Open column packing procedures

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

The urine of people who are heavy smokers contains mutagenic chemicals, chemicals that cause mutations in biological cells. Bioanalytical laboratories can analyze urine samples for these chemicals, but the samples must be cleaned up first prior to extraction with methylene chloride. The procedure for this cleanup utilizes an open column chromatography. Columns several inches tall and about an inch wide are prepared by packing them with an adsorbing resin that has been treated with methyl alcohol. The urine samples are passed through these columns as part of the sample preparation scheme. 

In a similar manner to the design process for packed columns, the physical characteristics and the performance specifications can be calculated theoretically for open tubular columns. The same protocol will be observed and again, the procedure involves the use of a number of equations that have been previously derived and/or discussed. However, it will be seen that as a result of the geometric simplicity of the open tubular column, there are no packing factors and no multi-path term and so the equations that result are far less complex and easier to manipulate and to understand. 

In a packed column the HETP depends on the particle diameter and is not related to the column radius. As a result, an expression for the optimum particle diameter is independently derived, and then the column radius determined from the extracolumn dispersion. This is not true for the open tubular column, as the HETP is determined by the column radius. It follows that a converse procedure must be employed. Firstly the optimum column radius is determined and then the maximum extra-column dispersion that the column can tolerate calculated. Thus, with open tubular columns, the chromatographic system, in particular the detector dispersion and the maximum sample volume, is dictated by the column design which, in turn, is governed by the nature of the separation. 

Columns should never be removed from the chromatograph while the packing material is still hot. The recommended procedure is to turn off oven and injector heaters, open the oven and allow to cool for approximately 30 min. The carrier gas is then turned off, the exit end uncoupled, and when no flow can be measured at the exit of the column, the inlet is disconnected. The two ends of the column should then be sealed with a suitable cap to prevent contamination of the packing material. 

Procedure Carefully add the Sample Preparation to the prepared column. Open the stopcock, and adjust the flow rate to about 2 mL/min, discarding the eluate. Rinse the sample beaker with 5 mL of chloroform, and add the rinsing to the column when the level drops to 2 cm above the silica gel. Never allow the column to become dry on top, and maintain a flow rate of 2 mL/min throughout the elution. Avoid interruptions during elution as they may cause pressure buildup and result in leakage through the stopcock or cracks in the silica gel packing. 

If the column can be opened at the feed injection side, which is not always possible for technical or regulatory (GMP) reasons, the first one or two centimetres of packing can be removed. The top of the bed is then re-slurried, fresh adsorbent added and the column re-compressed. Up to a column diameter of 200-300 mm this procedure might make it possible to avoid re-packing of a column and ensures the further use of the packed column for some time. Table 3.17 shows some CIP-regimes for silica-based adsorbents with different functionalities. 

Slowly pour the slurry into the column, ensuring that a minimum number of air bubbles are created. Open the tap of the column and allow the solvent in the slurry to steadily run into a suitably sized collection vessel (beaker/conical flask). Gently tap the sides of the column with a piece of thick rubber tubing to free any air bubbles that persist in the packed column and to ensure that the stationary phase is compacted into a uniform packed bed. It may be necessary to repeat the above procedure in order to pack all of the stationary phase that is required for the desired separation. If this is the case, allow the solvent in the slurry to run to approximately 1 cm above the level of the settling stationary phase. Turn the tap of the column to stop the flow of the solvent. Gently pour in the second batch of slurry and reopen the tap. Repeat the above process until all the stationary phase is loaded into the column. 

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