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Thus, more theoretical plates will be required to resolve the enantiomers and thus a longer column will be necessary. At a temperature of 5˚C and at an ethanol concentration of 5%v/v, the column need only be about 5 mm long(a length of column that is impractical to pack and operate). Contemporary columns, shorter that 2 cm are extremely difficult to operate efficiently.     Figure 24 The Minimum Column Length that will Produce the Required Efficiency The minimum column length that will provide the minimum analysis time for this particular separation is not in the practical range of column lengths normally available. Consequently, the optimum column length must be a compromise between, that which is theoretically desirable, and that which is practically possible, and thus the shortest column available would be chosen.    

the column length divided by the number of theoretical plates in the column) has, for obvious reasons, become termed the Height Equivalent to the Theoretical Plate (HETP) and has been given the symbol (H). However, it is seen that (H) is numerically equal to, , which is, in fact, the variance per unit length of the column. Thus, the function, , is the variance that the Rate Theory will provide an explicit equation to define and can be experimentally calculated for any column from its length and column efficiency. It follows that the equations that give a value for, (H), the variance per unit length of the column, have been termed HETP equations

matter. Sometimes it is inevitable that the detector sensor must be a significant distance from the column exit and, thus, a relatively long length of connecting tube will be necessary. Under these conditions, low dispersion tubing, such as serpentine tubing, may be the solution. Dispersion in the detector sensor is the second largest source of extra-column dispersion and can only be reduced to a satisfactory level by reducing the volume of the sensor cell to 2 ml or less. Reducing the cell length will reduce its response but maintain the same or similar noise level, thus, reducing the sensitivity (minimum detectable concentration). Maintaining the same length, but reducing the cell radius, will maintain the same response, but increase the noise level, also reducing the overall sensitivity. It is clear a compromise is necessary and this compromise will depend on the type of detector, UV absorption, fluorescence, etc., that is employed. Nevertheless, to realize the maximum column

. The safe maximum pressure for any tube can usually be obtained from the tube or column supplier. A slurry is made of the packing (110% of that needed to fill the column) and placed in the packing reservoir. The reservoir is rapidly connected to the pump (which must have both an adequate delivery rate and an adequate operating pressure - the delivery rate will depend on the column diameter and the applied pressure on the wall thickness and the column length). The pump is started and the column exit valve opened and the flow continued until it has been reduced to a constant value. The flow is then arrested and some packing will remain in the reservoir which ensures that the top of the column is tightly packed. The column is disconnected, the packing secured with a suitable frit, and then connected to the chromatograph. The major difference in equipment used for larger scale chromatography lies in the technology required to pack and maintain high-performance LC columns of

may need to be determined by experiment. During the initial pressure adjustment some of the packing passes into the column and forms a lightly packed bed at the bottom of the column. The exit valve is hen rapidly opened and the sudden flow of gas packs and compacts the bed at the same time. After packing, the reservoir is carefully removed so as not to loosen the top of the packing and connected to the sampling system. LC Columns If particle sizes in excess of 20 mm are used, then the column can often be dry packed, with appropriate tapping, or, even better, with longitudinal and radial sonic vibration. The variance per unit length obtainable from a preparative LC column should be less than 2 particle diameters (determined using analytical scale samples). It is worth remembering that (as already discussed) when designing preparative columns, it is better to obtain the necessary efficiency using a longer column packed with larger particles, than the converse. The long column

efficiency attainable at a given pressure. This is because, as the particle diameter is increased the column permeability is also increased allowing a longer column to be used. The permeability increases as the square of the particle diameter but the variance per unit length only increases linearly with the particle diameter. Thus, doubling the particle diameter will allow a column four times the length to be used but the number of plates per unit length will be halved. Consequently, the column efficiency will be increased by a factor of two. It is also seen that the higher efficiencies will be obtained with mobile phases of low viscosity and for solutes of low diffusivity. Solvent viscosity and solute diffusivity tend to be inversely proportional to each other and so the sensitivity of the maximum obtainable efficiency to either solvent viscosity or solute diffusivity will generally not be large. The approximate length of a column that will provide the maximum column efficiency