Solid phase extraction techniques for biological samples

  • Solid phase Extraction
  • Normal phase solid phase extraction
  • Reverse phase solid phase extraction
  • Ion exchange solid phase extraction

i)Cation exchange solid phase extraction

ii)Anion exchange solid phase extraction

  • Solid phase microextraction

Solid Phase Extraction:

Solid phase extraction is an extraction method that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. It is usually used to clean up a sample before using a chromatographic or other analytical method to quantify the amount of analyte in the sample. The general procedure is to load a solution onto the SPE phase, wash away undesired components, and then wash off the desired analytes with another solvent into a collection tube. Solid-phase extractions use the same type of stationary phases as are used in liquid chromatography columns. The stationary phase is contained in a glass or plastic column above a frit or glass wool. The column might have a frit on top of the stationary phase and might also have a stopcock to control the flow of solvent through the column. Commercial SPE cartridges have 1-10 ml capacities and are discarded after use. It is usually used to clean up a sample before using a chromatographic or other analytical method to quantify the amount of analyte in the sample. Solid phase extraction procedures are used not only to extract traces of organic compounds from environmental samples but also to remove the interfering components of the complex matrices in order to obtain a cleaner extract containing the analytes of interest. The SPE technique is widely applied for isolation of analytes from a liquid matrix and purified extracts.

Solid phase extraction is the very popular technique currently available for rapid and selective sample preparation. The versatility of SPE allows use of this technique for many purposes, such as purification, trace enrichment, desalting, derivatisation and class fractionation. The last few years have been chracterized by a wide interest in this technique and many publications describing SPE methods have been published.

This period is connected with the intensive development of research procedures for novel types of sorptive materials and lasted from the late 1960s until the beginning of the 1980s. The introduction of a wide spectrum of sorptive materials into analytical procedures gave a new stimulus for the development of SPE methodology.

The principle of SPE is similar to that of liquid-liquid extraction involving a partitioning of solutes between two phases. However, instead of two immiscible liquid phases, as involves partitioning between a liquid and a solid phase.

This sample treatment technique enables the concentration and purification of analytes from solution by sorption on a solid sorbent and purification of extract after extraction. The general procedure is to load a solution onto the SPE solid phase, wash away undesired components, and then wash off the desired analytes with another solvent into a collection on tube.

Mechanism of Solid Phase Extraction Process:

The selection of an appropriate SPE extraction sorbent depends on understanding the mechanism of interaction between the sorbent and analyte of interest. That understanding in turn depends on knowledge of the hydrophobic, polar and ionogenic properties of both the solute and the sorbent. The most common retention mechanisms in SPE are based on van der Waals forces.

Each sorbent offers a unique mix of these properties which can be applied to a wide variety of extraction problems. Four general theory interactions exist:Reversed phase involves a polar or moderately polar sample matrix and a nonpolar stationary phase. The analyte of interest is typically mid- to nonpolar. Retention of organic analytes from polar solutions onto these SPE materials is due primarily to the attractive forces between the carbon-hydrogen bonds in the analyte and the functional groups on the sorbent surface. These nonpolar – nonpolar attractive forces are commonly called van der Waals forces or dispersion forces. The nonpolar solvent, which can disrupt the forces between the sorbent and compound, is used to elute an adsorbed compound from a reversed phase SPE tube or disk. The following materials are used as reversed phase: carbonbased media, polymer-based media, polymer-coated and bonded silica media. Carbon-based media consist of graphitic, non-porous carbon with a high attraction for organic polar and nonpolar compounds from both polar and nonpolar matrices. Retention of analytes is based primarily on the analyte’s structure, rather than on interactions of functional groups on the analyte with the sorbent surface.

Polymer-based sorbents are styrene/divivinylbenzene materials. It is used for retaining hydrophobic compounds which contain some hydrophilic functionality, especially aromatics. Elution steps can be done with mid- and nonpolar solvents, because the polymeric packing is stable in almost all matrices. Polymer-coated and bonded silica media is a hydrophobic-bonded silica that is coated with a hydrophilic polymer. The pores in the polymer allow small, hydrophobic organic compounds of interest to reach the bonded silica surface, while large interfering compounds are shielded from the bonded silica by the polymer and are flushed through the SPE tube.

Normal phase solid phase extraction:

Normal phase involve a polar analyte, a mid- to nonpolar matrix and a polar stationary phase. Retention of an analyte under normal phase conditions is primarily due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface. These include hydrogen bonding, л- л interactions, among others. A compound adsorbed by these mechanisms is eluted by passing a solvent that disrupts the binding mechanism, usually a solvent that is more polar than the sample’s matrix

The bonded silica have short alkyl chains with polar functional groups bonded to the surface. The silica because of their polar functional groups, are much more hydrophilic relatively to the bonded reversed phase silica. As with typical normal phase silica, these sorbents can be used to adsorb polar compounds from nonpolar matrices.

