Liquid Extraction LLE

LLE is also called solvent extraction. It is used for both sample cleanup and concentration of the analyte. LLE is based on the phenomenon that a compound will distribute between two nonmiscible liquid phases. The equilibrium is strongly determined by the physicochemical parameters of the two liquids and can be advantageously used to concentrate some while dilute other components of the sample. 

Unsupported liquid membrane techniques with three phases involve an aqueous sample phase separated from another aqueous phase (called as receiver phase) by a layer of organic solvent (e.g., octane). Analyte components first diffuse from the sample into the organic liquid membrane and then back-extract out of the membrane into the receiving phase. At the same time, interferences do not diffuse into the organic membrane layer but stay in the original sample phase. 

Supported liquid membrane extraction techniques employ either two or three phases, with simultaneous forward- and back-extraction in the latter configuration. The aqueous sample phase is separated from the bulk organic or an aqueous receiver phase by a porous polymer membrane, in the form of either a flat sheet or a hollow fiber that has been impregnated with the organic solvent phase. The sample phase is continuously pumped, the receiver phase may be stagnant or pumped, and the organic phase in the membrane pores is stagnant and reusable [8-10]. 

When performed in the organic chemistry laboratory, LLE is a preparative technique, performed manually and employing separatory funnels. When performed in the pharmaceutical analysis laboratory, LLE can be either preparative-scale (hundreds of milliliters), using a separatory funnel, or analytical-scale (microliters to a few milliliters) and performed using test tubes, vials, and glass or polypropylene pipets. 

Liquid-liquid extraction is a very good sample cleanup technique for nonpolar or moderately polar analytes that can be deionized in aqueous solution by simple pH adjustment. Lrom a practical perspective, the best recoveries are obtained when an excess of organic solvent (3- to 10-fold excess) or multiple-step extractions are used. Multiple-step extractions have recently fallen into disfavor for routine use because they are time-consuming and labor-intensive. 

Method development for LLE is usually straightforward, as the approach can have a high probability of some success on the first attempt. The technique can, however, be labor-intensive, and it does not provide good recoveries for highly polar or zwitterionic species unless ion-pair reagents are added to the 

FIGURE 1 A pictorial representation of liquid-liquid extraction, including pH adjustment, extraction, phase separation, solvent evaporation and resuspension steps. 

For an electronically neutral analyte that is partitioning between water and organic liquid phases, the equilibrium process can be described as the distribution 

---

Usually the first step is an extraction of the desired compounds from plant material. This extraction can be done by different solvents, e.g., methanol [85], n-hexane [86], petroleum ether or solvent mixtures such as methanol/chloroform [87]. The use of a second liquid-liquid extraction (LLE) with 0.1 M NaOH after extraction with a non-polar solvent like n-hexane makes a separate analysis of acidic cannabinoids possible, which can be found... 

Grade extracts of different plants are often rich in lipophilic substances, such as plant oils, chlorophylls, and waxes and also highly polar components such as tannines or sugars. Because the complicated liquid-liquid extraction (LLE) procedures are... 

The most widely employed techniques for the extraction of water samples for triazine compounds include liquid-liquid extraction (LLE), solid-phase extraction (SPE), and liquid-solid extraction (LSE). Although most reports involving SPE are off-line procedures, there is increasing interest and subsequently increasing numbers of reports regarding on-line SPE, the goal of which is to improve overall productivity and safety. To a lesser extent, solid-phase microextraction (SPME), supercritical fluid extraction (SEE), semi-permeable membrane device (SPMD), and molecularly imprinted polymer (MIP) techniques have been reported. 

Depicted in Fig. 2, microemulsion-based liquid liquid extraction (LLE) of biomolecules consists of the contacting of a biomolecule-containing aqueous solution with a surfactant-containing lipophilic phase. Upon contact, some of the water and biomolecules will transfer to the organic phase, depending on the phase equilibrium position, resulting in a biphasic Winsor II system (w/o-ME phase in equilibrium with an excess aqueous phase). Besides serving as a means to solubilize biomolecules in w/o-MEs, LLE has been frequently used to isolate and separate amino acids, peptides and proteins [4, and references therein]. In addition, LLE has recently been employed to isolate vitamins, antibiotics, and nucleotides [6,19,40,77-79]. Industrially relevant applications of LLE are listed in Table 2 [14,15,20,80-90]. 

There are basically three methods of liquid sampling in GC direct sampling, solid-phase extraction and liquid extraction. The traditional method of treating liquid samples prior to GC injection is liquid-liquid extraction (LLE), but several alternative methods, which reduce or eliminate the use of solvents, are preferred nowadays, such as static and dynamic headspace (DHS) for volatile compounds and supercritical fluid extraction (SFE) and solid-phase extraction (SPE) for semivolatiles. The method chosen depends on concentration and nature of the substances of interest that are present in the liquid. Direct sampling is used when the substances to be assayed are major components of the liquid. The other two extraction procedures are used when the pertinent solutes are present in very low concentration. Modem automated on-line SPE-GC-MS is configured either for at-column conditions or rapid large-volume injection (RLVI). 

