Chromatography


Supported Liquid Extraction versus Liquid-Liquid Extraction: Extracting Acidic, Basic and Neutral Analytes from Plasma


James Edwards, Porvair Sciences, james.edwards@porvairsciences.com


Sample preparation is a key process when running any samples on a chromatography instrument. No matter how good the instrument is, to get the best sensitivity and reproducibility sample clean-up needs to be performed. A traditional method has been to use liquid-liquid extraction (LLE) which allows transfer of analyte from an aqueous solution to an organic solution. However, LLE can have issues around the time, irreproducibility and emulsions forming.


This application note discusses the advantages of using a supported liquid extraction (SLE) over an LLE method for extracting a range of analytes (acidic, basic and neutral) from pig plasma. The Microlute™ SLE plate offers an alternative method to LLE which follows the same principles of LLE methods. It improves recovery, reproducibility and speeds up sample preparation to allow a greater throughput of samples tested


Introduction


Liquid-liquid extraction is one of the oldest and most established sample preparation method. LLE was fi rst developed by the petroleum industry back in 1909 for the removal of aromatic hydrocarbons from kerosene [1].


The principles underlying LLE are well known and the number of publications simplifi es fi nding a method. LLE uses two different solvent phases which are immiscible with each other - typically an aqueous solution and a water immiscible solvent (e.g. dichloromethane, hexane, ethyl acetate) Shaking is then used to help drive the compound of interest from one phase to the other, usually from aqueous to an organic solution. The effi ciency at which this occurs is called the partition coeffi cient, Kp which is calculated using Equation 1.


thereby reducing the number of samples that can be processed at any one time. Both of these steps are also infl uenced by different lab users which can lead to variable results.


The Evolved Method


The alternative sample preparation based on the same principles of LLE is SLE. SLE uses diatomaceous earth as the support for the separation process to occur on. It is a naturally occurring, chemically inert porous material which has a high surface area. These properties allow water to easily load via capillary action onto the diatomaceous earth and adsorb to the surface of the diatom structures which make up the diatomaceous earth (Figure 2).


Equation 1. Equation to calculate the partition coeffi cient, Kp, where [A]1 is the concentration of analyte A in phase 1 and [A]2 is the concentration of analyte A in phase 2.


In chromatography the partition coeffi cient is known as ‘Log P of a molecule’. This applies the logarithm of Equation 1 when phase 1 is deionised water and phase 2 is n-octanol. This can provide information on how lipophilic/hydrophobic a molecule is, which is a useful measure for chromatography method development. Compounds with a low Log P are more hydrophilic which means they are more diffi cult to extract from aqueous solutions.


Due to the basic principles of solvent separation and easy access to common solvents, glassware, and equipment, LLE has become a popular extraction method in chromatography laboratories. However, this method has signifi cant disadvantages. To obtain effi cient partitioning between the phases, shaking is a crucial step. The action of shaking causes the surface area contact between the two solutions to become much higher and allow better transfer of analyte from one phase to the other. Insuffi cient mixing of the two solutions results in an ineffi cient LLE method. In contrast, too vigorous agitation can result in the formation of an emulsion - the formation of droplets of one solvent in the other which occurs when compounds are present which act as surfactants. The surfactant allows the two phases to interact with each other which causes an intermediate phase on the surface boundary (Figure 1).


The SLE process mimics LLE theory with two liquid phases interacting with each other. Phase one forms when the aqueous solution (water, plasma, serum etc.) is loaded and allowed to flow on via capillary action over several minutes. This allows the water to adsorb and create a phase on the diatomaceous earth’s surface which creates a very high surface area. The second phase is a water-immiscible organic solvent passed through the support bed under gravity. As the solvent flows past the adsorbed water, a partition forms with a very efficient phase boundary which acts like the shaking step in LLE. It allows sample clean-up by leaving unwanted compounds dissolved in the adsorbed water. These include compounds such as phospholipids or polar contaminants.


Figure 2. Diatomaceous earth (DE) is composed of naturally occurring silica-based mineral made from fossilised diatoms, a class of hard-shelled algae found in seas and oceans. Its small pore size and high surface area makes it the ideal material for absorption of aqueous solutions.


Figure 1. Diagram of a micelle which has formed part of an emulsion. The formation of them is due to the amphipathic nature of surfactants present which have both hydrophilic heads and hydrophobic tails.


LLE is also considered low throughput as samples are prepared serially rather than in parallel. Each step of the LLE process requires repeated agitation and transfer of solvent


SLE is generally considered a more reproducible method compared to LLE, from a sample-to-sample, experiment-to-experiment and analyst-to-analyst basis. It eliminates all the variable steps (shaking, manual handling and throughput) associated with LLE. Efficient interactions via solvent flow under gravity remove the need for shaking and manual extraction of the solvent layer. SLE can also be easily automated by combining 96 well plates and robotic samplers, allows a vast number of samples to be processed in just one day allowing for higher throughput of samples. In this application note we will demonstrate the advantages of SLE versus LLE for both time and performance.


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