Overloading by mass or volume as well as the wrong sample solvent can decrease the efficiency of the column significantly and therewith impair the results of the separation. The injection volume should not exceed 10% of the flow rate (e.g. 1 ml/min ? injection volume < 100 µL. Mass overloading is obtained as soon as the linear part of the adsorption isotherm is surpassed. This border is individual, but as a rule of thumb, no more than 0.01 mg sample should be loaded per ml column volume (e.g. max 22 µg on 4.6 × 250 mm column). The sample mass may however be increased if necessary for detection reasons. In the sample solvent, the amount of the strong mobile phase component (alcohol, acetonitrile in RP, or alcohol, ethyl acetate etc. in NP), should not exceed the amount in the mobile phase.
Poor plate counts can often be attributed to excessive dead volume. The dead volume is defined as the sum of the volume contributed by the sample injector, including the sample loop, the tubing connecting the sample injector to the column, the tubing connecting the column to the detector flow cell, the detector flow cell, plus any volume added by fittings, connectors, and in-line filters. Tubing with 0.010" ID is commonly found in HPLC systems. If you are using narrow bore (< 3.0 mm ID) columns, or, you can enhance your system´s performance by replacing the 0.010" ID tubing with 0.007" ID tubing to reduce system dead volume. Tubing with 0.005" ID can further reduce dead volume, but at a cost of inconvenience. Tubing with IDs less than 0.007" restricts flow rate and may have problems with buffer salt precipitation. If you use a variety of brands of HPLC columns, make sure that the fittings used to connect to the column are properly matched. If your system has stainless steel tubing and fittings, select the proper fittings for each brand of column that you use and install those fittings prior to connecting the column. If your system has PEEK tubing and fittings, it is a good idea to install a new PEEK fitting on a freshly cut piece of PEEK tubing with each column change to insure a proper fit.
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