Before any analysis by HPLC the solvents for the mobile phase are prepared. In every HPLC it is possible to identify the solvent system by the reservoirs and the Teflon tubing leading from them into the gradient proportioning valve in the pump (a low pressure mixing type), which should be marked clearly as A,B, C, D (according to the number of reservoirs available). One tubing only comes out of the pump, in which the final composition flows constantly.
• It is important that on every reservoir will be clearly labeled!
• In every bottle’s end of the tubing there is a very fine filter that prevents from colloids such as algae and dust to enter the column. The filter should be protected from microorganism’s growth and take care of it from time to time (at least once a month when aqueous buffers are used) by sonication in 10%-20% HNO3 in water.
Selecting water for HPLC by Millipore
using different buffers. In this case water should be used in
• All types of solvents should be filtered before any use (or verify that they are sold already filtered), to prevent clogging of the column. Filtration needs to be performed with a special solvent filtration system that should exist in every analytical lab that uses HPLC. There are 3 categories of membranes for filtration, compatible with water and compatible for organic solvents and compatible with both. Pore diameter in these membranes are 0.2 – 0.45 um
• In many instrument there might be an additional in-line filter between the pump and the injector tha tcleans the solvents from un-washable impuritites in the mobile phase. In special cases (full aqueous mobile phases) it is also recommended to add a small silica-gel column that will saturate the mobile phase before its arrival to the reversed phase column with silica, by that will reduce the hydrolysis of its packing and prolong its life.
• After the preparation of the solutions and solvents a protocol is decided, i.e., the composition of the mobile phase during the run. For this purpose it there is a need to mix various solvents.
• It is imperative to verify whether the solvents mix easily with each other before using them. Mixing solvents that are not compatible with each other during a separation program is strictly forbidden because the pump, the column and the detector cell can be damaged! For example, chloroform is entirely wrong for reversed phase chromatography when water is used in the mobile phase as a bulk solvent. Hexane does not mix well with methanol and acetonitrile. Isopropanol mixes with water, acetonitril and methanol (reversed phase) on one hand, and on the other it also mixes with hexane and/or chloroform (normal phase). Therefore, isopropanol is used as a mediating solvent in the switch between reversed phase and normal phase modes of chromatography.
• It is important to note that not every solvent is appropriate for HPLC use, for example pentane is too volatile and bubbles are release from it during high pressures changes. Isopropanol or ethanol are very viscous, and cause high pressures in the column when they are used at high percentage. A MIXTURE OF 50:50 METHANOL WATER is very viscous and creates high pressures! Make sure to use a temperature over 40 when using it.
• Another important thing to remember that acetonitril and methanol can be used as mediators between most aqueous non-buffer type of mobile phases, therefore it is recommended to leave the system in a mixture of water-methanol-or-acetonitril or pure methanol-or-acetonitril in the end of work.
• Degassing of solvents is removing all air in them, and it is imperative in most HPLC methods. There are several ways to degass solvents, the most common one currently is by using in-line vacuum degasser. The other method is by sparging helium in the reservoir. The vacuum degasser contains vacuum chambers, one for every reservoir, where the solvents enter, effuse all the gasses from them instantly, and proceed degassed. When using an in-line vacuum degasser there is a need to verify that the chambers are washed thoroughly during the purge stage of the sustem. In some instruments the volume of such chambers can get up to 12 ml.
• When Helium is bubbled through the solvents it is important to make sure that the Helium cylinder is always opened to maintain positive pressure in the tubings leading to the instrument. The Helium is opened/shut by a special valve at the HPLC system itself. It is important to have pressure regulator to every one of the solvents due to the differences in their viscosity and solubility of gasses in them. Helium is an inert gas and its solubility is so small that it removes the dissolved air and saturates the solvents, and then bubbles are not created during mixing of the solvents and the change in mobile phase composition during the run (gradient). Helium degassing should be used only in system under fume hood, because Acetonitril and Methanol are toxic!
• When the solvents are ready for work the system is purged with the degassed solvents to wash the tubing all the way to the column’s connection. This is done to drive out previous solvents and/or air from the system. The void volume of the system should be accounted for, including the tubings from the reservoirs, which could be as high as 5 ml. This wash can be done at very high flow-rates, up to 10 ml/min for 2-3 minutes for every reservoir. In every HPLC system there is a special valve for this purpose, which diverts the solvents out in between the pump and the injector.
• It is important to purge all the solvents in the system, not just those used immediately, because sometimes the user can make a mistake and select the wrong solvent and introduce air to the system.
• When switching solvents it is imperative to notice that they can readily dissolve in each other even in the purge stage, because they all pass through the pump, which is the meeting point to all solvents. When Normal Phase is used it is especially important to switch between solvents according to their ability to mix readily.
Copyright (C) since 1997: Dr. Shulamit Levin