John W. Dolan, LC Resources, Walnut Creek, California, USA.

Occasionally I get questions from readers regarding how to reduce solvent consumption for liquid chromatography (LC) methods. A specific question recently concerned recycling the mobile phase. It has been more than 10 years since solvent recycling has been the main subject of an "LC Troubleshooting" instalment,^1,2 so this is a topic worth covering again. In addition to recycling, this month's discussion covers conservation through reduced solvent use and considers the economics of mobile phase use.

For the present discussion, I am going to focus on the consumption of the organic solvent used for LC and consider acetonitrile the selected solvent. I just checked the list price of acetonitrile from one major supplier: $39/L when purchased in cases of 4 × 4 L bottles; many of you will get discounted prices. If we assume that the "average" mobile phase is 50:50 aqueous–organic, the cost is approximately $20/L for the organic. The cost of the aqueous portion of the mobile phase is small, because most workers use high performance liquid chromatography (HPLC)-grade water prepared by a water purification system at a negligible cost after the system is purchased, and dilute buffers are relatively inexpensive. Another, often hidden, cost is disposal of the mobile phase waste stream. One laboratory I know pays approximately $2.50/L to dispose of mixed aqueous/organic mobile phase waste. Combining all these costs: $20/L for the organic + $2.50/L disposal + a small amount for water and buffer + 10% nonproductive use of the LC system, I will use $25/L as the overall cost of the mobile phase for a typical reversed-phase LC run.

The simplest way to conserve mobile phase is to reuse all of it. Just take the waste line from the detector and direct it back into the mobile phase reservoir. Of course this only works for isocratic methods, for which the mobile phase composition is constant. At first this might seem like you would be contaminating the mobile phase and generating spurious peaks in the chromatograms, but closer examination of the process will expose the error of this assumption. Each injection will add a small amount of the sample to the mobile phase reservoir — at most a few micrograms. If the total mobile phase volume is relatively large (I recommend 1 L) and stirred to maintain homogeneity, the percentage change in the mobile phase additive (the sample in this instance) is negligible. If a chemical is present in the mobile phase at a constant concentration, it will not generate a peak in the chromatogram, even if it would if injected alone. We know this from practical experience — buffers added to the mobile phase do not cause peaks in the chromatogram, but if the buffer were injected directly, a peak might be observed. Similarly, a constant small amount of sample compound in the mobile phase will not generate a peak either.

Three precautions need to be considered if you choose to undertake this simple recycling technique. First, mobile phases will not last forever. Gradually, the more volatile components of the mobile phase will evaporate, resulting in a change in mobile phase composition. If you limit the use of a batch of mobile phase to a week or two and keep the container almost sealed (e.g., leave a 1 mm vent hole in an otherwise tightly sealed cap), evaporation should be minimal.

A second problem is that some mobile phases support microbial growth, so extended use of the mobile phase will allow a build-up of biological contaminants. This is usually not a problem when more than 25–30% organic solvent is used in the mobile phase. If you see any cloudiness develop in the mobile phase, discard it and replace it with a new batch. Don't forget to clean or replace the solvent reservoir frit to avoid contamination of the next batch of mobile phase.

The third problem relates to a build-up of contaminants in the mobile phase. Eventually, as sample contaminants accumulate, the baseline can become noisier. For most methods, this should not be a problem, because the mobile phase will be discarded for one of the previous reasons before enough sample-related materials accumulate.