Salting-out Liquid-Liquid Extraction (SALLE) -

Salting-out Liquid-Liquid Extraction (SALLE)

Jul 1, 2009
By: Ronald E. Majors
LCGC North America

Ronald E. Majors

Liquid–liquid extraction (LLE), also known as solvent extraction, has long been an effective method of separating compounds having different solubilities in two immiscible liquids (1). The two liquids are typically water, perhaps with some additives, and a nonpolar organic solvent such as isooctane. Typically, polar compounds prefer the aqueous layer while nonpolar compounds are extracted into the organic layer. A drawback of the use of nonpolar, water-immiscible organic solvents is that due to their low dielectric constants, they are relatively poor at the extraction of very polar or highly charged solutes, particularly for highly water-soluble pharmaceuticals that may require extractions at very low or very high pH values. More-polar solvents (such as acetonitrile) that provide solubility for these more-polar compounds are frequently water-miscible and, thus, cannot be used for conventional LLE.

It has long been known that the addition of an inorganic salt into a mixture of water and a water-miscible organic solvent causes a separation of the solvent from the mixture and the formation of a two-phase system (2). Sometimes this phenomenon is referred to as "salt-induced phase separation." Observations of this "salting-out" phenomenon were made for a number of water-miscible organics such as acetone, methanol, ethanol, and acetonitrile. Different salts and different salt concentrations will cause different degrees of phase separation. The high-polarity, water-miscible solvents used in salting-out systems have been investigated for extraction or concentration of many analytes that cannot be extracted by conventional LLE methods. This salting out often occurs at high salt concentrations. In some cases, the "salting-out" (or perhaps better termed "sugaring out") effect also can be achieved with high concentrations of saccharides (3).

The purpose of this installment of "Sample Prep Perspectives" is to report on the use of salting-out extraction to isolate compounds of interest. Some pertinent applications will be cited. The extracts are not particularly clean relative to other, more intensive sample preparation procedures. This simple extraction process is useful especially when very selective detection is used in gas chromatography (GC) or high performance liquid chromatography (HPLC).

Background

Although the salting-out phenomenon has been known for many years, some systematic studies have been carried out to help choose the optimum solvent systems and salt additives. Matkovich and Christian (4) investigated the salting out of acetone from water as a solvent-extraction system for metal chelates. They were interested in finding a solvent system that would enhance the sensitivity of flame emission and atomic absorption spectrometry measurements for metal ions. By extraction of the metals as their chelates into the acetone phase in the absence of water, sensitivity could be enhanced. Their investigations included 79 salting-out agents, and of those found for this water–acetone system, calcium chloride, magnesium chloride, and sucrose were most effective.

Table I: Recoveries of nitroaromatics, nitramines, and nitrate esters from water by salting-out extraction (%)*

Salting out extraction has been used for the preconcentration of neutral polar organics from water. Leggett and coworkers showed that high recoveries of trace explosive compounds can be achieved from water using acetonitrile–sodium chloride salting-out extraction (5). They initially used conventional LLE with methylene chloride as an extraction solvent and, as reported in Table I, for some nitroamines such as HMX and RDX, extraction efficiency was rather poor. Addition of sodium chloride to the water improved the situation a bit but the salting-out extraction with acetonitrile gave the greatest overall recoveries. In fact, the U.S. Environmental Protection Agency method 8330A for the isolation of nitroaromatic and nitramine explosives from water at the nanogram-per-liter level is based upon a salting-out extraction method (6). The method involves the addition of 251 g of sodium chloride to a 1-L volumetric flask containing 770 mL of water sample. The addition of 164 mL of acetonitrile with stirring is followed by a phase separation step. Removal of the acetonitrile upper layer is followed by some additional small volume extractions and a back extraction. Finally, an aliquot is injected into a reversed-phase HPLC column with a further confirmation on a cyano column.

Extractions using the salting-out effect began to be used more extensively in the area of pesticide analysis. First, nonionic pesticides in various vegetable food samples were extracted using water-miscible organic solvents like acetonitrile and methanol. The Luke method showed that acetone–water (65:35, v/v) also could be used for this purpose (7). A multiclass, multiresidue method (MRM) was used by Mills and coworkers to extract nonpolar pesticides from various food samples of vegetal origin using acetonitrile (8). The addition of sodium chloride and water to the acetonitrile extract allowed partitioning into a very nonpolar solvent, petroleum ether. The use of acetonitrile followed by salting out proved to be better suited for the extraction of both nonpolar and polar pesticides from vegetable samples and has been adopted today by several regulatory bodies (9). However, the extracts were still rather "dirty."

In 1993, Anastassiades, Lehotay, and coworkers from the U.S. Department of Agriculture in Wyndmoor, Pennsylvania (10) further refined the acetonitrile extraction by using sodium chloride salting out plus the addition of a drying agent (magnesium sulfate) to remove the water and force the pesticides into the acetonitrile phase. Termed the QuEChERS technique, pronounced "catchers," standing for quick, easy, cheap, effective, rugged, and safe, the technique has caught on quickly (11). Following the acetonitrile extraction step, the introduction of a dispersive solid-phase extraction (SPE) step in which a portion of the raw extract is mixed with bulk SPE sorbent such as C18, primary-secondary amine, and graphitized carbon helps to further clarify the extract and, after centrifugation, allows the supernatant to be injected directly into a GC–mass spectrometry (MS) or LC–MS or LC–MS-MS system. Hundreds of pesticide residues in a wide variety of fruits and vegetables are now done routinely using QuEChERS cleanup. Official regulatory methods are now available (12).

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