High Resolution Recycling Gel Electrophoresis

 

In order to improve the separation power of an electrophoretic system it is necessary to increase the efficiency of the apparatus (i.e. to increase the number of theoretical plates). The theoretical plate number of the gel or column can be improved by increasing the length of the gel or column, or by recycling the sample repetitively through the gel or column until the required separation is achieved. However, long columns are difficult to pack and manage and possible deformation of the gel places a limit on the length of the gel that can satisfactorily employed. In addition the column must contain large quantities of expensive separation media and will consume large volumes of buffer to effect a single separation.

 

It follows, that the most effective and economic way of improving the resolution of closely migrating pairs is to recycle them through the column. On each pass the individual components of the mixture will be moved further and further apart. In addition the column can be relatively short and, thus, relatively inexpensive to operate and in a recycling development procedure the same buffer is continually pumped through the system and is thus, very economic in buffer consumption. As will be seen in due course, however, the system has a serious limitation due to the restricted peak capacity of the column.

 

A diagram of a basic recycling gel electrophoresis instrument is shown in figure 22. It consists of a column, peristaltic pump, fraction collector, detector, computer and a printer/monitor system. It also includes a very important six-port two-way valve a diagram of which is shown in figure 23. The sample enters the column after passing through the detector and out to the peristaltic pump. The flow from the pump passes to a six-port two-way valve where the flow can either be directed to a fraction collector or back through the detector. The output from the detector passes to an A/D converter thence to a computer and the output displayed on a printer or monitor.

 

 

 

Figure 22. A Diagram of a Recycling Gel Electrophoresis Instrument

 

The valve connections shown in figure 23 allow the system to function in the following manner. In the position shown on the left buffer containing some sample is drawn through the detector, then through the column to the pump and from the pump to the fraction collector. After the sample enters the column (as monitored by the detector) the valve is switched to the position shown on the right. 

 

The outlet from the pump now enters the detector via the sample loop, as shown. Thus, the flow cycles from the pump to the detector-to the column back to the pump. When a satisfactory separation has been achieved the valve is turned back to its first position and the solute(s) collected in the fraction collector.

 

This recycling system is the basic form of the recycling device and relatively simple. However, extremely complicated instruments with automatic control are now commercially available although somewhat expensive are probably more economic to operate.

Figure 23. A Six-Port Two-Way Valve

 

An example of a separation achieved by recycling, including the deleterious effect of limited of limited column  peak capacity is shown in figure 24.

Figure 24. A Gel Electrophoretic Separation Achieved by Recycling

It is seen that up to recycle 4 the separation has become established and is sufficient for analytical purposes or for fraction collection, However, it is also seen that, subsequent to cycle 4 (i.e. from cycle 5 onward) the separation deteriorates, the peaks widen and there height is seriously reduced. This is because during the recycling process the peaks broaden and after an optimum number of cycles the two solutes of interest are completely spread through the column; viz the column has a limited peak capacity and as a result the back of the second peak becomes merged with front of the first peak. Consequently, the two solutes are merged together again. Thus, the recycling process has a limit and this limit will depend on the column peak capacity, which, in turn, will be a function of the total column volume. Nevertheless the use of recycling is o++ften the best and most economic method of resolving closely migrating pairs of solutes.

 

The optimum recycling number can be derived theoretically.

 

Let the two closely eluting substances have migration volumes of () and (). after the optimum number of cycles (n) have been run.  At this stage the slower component with be equidistant from the () and () peak of the faster eluting substance. 

 

  Thus,

Hence,

 

Unfortunately, () a calculated from the equation is rarely an integer and so the optimum value will be the integer before the actual calculated value of ().

 

It follows that the final resolution () after passing the optimum number of cycles will be

 

 

 

                                          where            ()