Sample Preparation

Liquid/Liquid Extraction Techniques

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Aims and Objectives

Aims and Objectives

Aims

To describe the basic principles of liquid/liquid extraction

To give an overview of limitations and drawbacks of liquid/liquid extraction
protocols and give specific advice for various problems that are routinely
encountered
To give an overview of emulsions and how to deal with them
To present support-assisted liquid/liquid extraction (SALL) as a useful variation
on standard liquid/liquid extraction

Objectives
At the end of this Section you should be able to:

Maximise analyte recovery by optimising liquid/liquid extraction conditions

Maximise extract cleanliness by optimising liquid/liquid extraction conditions
Minimise the impact of some common problems in liquid/liquid extraction
Recognise practical aspects of support-assisted liquid/liquid extraction

Content
Liquid / liquid Extraction I
Liquid / liquid Extraction II
Drawbacks of Liquid/liquid Extraction
Emulsions
Support-Assisted Liquid / liquid Extraction I
Support-Assisted Liquid / liquid Extraction II

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Liquid / liquid Extraction I

Liquid/liquid extraction is one of the most widely employed and useful techniques in
pharmaceutical sample preparation. This is due to a number of characteristics, including
simplicity, rapid method development, and reasonable selectivity.
Liquid/liquid extraction involves adding a solvent to the sample that is immiscible, followed
by selective partitioning of analytes versus contaminants between the two phases. In the
interest of extraction completeness, it is necessary to use an adequate amount of
extracting solvent to capture all of the analytes from the original sample. In most cases, a
volume of extracting solvent equal to or less than the original sample volume is adequate,
assuming reasonable analyte solubility in the solvent. This extracting solvent is added to
the sample, then the two phases are agitated, by vortexing or shaking, to bring about
substantial physical mixing. After agitation, the phases are allowed to separate.

Complete and incomplete analyte extraction

After the two phases separate, the phase containing the analytes is removed, either by
careful pipetting, or by freeze-pour. Freeze-pour involves placing the samples in a
freezer at a sub-zero temperature to freeze the aqueous layer, after which the organic
layer may be simply poured off. This approach works best for extracting solvents less
dense than water, so the non-frozen organic layer is on top.

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Organic layer isolation

Since most pharmaceutical samples are aqueous, the extracting solvents used are
typically non-polar organics. Common solvents used for liquid/liquid extraction are ethyl
acetate, methyl tertiary-butyl ether (MTBE), methylene chloride, hexane, and mixtures
thereof.

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Liquid / liquid Extraction II

After collection of the extraction solvent, the solvent may be dried down and reconstituted,
in most cases with a different solvent. Since most LC-MS mobile phases are aqueous in
nature, and therefore incompatible with most extracting solvents, dry down and
reconstitution allows transfer of the purified analytes into a more compatible solvent. In
many cases, the reconstitution solvent used is the LC mobile phase.

If the analytes are not yet adequately pure, a second extraction of the organic solvent
using an aqueous phase is sometimes used (referred to as a back-extraction). This
approach works best with basic compounds, which can be neutralized (making them more
organic-soluble) by raising the pH of the original sample, extracting into an organic
solvent, then back-extracting into an aqueous phase using an acidic buffer (ionizing the
analytes, thus making them more water-soluble).
Another approach to liquid/liquid extraction for highly water-soluble analytes is extraction
of non-polar, organic-soluble interferences from the sample, leaving the analytes behind in
the aqueous phase. This approach has the specific drawback that the low-volatility
aqueous phase is not readily concentrated, as is an organic solvent.

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Drawbacks of Liquid/liquid Extraction

Liquid/liquid extraction has a number of drawbacks that limit its use as a sample
preparation technique. These include limited selectivity, difficulty of automation, and
emulsions.
Liquid/liquid extraction works via solubility characteristics of the analytes, and relies on
differential solubility of the analytes versus other sample matrix components. Since the
solvents used are typically non-polar organics, hydrophobic analytes are extracted into the
organic layer, but other non-polar interferences (for example, serum lipids) are often coextracted. Not only does this give a less pure extract than desired, but the co-extractants
may accumulate on the analytical column, typically a reversed phase column.
For highly water-soluble analytes, in which the organic phase extracts the contaminants
versus the analytes, other water-soluble matrix components may stay in the aqueous
phase with the analytes, notably peptides and proteins. Again, proteins may accumulate
on the analytical column over time, causing back-pressure and other analytical difficulties.

Liquid/liquid extraction drawbacks

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Another drawback of liquid/liquid extraction for large numbers of samples is that it is not
well-suited to automation. Other approaches, SPE in particular, are much easier to
automate.

Emulsions
One major drawback of liquid/liquid extraction is the formation of emulsions. Emulsions
occur when the sample contains a high level of surfactant-like compounds (notably
phospholipids), that prevent clean separation of the two phases. With emulsions, a midzone between the two phases is created, having intermediate solubility in each of the two
phases, and making it difficult to quantitatively collect one phase or another.

Emulsion formation
Emulsions often occur with samples where the animal (or human) diet is high in fats.
Thus, emulsions sometimes appear when passing from pre-clinical trials, with animals on
low-fat, controlled diets, to clinical trials, with humans who may be on high-fat diets. This
characteristic problem makes liquid/liquid extraction a less dependable procedure if it is
expected that the same extraction protocol will be used for both pre-clinical 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.
Emulsions may sometimes be disrupted, or broken-up, by the addition of salt to the
emulsion. The salt changes the capacity of the aqueous phase to accommodate the
marginally-soluble components in the system, thus driving them into the organic phase.

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Support-Assisted Liquid / liquid Extraction I

As discussed (please refer to the Primary Sample Preparation Techniques module from
the Sample Preparation Channel), support-assisted liquid/liquid extraction (SALL) is a
useful variation on standard liquid/liquid extraction. The chemistry of this technique is
virtually identical to liquid/liquid extraction, but the physical nature of the technique offers
distinctive benefits unique to the approach.
In SALL, an aqueous sample is applied to a high-surface area matrix such that the sample
is dispersed over the surface of the matrix, creating a potential interface for extraction.
This is most often executed in a syringe barrel-type column format, similar to those used
in SPE. The most common matrix employed for sample dispersion is purified
diatomaceous earth. A water-immiscible organic solvent is subsequently passed over the
matrix holding the aqueous layer, and analytes of interest partition into the organic phase.
The amount of matrix required is directly proportional to the volume of aqueous sample to
be extracted. Therefore, most commercial suppliers of SALL products specify products
based on the sample size for which they are to be used.

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Support-Assisted Liquid / liquid Extraction III

Some commercial SALL products are available pre-buffered. This allows for the pH of the
sample to be changed simply by applying the sample to the column. The approach is
useful for analytes where pH can improve extraction into the organic phase (either basicbuffered for amines, or acidic-buffered for acids).

The organic solvents most commonly used are the same solvents used in conventional
liquid/liquid extraction; ethyl acetate, methyl tert-butyl ether (MTBE), methylene chloride,
hexane, and mixtures thereof.
Another advantage of SALL is that the nature of the column technique allows for superior
automation as compared to conventional liquid/liquid extraction.

Finally, the SALL approach is essentially free of the emulsion problems common to
conventional liquid/liquid extraction. In pharmaceutical applications, in particular, this
allows for the use of the technique across the range of samples from pre-clinical to
clinical, with reduced concern for method ruggedness.
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