be longer and that the linear dynamic range (see p. 210) may be restricted. In contrast. 

The preferred method for packing silica gel and alumina columns is the slurry method, whereby a slurry of the adsorbent and the first eluting solvent is made and poured into the column. When nothing is known about the mixture being separated, the column is prepared in petroleum ether, the least polar of the eluting solvents. 

The liquid feed can also be introduced into the column at an intermediate position which separates the column in an enriching and a stripping section. It is possible to create an internal reflux by temperature change in the upper section of the column or an external reflux for improving the separation efficiency. More details on column operation and some application samples are given in a recent review article [5],... 

The heart of every preparative chromatographic system is the column packed with the adsorbent. If all other parts of the equipment are well designed with regard to minimum hold-up volume, the column is responsible for the axial dispersion of the separation. The column has therefore to be designed in an optimal way. Tremendous work has been done to obtain good preparative columns. Typical column design is of... 

The mass transfer equations discussed above are now combined with a material balance on the transferred component to calculate the column or packing height required for a given separation. The column cross-sectional area A is assumed known at this point although in a complete column design A must be determined based on pressure drop considerations. The column, which is in countercurrent flow with only liquid feed and vapor product at the top, and vapor feed and liquid product at the bottom (absorber, stripper, column section), is deflned as follows ... 

---