The polar adsorption material is modified silica gel commonly used as the base of all of the bonded phases. The functional groups that are involved in the adsorption of compounds from nonpolar matrices are the free hydroxyl group on the surface of the silica particles. That may be used to adsorb polar compounds from nonpolar matrices with subsequent elution of the compounds in an organic solvent more polar than the original sample matrix.

Reverse phase solid phase extraction:

Reversed phase separations involve a polar or moderately polar sample matrix and a nonpolar stationary phase. The analyte of interestis typically mid- to nonpolar. Several SPE materials, such as thealkyl- or aryl-bonded silicas are in the reversed phase category. Here, thehydrophilic silanol groups at the surface of the raw silica packing(typically 60Å pore size, 40μm particle size) have been chemically modified with hydrophobic alkyl or aryl functional groups byreaction with the corresponding silanes.

CH3CH3

Si-OH + Cl-Si-C18H37® Si-O-Si-C18H37 + HCl

CH3CH3

Retention of organic analytes from polar solutions onto these SPE materials is due primarily to the attractive forces between the carbon-hydrogen bonds in the analyte and the functional groups on the silica surface. These nonpolar-nonpolar attractive forces are commonly called van der Waals forces, or dispersion forces. To elute an adsorbed compound from a re-versed phase SPE tube or disk, use a nonpolar solvent to disrupt the forces that bind the compound to the packing. LC-18 and LC-8 are standard, monomerically bonded silicas. Polymericallybonded materials, such as ENVI-18 and ENVI-8, result in a more complete coverage of the silica surface and higher carbon load-ing. Polymeric bonding is more resistant to pH extremes, and thus is more suitable for environmental applications for trapping or-ganic compounds from acidified aqueous samples. All silica-based bonded phases have some percentage of residual unreacted silanols that act as secondary interaction sites. These secondary interactions may be useful in the extraction or retention of highly polar analytes or contaminants, but may also irreversibly bind analytes of interest.

Ion exchange solid phase extraction:

Ion exchange SPE can be used for compounds that are charged. Anionic (negatively charged) compounds can be isolated on LC-SAX or LC-NH2bonded silica cartridges. Cationic (positivelycharged) compounds are isolated by using LC-SCX or LC-WCX bonded silica cartridges. The primary retention mechanism of the compound is based mainly on the electrostatic attraction of the charged functional group on the compound to the charged group that is bonded to the silica surface. In order for a compound to retain by ion exchange from an aqueous solution, the pH of the sample matrix must be one at which both the compound of interest and the functional group on the bonded silica are charged. Also, there should be few, if any, other species of the same charge as the compound in the matrix that may interfere with the adsorption of the compound of interest. A solution having a pH that neutral-izes either the compound’s functional group or the functional group on the sorbent surface is used to elute the compound of interest. When one of these functional groups is neutralized, the electrostatic force that binds the two together is disrupted and the compound is eluted. Alternatively, a solution that has a high ionic strength, or that contains an ionic species that displaces the adsorbed compound, is used to elute the compound.

Solid phase microextraction:
Solid-phase microextraction is a simple and effective adsorption and desorption technique, which eliminates theneed for solvents or complicated apparatus, for concentrating volatile or nonvolatile compounds in liquid samples orheadspace. SPME is compatible with analyte separation and detection by gas chromatography and high-performance liquiqchromatography, and provides linear results for wide concentrations of analytes. By controlling the polarity and thickness ofthe coating on the fibre, maintaining consistent sampling time, and adjusting other extraction parameters, an analyst canensure highly consistent, quantifiable results for low concentration analytes. To date, about 400 articles on SPME have been

published in different fields, including environment, food, natural products, pharmaceuticals, biology,toxicology, forensics and theory. As the scope of SPME grew, new improvements were made with the appearance of newcoatings that allowed an increase in the specificity of this extraction technique. The key part of the SPME fibre is of coursethe fibre coating. At the moment, 27 variations of fibre coating and size are available. Among the newest are a fibre assemblywith a dual coating of divinylbenzene and Carboxen suspended in, and a series of 23 gauge fibresintended for specific septumless injection system. The growth of SPME is also reflected in the expanding number of theaccessories that make the technology even easier to use Also available is a portable field sampler which is a self-containedunit that stores the SPME fibre after sampling and during the shipment to the laboratory. Several scientific publications showthe results obtained in inter-laboratory validation studies in which SPME was applied to determine the presence of different

organic compounds at ppt levels, which demonstrates the reliability of this extraction technique for quantitative analysis.

References:

  1. Hennion, Marie-Claire (1999). "Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography".Journal of Chromatography A856(1-2): 3–54.doi:10.1016/S0021-9673(99)00832-8
  2. A. Żwir-Ferenc, M. Biziuk (2006). “Solid Phase Extraction Technique – Trends, Opportunities and applications”. Polish J. of Environ. Stud. Vol. 15, No. 5 (2006), 677-690.