Diffusive sampler Membrane extraction (MESI) Liquid-liquid extraction (LLE) Solid-phase extraction (SPE) SPE-PTV-GC Solid-phase microextraction (SPME) Headspace GC (SHS, DHS) Large-volume injection (LVI) Coupled HPLC-GC Membrane extraction (MESI) Difficult matrix introduction (DMI) Conventional solvent extraction methods 1 Pressurised solvent extraction methods Headspace GC (SHS, DHS) Thermal desorption (TD, DTD) Pyrolysis (Py) Photolysis Photon extraction (LD) Difficult matrix introduction (DMI)... 

Recent Developments in Liquid-Liquid Extraction (LLE) for Clean-Up... 

Application of SPE to sample clean-up started in 1977 with the introduction of disposable cartridges packed with silica-based bonded phase sorbents. The solid phase extraction term was devised in 1982. The most commonly cited advantages of SPE over liquid-liquid extraction (LLE) as practiced on a macroscale include the reduced time and labor requirements, use of much lower volumes of solvents, minimal risk of emulsion formation, selectivity achievable when desired, wide choices of sorbents, and amenability to automation. The principle of operation consists of four steps (1) conditioning of the sorbent with a solvent and water or buffer, (2) loading of the sample in an aqueous or aqueous low organic medium, (3) washing away unwanted components with a suitable combination of solvents, and (4) elution of the desired compound with an appropriate organic solvent. 

Due to the predicted and previously detected low concentrations of pesticides in environmental samples (usually around the nanogram per liter level), a preconcentration step of the water samples is necessary prior to measurement. In this way, a preconcentration factor of several orders of magnitude (200-1,000-fold) is mandatory to reach the low detection limits necessary for the identification of pesticides, especially in complex wastewater samples. Also, the use of surrogate standards (e.g., triphenyl phosphate) added before the extraction step is a common practice in order to account for possible errors during the extraction process and for quantitative purposes. The commonly used extraction methods for polar compounds from water matrices involve isolation using liquid-liquid extraction (LLE) and solid-phase extraction (SPE), which are commented on below. Other methods such as semipermeable membrane devices (SPMD) are also mentioned. 

The complex composition of aqueous environmental sample matrices, especially sewage and marine water samples, and the low concentrations in which the surfactants are generally found, have made it necessary to perform an initial stage of concentration and purification of the analytes prior to its analysis. Traditionally, such steps were carried out off-line with procedures based on liquid—liquid extraction (LLE), sublation or steam distillation, followed by chromatographic clean-up steps. 

Zweigenbaum and co-workers [11] used high flow rates and an isocratic system using a Mac-Mod Rapid Resolution column (15 mm x 2.1 mm, 3 pm) to perform the fast separation of six benzodiazepines isolated from human urine using a 96-well liquid-liquid extraction (LLE). 

A pivotal step in the analytical process is sample preparation. Frequently liquid-liquid extractions (LLEs) are used. Solvents, pH, and multiple back extractions are all manipulated to increase selectivity and decrease unwanted contaminants before injection on the GC system. Solid phase extraction (SPE) is more convenient than it used to be because of an increase in commercially available SPE columns. SPE columns are packed with an inert material that binds the drug of interest, allowing impurities to pass through. As with LEE, solvent choices and pH affect retention and recovery. There are three commercially available types of SPE columns, diatomaceous earth (which uses the same principles as LLE), polystyrene-divinylbenzene copolymer, and mixed mode bonded silica (Franke and de Zeeuw, 1998). 

Fentanyl, a potent synthetic narcotic analgesic, and norfentanyl were determined in postmortem samples nsing liqnid-liquid extraction (LLE) and HPLC-ESI(h-)-MS-MS enabling the authors to determine the distribntion of parent compound and metabolite in tissnes and organs of a suicidal fatality case [36]. However, matrix effects were not investigated. 

In the previous section (2.1.2) we were concerned with phase transitions between liquid and vapor and discussed the various techniques for effecting such changes. In this section we will look at transferring solute components from one liquid phase to a second liquid phase. This technique is referred to as liquid-liquid extraction (LLE). The main restriction on this separation technique is that the two phases must be immiscible. By immiscible liquids we mean two liquids which are completely insoluble in each other. A little reflection will reveal it is very difficult to have two liquids that are mutually insoluble. If such a system were achievable, then the total pressure, P, of the system would be defined by. 

High-performance LC determination is also compatible with the extraction of environmental water with an organic solvent such as methylene chloride or ethyl acetate (31,32) and solid-phase extraction (SPE) (33,34). Solid-phase extraction and liquid-liquid extraction (LLE) have been compared with respect to their ability to preconcentrate pesticides prior to HPLC analysis. The reproducibility of the method is better when C,8 cartridges are used than with conventional LLE, but LLE sometimes gives better recoveries, for example, for dimethoate, chlor-pyriphos ethyl, and carbofenothion (35). 

---