Tips for Troubleshooting Liquid–Liquid Extractions

December 1, 2017
Dawn Wallace Watson
LCGC North America
Volume 35, Issue 12
Page Number: 886

A common challenge in LLE is the formation of emulsions. Several approaches can address the problem. Or, one can try an alternative technique.

Liquid–liquid extraction (LLE) is the most widely used extraction technique for liquid samples. The information provided in this article focuses on LLE using a separatory funnel. Some of the potential issues that may require troubleshooting when using LLE include (1):

  • Emulsion formation

  • Analytes strongly adsorbing to particulates

  • Analytes binding to high-molecular-weight compounds (protein–drug interactions)

  • Mutual solubility of the two phases

  • Potential for solvent mediated decomposition

  • Method robustness and transfer issues because of manual processing

The formation of emulsions is at the top of the list because it is a very common problem. Let's focus on how to troubleshoot and avoid this issue, as well as offer an alternative extraction technique that precludes emulsion formation.

Emulsions commonly occur when a sample contains a high amount of surfactant-like compounds (that is, phospholipids, free fatty acids, triglycerides, proteins, and so on). These surfactant-like molecules are large and will have mutual solubility in the aqueous and organic solvents which results in the formation of an emulsion in a mid-zone between the two phases. This intermediate solubility in each of the two phases makes it difficult to quantitatively collect one phase or another. Furthermore, the emulsion can also trap some of the analyte of interest, leading to quantitative problems. Emulsions often occur with samples where the animal (or human) diet is high in fats. Thus, emulsions sometimes appear when passing from preclinical trials with animals on low-fat controlled diets to clinical trials with humans who may be on high-fat diets. This characteristic problem makes LLE a less dependable procedure if it is expected that the same extraction protocol will be used for both preclinical and clinical samples. If this problem is anticipated, it is worth trying high-fat samples during method development in addition to the standard test matrices.

There are a few tricks of the trade to either stop emulsions from forming in the first place or to disrupt them if they do form. As a general rule, it is easier to prevent emulsion formation than to break it after one has formed. The simplest way to prevent the formation of an emulsion is to gently swirl instead of shake the separatory funnel. By swirling the separatory funnel the agitation that can cause the emulsion to form is reduced, but the surface area of contact between the two phases is maintained to allow for extraction to occur.

Emulsions can be disrupted by the addition of brine or salt water, which increases the ionic strength of the aqueous layer and facilitates separation of the two phases by forcing the surfactant-like molecule to separate into one phase or the other-this technique is known as salting out.

The individual layers or emulsion can often be separated via filtration through a glass wool plug (to remove the emulsion) or a phase separation filter paper (to isolate a specific layer). Phase separation filter papers are highly silanized and, depending on the type of paper, will allow either the aqueous or organic phase to pass through and be isolated. Centrifugation of the separation can also be used to isolate the emulsion material in the residue of the centrifugation.

Addition of a small amount of a different organic solvent will adjust the solvent properties of the separation and can result in the surfactant-like molecule being solubilized in either the organic or aqueous layer to a greater extent, which breaks the emulsion.

Supported liquid extraction (SLE) is a technique that can be used for samples that are prone to emulsion formation (2). Analytes are separated based on differential solubility. The aqueous sample may be pretreated-for example, the pH can be adjusted so that the analytes are in a suitable form to be extracted into an organic solvent. Following this adjustment, the sample is applied to a solid support (often diatomaceous earth), which creates an interface for extraction. A small volume of water-immiscible organic solvent is subsequently passed over the matrix holding the aqueous layer and the analytes partition into the organic phase. The extraction solvent is allowed to percolate by gravity; sometimes a gentle vacuum or pressure is applied. Organic solvents that are commonly used include ethyl acetate, methyl tert-butyl ether (MTBE), dichloromethane, hexane, and mixtures thereof.