Extra Column Dispersion - Separations on Small Bore Columns 2m Long > Page 68

The column was operated close to its optimum velocity and, even with a 2 m column the analysis time extended over 40 hr. The number of peaks disclosed by the chromatogram is about 150. The separation was developed isocratically by a 75% v/v acetonitrile/water mixture. It is seen that there is now a clear resemblance to a GC separation carried out on a capillary column. Unfortunately the analysis times are far from comparable.

It is seen that extra-column dispersion can arise in the sample valve, unions, frits, connecting tubing, and the sensor cell of the detector. The maximum sample volume, i.e., that volume that contributes less than 10% to the column variance, is determined by the type of column, dimensions of the column and the chromatographic characteristics of the solute. In practice, the majority of the permitted extra-column dispersion should be allotted to the sample volume, as a large sample volume may be necessary to handle a particular sample type for successful analysis. In any event, the chromatograph should always be designed so that the dispersion from other parts of the system is kept to the absolute minimum. Dispersion in sample valves can be minimized by mechanical design (internal loop valves tend to provide the minimum dispersion). Dispersion from unions can be minimized by using drilled-out unions or low dead volume unions.

Stainless steel frits provide very little dispersion and can be employed without great concern for their contribution to the overall dispersion of the system. Connecting tubes are one of the major sources of extra-column dispersion and should be kept as short as possible and the radius reduced to a minimum commensurate with the tube not becoming blocked. Tubes 0.005 in. in diameter are recommended as the minimum diameter that is unlikely to become blocked. In exceptional cases 0.003 in. I.D. can be used but all mobile phases and samples must be carefully filtered free of solid 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 efficiency and, thus, the maximum resolution, very small detector cell volumes are essential.