HPLC Products

Normal

 

First, note that this means a shoulder on the trailing edge of every peak, or every peak is split into two!

By far the most likely cause is a column void:

Void in the column.

If the inlet bed of the column has dissolved, the particles become smaller and smaller until they drop down between the bigger particles, leaving a void at the top of the column and restricting the flow, giving rise to an increase in back pressure. Removal of the top fitting to the column may confirm that this is the case. If the top bed of the column is not flat, then a part of the sample starts a little further down the column than the rest, and hence all the peaks are split or have a shoulder.

The symptoms of a column void are broad tailing peaks and increased back pressure. If there is a void, the normal procedure is to replace the column. Should you be on a desert island, or working in a lab where the lack of money makes you feel like you are on a desert island, you may like to try topping up the column with spare packing material. The easiest way to do this is to remove the top end fitting from the column, thereby exposing the void. As quickly as possible, transfer some wet packing material from the bottom of an old column of the same type, leaving the silica a little proud of the end of the column. Replace the inlet fitting on the column that had the void, preferably using a new frit. This should correct the poor peak shape, although the increased back pressure will remain. For further instructions on how to do this, please call the helpline number below, or book on the HPLC Troubleshooting Course!

Silica dissolves when the pH goes over 7, at elevated temperatures, in highly aqueous mobile phases, and at high buffer concentration. If your method uses any of these, then precautions need to be taken against silica dissolution, which can include:

• Use a column which is stable at higher pH, such as Reprosil Pur Basic from Dr Maisch
• Use a polymer-based column where there is no silica to dissolve, such as Shodex ODP2-HP
• Use a pre-column before the injector to saturate the eluent with silicate anions.

If this is not the problem, the only other likely cause is a blockage in the end-fitting or the inlet frit, causing a split injection.