Preparation of the Column

Subsequent to this point separate procedures must be followed for large and small pored gels. With small pored gels that are quite rigid, the gel particles are permitted to settle 5 to 10 minutes with the column outlet closed. The outlet is then opened, permitting excess eluent to drain away. 

An understanding of hydrostatic pressure is required prior to discussing the packing of a column with large pored gels. Hydrostatic pressure is 

Fig[ure 5-7. Operating or hydrostatic pressure for various column assemblies. A and B pressure is measured between the free eluent surface in the column or the reservoir and the end of the outlet tubing. C and D pressure is measured from the bottom of the air inlet tube in the Mariotte flask to the end of the outlet tubing, no matter whether the flow is downward (C) or upward (D). 

Fignre 5-8. Schematic representation of a Mariotte flask in operation. 

Procedures for pouring a column of large pored gel must account for the compressibility of these soft gels. Fundamental differences between these procedures and those for small pored gels are the precautions taken to ensure that the hydrostatic pressure on the column bed never exceeds that recommended in Table 5-5 during packing and chromatography. Pressures in excess of those indicated will compress the soft gel and seriously reduce effluent flow rates. The freshly poured gel is first allowed 

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Classical gels had a low degree of cross-linkage and were of a large particle size. This resulted in that modest flow rates could only be applied and the separation time was typically 10 hr, which at that time was perfectly acceptable, keeping in mind that preparation of the column could take up to 2 days or more. After the introduction of Sephadex, new materials have been introduced continuously on the market, and still, 30 years after the introduction of the first commercial material, new media are still introduced, also from the originators of Sephadex. What are the driving forces behind this development and what are the features of these new media ... 

The field of aqueous SEC is of growing interest and also represents an area of active development by several companies, with Polymer Laboratories included. In this area, contrary to the organic SEC scenario, a variety of polymer chemistries are employed in the preparation of the column packing materials. This fact alone makes the practice of aqueous SEC more difficult than its organic counterpart and requires the suppliers of such columns to offer a good level of technical support to the column user. This chapter outlines the characteristics and applications of PL aquagel-OH aqueous SEC columns. 

Deactivation of silica gel and preparation of the column is carried out as in Note 6, except that the checkers consider 20 g. of silica gel per gram of crude product to be adequate in this case. Running through a gradient of petroleum ether containing increasing amounts of ether, the submitters found that the product was eluted with 15% vjv ether, and the checkers found that 25% vjv ether was required. 

Equation (11) shows that ko increases rapidly with increasing porosity (i/). For columns used in HPLC, e is usually about 0.40 and seems not to depend on the material and the packing technique used for the preparation of the column (22). For e = 0.40 the equation predicts that A — I x I0" in agreement with experimental observations. In order to achieve the very homogeneous packing necessary for a good colunin efUciency, (he... 

Preparation of the Columns. Columns were manufactured from 3l6 SS steel tubes l/U" x U mm diam. Swagelok 1/8" x 1/U" 3l6 SS... 

Adsorbents such as silica gel and molecular sieve are heated to approximately 300°C overnight. This releases any substances that may have been adsorbed during preparation of the column and... 

After preparation of the column, the bed is equilibrated with mobile phase, wbidi consists of approximately 59% toluene and 41% methanoL The b will rapidly become neariy completely transparent Now, a suitable dye dissolved in the mobile phase can be injected, and the band can be observed moving through the transparent bed. One can also generate opaque bands by injecting methanol or other solvents. 

The cis and trans isomers of linolenic acid were analyzed as their phenacyl derivatives on a silver-impregnated silica column (2 = 242 nm). The method for the preparation of the column is discussed. A 49.75/49.75/0.5 1,2-dichloroethane/ DCM/acetonitrile mobile phase was used and elution was complete in under 40 min [763]. Peaks were well resolved and peak shapes were good. A linear concentration range of 0-200 pg was reported. 

The following methods are used to avoid problems resulting from uneven packing and column irregularities. These procedures should be followed carefully in preparing a chromatography column. Failure to pay close attention to the preparation of the column may well affect the quality of the separation. 

Preparation of the columns is based on a two-step procedure, namely 1) preparation of a slurry where the adsorbent (graphitized carbon black) and the liquid phase (for instance, SP-1000) are mixed together in a suitable solvent and sonicated 2) coating of the capillary columns by means of a static method using the prepared slurry. 

The preparation of the column is illustrated by a routine established for the isolation of small amounts (up to 1 mg) of bile acids and conjugates from biological fluids such as blood (20). About 5 ml of the anion-exchange resin is pipetted into a chromatographic column 1.0 cm in diameter. The resin is washed with 50 ml of 1 jV NaOH and 50 ml of 1 A NaOH in 80% ethanol, followed by distilled water until the effluent is neutral. The column is now ready for use. 

Why should care be exercised in the preparation of the column to prevent air bubbles from being trapped in the adsorbent ... 

For the preparation of the columns the liquid crystalline melts are simply deposited on porous packing material or on capillary tubes [128,131]. It is important to maintain a uniform temperature over the whole column during the measurement. 

Gross and Pitt-Rivers (18) described a technique for the separation of TRITh and Tx on a column of kieselguhr by a modification of the Gronk-vist-Hellberg (13) procedure. The stationary phase used is 0.5 N NaOH and the mobile one is n-butanol mixed with 20% chlorofonn. The preliminary treatment of the adsorbant, the preparation of the column, and the fractionation of the effluent are carefully described in the originai paper. The samples separated by an automatic fraction collector must be tested for radioactivity in order to locate and, eventually, identify the labeled constituents of the mixture. Radiochromatography of some of the fractions may be necessary to control their homogeneity or for identification purposes. 

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