Packed columns minimum

The combined acetone extracts were extracted six times with one-fourth volume of ethylene dichloride and the ethylene dichloride extract was evaporated under vacuum to leave the steroid residue. This steroid residue was taken up in a minimum of methylene chloride and applied to the top of a column packed with 30 grams of silica which had been previously triturated with 21 ml of ethylene glycol. Then various developing mixtures, saturated with ethylene glycol, were passed over the column. Cuts were made as each steroid was eluted as determined by the lowering of the absorption of light at 240 mp on the automatic chromatographic fraction cutter. 

Thus, the minimum value of (a) for any pair of solutes can be calculated for any given column. The minimum values of (a) required for a pair of solutes that will be separated on a column having 25,000 theoretical plates (the efficiency of the standard ASTEC column 25 cm long, 4.6 mm in diameter and packed with spherical particles 5 p in diameter) is shown plotted against the (k ) of the first eluted solute is shown in figure 5. 

II Given these conditions, what would be the minimum diameter of a 25 cm column, packed with 5 /an particles, if the column is to show infinite diameter behaviour ... 

The HETP curve clearly shows, that for a packed column, the particle size has a profound effect on the minimum value of the HETP of a column and thus the maximum efficiency attainable. It would also appear that the highest efficiency column would be obtained from columns packed with the smallest particles. This will in due course be shown to be a fallacy, but what is true, is that the smaller the particle diameter the smaller will be the minimum HETP and thus, the larger the number of plates per unit length obtainable from the column. At this time it will suffice to point out that the total number of theoretical plates that can be obtained will depend on the length of the column which, in turn, must take into account the available inlet pressure... 

Employing the conditions defined in the three data bases and the appropriate equations derived from the Plate and Rate Theories the physical properties of the column and column packing can be determined and the correct operating conditions identified. The precise column length and particle diameter that will achieve the necessary resolution and provide the analysis in the minimum time can be calculated. It should again be emphasized that, the specifications will be such, that for the specific separation carried out, on the phase system selected and the equipment available, the minimum analysis time will be absolute No other column is possible that will allow the analysis to be carried out in less time. 

To separate the components of this reaction mixture, the crude product is dissolved in a minimum quantity of petroleum ether. The solution is then passed through a 2 x 20 cm chromatography column packed with aluminum oxide. The nitro and azo compounds are eluted from the column first with sufficient petroleum ether. The azoxy compound is eluted with petroleum ether containing 1 % of methanol. The eluting solvent is evaporated and the residual product is recrystallized from ethanol, m.p. 117°-118°C, nematic-liquid transition point 134°C. 

The column packing consists of Vydac reversed-phase material (chemically bonded ODS). The fluorescamine derivatives are separated by gradient elution with 10% methanol in buffer (pH 8.0), followed by a linear increase in proportion to 40% methanol in buffer (pH 8.0). The separation of several diamine derivatives with this system is shown in Fig.4.51. The limits of detection are ca. 2S-S0 pmoles of amine using a fluorimeter with a microflow cell. Both amino groups of the diamines react with fluorescamine so that a minimum of a 2 1 molar ratio of fluorescamine to diamine is required for the best results, a ratio of 3 1 or 4 1 is preferred. 

Column packing was SE30 on Chromosorb W(100-120 mesh). Retention time at 235° is given as 3.7 minutes, and the minimum detectable amount at attenuation X20 is 0.2 J.g in 1 Hi of ethanol. 

The heart of every preparative chromatographic system is the column packed with the adsorbent. If all other parts of the equipment are well designed with regard to minimum hold-up volume, the column is responsible for the axial dispersion of the separation. The column has therefore to be designed in an optimal way. Tremendous work has been done to obtain good preparative columns. Typical column design is of... 

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