The Column Technique

With almost all adsorption materials, the column chromatography is superior to the batch method. Commercial columns (Pharmacia, Bio-Rad) are preferable to makeshift contraptions made from syringes, pipettes, and rubber plugs. The required time and the chance of misfortune increases with homemade devices. And in the end, it is not the doctoral candidate s money that is spent on proper columns but her own time that is saved. 

For many protein purifications, inexpensive low-pressure columns suffice, which are run with tube pumps. However, low-pressure separation methods are sometimes too inefficient and/or too slow (e.g., with proteins that must be purified about 1,000-fold and for which no affinity column exists). 

In these cases, it pays to do a few trials with HPLC (high-performance liquid chromatography). It offers high reproducibility and—with peptides and small stable proteins—high yields. 

Reversed-phase HPLC with water/acetonitrile gradients is the method of choice for peptide purification (e.g., from tryptic digestion). However, it can also efficiently separate small stable proteins (e.g., toxins). Reversed-phase HPLC columns use picomolar amounts of sample. You can immediately use the peaks for Edman sequencing (see Chapter 7) or for mass determination via MALDI-TOF (see Section 7.5). A typical run takes an hour. 

For peptides, you use silica particles with pore dimensions of 100 to 300 A. For proteins with 300 to 4,000 A, the pore diameter should be 10 times bigger than the peptide or protein. The diameter of the silica particles lies between 3 and 5 pm. Generally, the smaller the diameter the better the separation. The sample is normally eluded with a rising acetonitrile gradient. Methanol or 2-propanol also serve well, the latter being the stronger eluens. 

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If the solution were removed from Tank 1 and added to Tank 2, which also contained 1 eq of resin in the X ion form, the solution and resin phase would both contain 0.25 eq of Y ion and 0,75 eq of X ion. Repeating the procedure in a third and fourth tank would reduce the solution content of Y ions to 0.125 and 0.0625 eq. respectively. Despite an unfavorable resin preference. using a sufficient number of stages could reduce the concentration of Y ions in solution to any level desired. This analysis simplifies the column technique, but it does provide insights into the process dynamics. Separations are possible despite poor selectivity for the ion being removed. Most industrial applications of ion exchange use fixed-bed column... 

Two approaches are considered the batch technique and the column technique. In the former, the stationary phase (medium) is dumped into the sample in a Hquid form, usually in an aqueous environment. The stationary phase is allowed to contact the sample for a period of time and then is removed by filtration or by pouring off the liquid phase, leaving behind the compound of interest sorbed onto the stationary phase or contained in the liquid phase. The batch technique is slower than the column approach, but it is very easy to perform. 

The column approach—in which the sample is passed through the medium— is more widely used. Although dilution is a possible consequence, the column technique is more useful for removing traces of the analyte of interest. Convenient prepacked sample preparation columns are readily available. A newer form of medium is the flow-through membrane disk, which offers less flow resistance than a column yet provides similar sample capacity. Majors and Hardy have provided a thorough review of the range of methods in use [29]. 

The column technique has been used for the separation and determination of the purity of camphor. 

Practically every type of separation that has been done by the column technique can also be carried out by thin-layer chromatography. Several papers and reviews were published on the various aspects of the technique. In addition to the books on chromatography [17,26-301, an overview of ion-exchange application of TLC was presented by Devenyi and Kalasz 311. Recent results on the separation of enantiomers have been reviewed by Mack, Hauck and Herbert (32.33) (enantiomer. separation on an RP-18 plate, impregnated with copper salt and proline derivative as chiral selectors) and Lepri, Coas and Desideri, using a microcrystalline triacetylcellulose stationary phase, or modified beta-cyclodextrins in the mobile phase 134.35). 

The procedures of partial wetting, evaporation and condensation depend on several factors and basically influence both the separation of the analyte spots and the speed of the mobile phase movement 39,40. A beneficial effect of the evaporation of the running mobile phase from the thin-layer plate and wetting the stationary phase with the mobile phase is that spots with Rf values over 0.5 are concentrated. Thereby, the efficiency of the TLC system can be highly increased in comparison with the column technique. 

Thin Layer Chromatography-.f This name (abbreviated TLC) is applied to the thin layer versions of the column techniques described in Secs. 2.6.1 to 2.6.3, other than paper chromatography. The stationary phases (except cellulose) mentioned in these sections lack the strength of paper and require the support provided by a glass plate or polyester film. 

Preconcentration and removal of Co, Cu, Zn, Cd, and Hg by silica gel modified with pyridinium ions was developed and applied by using the column technique. Selective elution of these metals was accomplished by a... 

The interaction of sUica gel phases-modified-diethylenetriamine, with naphthaldehyde and sahcylalde-hyde, was found to produce four new sotid-phase extractors. Characterization of these modified silica gel phases and their capability toward selective extraction and separation of six metal ions Fe(lll), Ni(ll), Cu(II), Zn(II), Cd(ll), and Pb(ll), were studied and evaluated by both the batch and the column techniques as a fimction of pH and time of contact The reactivity of metal ion soqition was discussed on the basis of effects of bulk as well as orientation of immobilized chelate on sorbent reactivity. Other aspects of synthesis. 

The column technique has been applied to lymphocytes from non-im-munized mice as well (Wigzell and Makela, 1970). A mixture of normal lymph node, spleen and bone-marrow cells was passed through an OA-coated column. Column passed cells as well as control cells were transferred to lethally irradiated mice (10 cells per mouse) and followed by immunization with OA and BSA. The antibody content in the sera was detected by the Farr assay. There was no response to OA, while the response to BSA was normal. Control cell suspensions which were not subjected to the column passage resulted in a good response to both OA and BSA (Table 5). 

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