Packing a column

This is the method I use for packing a column with 5 pm bonded phase silica ... 

Sometimes it is necessary to pack a column under pressure (5 to 10 psi). This leads to a tightly packed bed that yields more reproducible results, especially with gradient elution (see below). 

Pack a column containing an immobilized iminodiacetic acid support (Pierce). The column size should be no less than 1.5 times that required to bind the anticipated amount of conjugate to be applied. The maximal capacity of such a column for binding antibody can be up to 50 mg/ml gel however, best results are obtained if no more than 10—20 mg/ml of conjugate is applied. 

As a result of certain drawbacks attached to coated silica gel CSPs, they could not be used for preparative separations. However, the literature provides some reports on the preparative chiral separations on the bonded CSPs. The bonded CSPs are suitable for use at a preparative scale but the yield is very poor due to poor loading capacities. Davankov et al. [73] packed a column with 300 g of the L-hydroxyproline-type resin and used the chiral separation at a preparative scale. 

FIGURE 9.6 Renewable separation-column approach for automatically packing a column on-line, localizing the column packing between a pair of 2-position valves. 

The germ reduction of chromatographic resins is conventionally carried out by chemical treatments before or after packing a column. The choice of the sanitizing agent is largely dependent on the sensitivity of the resin. 

Select a pre-packed desalting column from the table below or pack a column. 

Quantity of gel = estimate amount of gel required to bind the sample, use five times this amount to pack a column. 

Pack a column or a syringe (remember to put glass wool or similar at the bottom) with 2 mL of rProtein G Agarose (see Note 6). Connect column to the pump. 

A beginner mixed a measured amount of cough syrup into Celite and packed a column. The recoveries were low. What might have caused this ... 

Pre-swell the beads before packing a column. Dry beads swell when placed in water, especially the 2X and 4X beads. The higher the cross-linking, the less the swelling will be. Soak them in water for several minutes or until their volume appears to stop increasing. This also will remove some trapped air and permit a quality column packing. 

Use a wash bottle filled with petroleum ether and make a slurry out of the alumina. This is called "wet packing" a column. 

Another concern with packed-bed reactors is reproducibility. There are two parts to reproducibility can multiple packed-bed reactors be made reproducibly and what is the stability of the reactor The first question concerning reproducibility can be answered as follows. The art of packing a column is at times difficult and each individual analyst will have different success. However, all packed-bed reactors can be calibrated in the FIA system. A variation in the performance level between reactors of less than 10% is acceptable. The second reproducibility question concerns the loss in reactivity due to reagent degradation or saturation of the reactive sites. In the ideal case the reactivity of the column should not change with respect to time, thereby producing a reproducible signal for the same concentration of analyte. This is a more difficult problem. Ideally, the analyst should find the reactor conditions that will minimize loss of activity. In practice, especially with enzymes, this condition will not be met. Frequent calibrations will be needed to insure the most accurate results. 

Sluny packing is the oldest and most reliable way for the novice to pack a column. It is the only packing mode available for materials that swell in the mobile phase, such as the carbohydrate packings. The desired amount of adsorbent is placed in a beaker and a solvent added see Fig. 12). The mixture is stirred and additional solvent added if needed, to obtain a pourable slurry. The slurry must not be so thick that air bubbles are trapped in the column or so thin that the column cannot be packed in one pour. For adsorbents that swell, sufficient time has to be allowed for the adsorbent to be fully solvated. Typically, one might add enough solvent to wet the adsorbent and produce a thick slurry and then wait overnight before adding additional solvent to produce the pourable slurry. 

Dry packing is another very efficient way to pack a column. It is most often used in conjunction with regular or bonded silica gel. If done well, a dry packed column will give excellent resolution. As the name implies, dry packing... 

To increase the surface-to-volume ratio in the preconcentration channel without the need for particles, the channel can be filled with a polymeric rod. These are formed by in situ polymerization, during which the polymer material also reacts with the wall of the channel. As a result, no frits are needed to hold the material in place. Columns filled with a polymeric rod, so-called monolithic columns, were originally developed for conventional liquid chromatography. They are made by sol-gel technology, " which enables the formation of a highly porous material containing macropores and mesopores in its structure. The use of a monolithic phase circumvents the problems encountered when packing a column with particles. 

There are a number of techniques available to the chromatographer that can be used to pack a column. The best and most appropriate method will depend on the particle size of the packing, the scale of the separation and the character of the material to be separated. However, contrary to popular belief, there is no magic associated with column packing, but the procedure does need a little experimental skill, patience, and some... 

IX. PROTOCOL 1 PACKING A COLUMN WITH A THEOPHYLLINE-IMPRINTED POLYMER... 

Note 7. A well-packed column is essential to achieve high-efficiency elutions. There are two different methods to pack a column the dry and the wet method. In the first case the column is filled with the dry stationary phase, closed and the mobile phase is then pumped in. This method is suitable for silica-based beads, but not for polymeric beads, because this kind of material is prone to swelling when wet. Thus, high column back pressures, preferential channels and flow limitations due to a nonhomogeneous wetting of the stationary mobile phase could deteriorate the chromatographic performances of the column. When the wet packing method is used, the stationary phase is swollen and suspended in a solvent of similar density, and introduced into the column as a slurry. In this case the main drawback is the necessity to provide a reservoir for the column to keep the beads in suspension. 

Particle size is one of the most important parameters acting on efficiency. The smaller the LC packing particle size, the higher is the efficiency [5]. The plate height, H, of a well-packed column can be as low as twice the particle diameter. A 10 pm plate height (100,000 plate/m) should be obtained when 5 pm particles are used to pack a column. 

To pack a column, the packing material as a sonicated slurry is poured into the slurry reservoir of the packing rig, the column connected and the pressure applied. Calculation of the correct quantity of packing material is carried out by multiplying the volume... 

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