Slurry column packing

The procedure chosen for column packing depends chiefly on the mechanical strength of the packing and its particle size. Particles of diameter > 20 jum can usually be dry-packed, whereas for particles with diameters <20 /xm slurry... 

No expensive equipment is required for OCC however, the separation efhciency depends on the analyst s experience since a new column has to be packed for each analysis. In addition, depending on the packing type (powder or slurry), stationary phase, and purpose of the separation, the separation can take from 30 min to 4 hr. The AOAC official method for the determination of carotenoids still uses OCC." Separation of carotenoids from many foods was developed on a column packed with a mixture of MgO and HyfloSupercel (or celite or diatomaceous earth) at 1 1... 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

A calcium hydroxide column slurry packed in the laboratory was used to evaluate the distribution of all-trans-, l3-cis-, and 9-cis- isomers of P-carotene in fresh and processed vegetables and fruits. Elution order was reported to be l5-cis-, 3-cis-, sil-trans-, and 9-cw-P-carotene, using 2% p-methylanisole or 2% acetone in hexane as mobile phase, in a 35-min run. However, a column packed with calcium hydroxide as the stationary phase is not commercially available. 

Reversed-phase silica gel column Place a cotton wool plug at the bottom of a glass chromatography column. Pack 5 g of reversed-phase silica gel slurried with a solvent mixture of n-hexane-benzene-methanol (80 20 0.4, v/v/v) into the glass column. Place an anhydrous sodium sulfate layer about 1 -cm thick above and below the silica gel bed Bell jar-type filtering apparatus Buchner funnel, 11-cm i.d. 

Analytical hplc these days is nearly always done with microparticulate column packings, which are small porous particles, usually spherical or irregular silica, with nominal diameters of 3,5 or 10 fxm. They combine the best features of the other two types, having high efficiency as well as a large surface area. In bulk, the appearance of a microparticulate silica resembles that of a fine talcum powder. With microparticulates, dry packing methods result in column beds that are unstable under pressure, so they are packed into columns using a slurry of the material in a suitable solvent and under considerable pressure. 

Because the halogenated hydrocarbons that have to be used for this are both toxic and expensive, the use of balanced density slurries for packing columns is declining. 

Tang et al. used columns packed with a slurry of beads suspended in supercritical C02. This packed column was filled with a dilute sol solution prepared by hydrolysis and polycondensation of tetramethoxysilane and ethyltri-methoxysilane precursors. The column was dried using supercritical C02 and heated first to 120 °C for 5 h followed by another 5 h at a temperature of 250 °C [108-110]. Column efficiencies of 127,000 and 410,000 plates/m were reported... 

Fig. 9. Apparatus for sluijy packing of columns. After the properly fittul column tube is attached to the bottom of the reservoir, both are filled up with the slurry ofthe micropartic-ulate stationary phase. Thereafter a displacement liquid is pumped into the reservoir by the constant pressure pump, e.g., Haskel Model DST>I00, which is driven by preuurized air. Upon displacement, the slurry from the reservoir is filtered over the porous etal frit at the bottom of the column tubing which becomes densely packed with the partic s. By intermit-tently operating the liquid shut-off valve between the pump and the reservoirpressure waves can te generated in order to flirther compact the column packing. Reprinted from Bakalyar et at. U05) with permission from Spectra-Physics.

Before a gel slurry is packed into the column, it should be defined and deaerated. Defining is necessary to remove very fine particles, which would reduce flow rates. To define, pour the gel slurry into a graduated cylinder and add water equivalent to two times the gel volume. Invert the cylinder several times and allow the gel to setde. After 90 to 95% of the gel has setded, decant the supernatant, add water, and repeat the settling process. Two or three defining operations are usually sufficient to remove most small particles. 

Method. The stationary phase consists of the appropriate amount of picric acid dissolved in a citrate buffer system (Titrisol Merck). The pH of these solutions are checked and when necessary are adjusted to the desired pH with 5 N sodium hydroxide. The columns are packed with a slurry of silica gel by means of the equal-density procedure described earlier [54]. For other adsorbents, a new technique [55] is used for packing the slurry. After packing, twice the dead volume of chloroform is pumped through the column. The column is then heated at 180 °C for 2 h and flushed simultaneously with a gentle stream of nitrogen. The columns (usually 10 cm long) are treated with ca. 10 ml of the stationary phase at a flow-rate of 0.5 ml/min, and are then flushed with 20-40 ml of hexane. The column is equilibrated with chloroform at a flow-rate of 0.2 ml/min for various times. The samples are injected as ion pairs on to the column. Formation of the scopolamine and hyoscyamine ion pairs occurs in 5 ml of buffer solution (pH 5 or 6) to which a solution of picric acid is added containing 40 mg of picric acid in 5 ml of buffer. 

A number of commercial column packing apparatuses are available. One type, the ascending type, is a stirred can that pumps slurry upward into the pressure line and then down into the column. The descending type of packer is simply a slurry reservoir that attaches in place of the inlet end-cap and frit and is equipped with a pump connection at the top (Fig. 5.3). Manufacturers use 20,000-psi pumps to drive slurry into the column, but most laboratory packing apparatuses rely on pumps that reach a maximum of only 6,000-10,000psi. The pumps are run fully open until the pressure stabilizes. 

First, the target compound to be bound to the column must be identified, isolated, and activated. In some cases, the column packing is purchased already activated. Once the target is chemically bound to the column, the packing must be slurry packed into the column. Fortunately, these columns concentrate and bind the substrate so they can be broad and narrow like an ideal ion exchange column and are fairly easily packed. 

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