Solid phase extraction SPE

SPE is currently used for the purification or concentration of a crude extract prior to the measurement of one or several of its constituents. SPE is a clean analytical procedure which has drastically changed the classical approaches of solvent extraction as liquid/liquid extraction in a separating funnel, comparatively slower and that use relatively large amounts of liquid organic solvents. 

The solid sorbents closely resemble to that of the solid stationary phases of liquid chromatography. Bonded silica gels (of reverse phase polarity) with a particle size of between 4 and 100 p.m, allow a percolation of faster flow rate. Other adsorbents, containing graphite or co-polymers such as styrene-divinylbenzene of large functional group bonded surfaces, are more stable in acidic solutions. 

Non-polar compounds are isolated from a polar matrix with a cartridge or a disc of C-18 derivatized silica gel. Polar compounds are isolated Ifom a non-polar matrix with a cartridge filled with a normal phase. Charged species can be isolated on an ion exchange phase. These three situations are well adapted to analysis by HPLC. 

A current research theme concerns the preparation of adsorbents having a marked affinity for a particular type of compound. For this, polymers are synthesized which possess recognition sites in the form of surface deformations, appropriate for trapping molecular targets. The principle resembles that of immuno-extraction, which is even more selective, as explained in the next paragraph. 

SPE is an exhaustive and almost solvent-free sample preparation technique. Typically, a tube is filled with a sorbent, which can be porous particles or a polymerized monolith. Various interactions are used to extract analytes from complex samples. Many of the commercially available SPE systems are for single use, but some, like RAM (restricted access materials) and MIP (molecular imprinted polymers), are typically obtained as reusable extraction devices. As will be discussed in detail, the extraction sorbents mainly function as normal phase, reversed phase, cation exchange, anion exchange, or a combination of these. 

An important characteristic is the amount of analyte that can be extracted without saturating the sorbent, defined as the capacity of the sorbent. The capacity depends on, as with all chromatographic techniques, the type of sorbent (with or without a bonded phase) and the surface area. The larger the surface area, the higher the capacity. When testing the capacity, this should be done with a realistic sample matrix, since capacity does not depend only on the analyte as other compounds can also occupy functional groups (and thereby lower the capacity for the analyte). 

As a rule of thumb for particle-packed normal-phase and reversed-phase cartridges, the capacity is approximately 5% of the mass of the packed material. This means that not more than 5 mg of analytes should be applied on a 100 mg packed SPE cartridge. 

The capacity of other materials, such as weak ion exchangers, should be found experimentally. 

4) rinsing the sorbent with solvent of appropriate elution strength, and 

Using SPE disks or small bead-volume cartridges for miniaturisation of sample volumes and robotics with 96-well plates, automation is facihtated for large numbers of plasma and urine samples, especially in combination with LC/MS/MS detection [53, 54]. On-Hne sample concentration by trapping-columns for automated LC/MS-analysis will be discussed in Section 10.2.6. 

Immunoaffinity extraction (lAE) is probably the most effective way of extracting trace amounts of analytes from biosamples, espedaUy if coupled directly to LC/ MS/MS. Henion et al. published a method for automated lAE-LC/LC-MS/MS-anal-ysis for the detection of LSD metabolites and beta-agonists in urine, and benzodiazepines from chemical libraries [55, 56], whereas Maurer et al. used lAE for the determination of amanitines in urine [57]. Approximately 20-fold higher sensitivity was achieved in these studies by lAE compared to the standard SPE method. However, a disadvantage of lAE is the narrow linear range due to the fact that the antibodies are easily overloaded. 

Of course such ideal conditions are seldom if ever fully satisfied and SPE can give incomplete recovery of analytes (monitored by use of internal standards) and incomplete removal of potential interferences. These problems can be alleviated by adjusting the conditions 

More sophisticated fully automated multichannel versions of this on-line approach are also available from several manufacturers. The current ultimate in automated multiplexed SPE for high throughput operation uses 96-well plates (or even 384-well plates) with robotic addition of extracts and addition/removal of solvents a recent article (Majors 2004) describes developments in this technology. However, in most instances of rigorously validated methods, aU critical pipetting steps (sample aUquoting and addition of standards) is done manually 

Cleaning Wash. To selectively remove the nonretained compounds, wash the SPE phase with the same volume of noneluting solvent (e.g. aqueous buffer for reverse phase systems) as that used in the pre-conditioning step. Some analyte might be washed out also in this step so this eluant can be retained for secondary SPE treatment if desired however, a suitable SIS (Table 2.1) will correct for any such losses so this eluant is often 

Naturally details wiU vary with the nature of both analyte and matrix, and with the overall purpose of the analysis, but the principles underlying these five steps are important in all cases. 

The conventional process of solid-phase extraction involves passing a liquid sample over a solid surface in order to separate target analytes from the sample matrix. The attractive interactions of the analyte with the solid 

At the time of this writing, there are at least six main suppliers of solid-phase extraction equipment and supplies. At least three of the suppliers offer technical guides and general solid-phase extraction literature.1-4 Table 1 lists the companies and their Internet sites. Thurman and Mills provide a comprehensive list of SPE product suppliers in their book Solid Phase Extraction (Wiley, 1998).5 

Chemically bonded silica gels with cyanopropyl, aminopropyl, and diol functional groups are also available and each has been used for normal-phase as well as reversed-phase separations. Polymeric-based packings are available and can be used for ion-exchange or reversed-phase applications. Cross-linked polymeric based packings have been combined with ion-exchange materials to create a more pH-stable stationary phase. Graphitized carbon 

TABLE I Solid-Phase Extraction Equipment and Suppliers 

TABLE 2 Commercially Available Sorbents for Solid-Phase Extraction (compiled from refs. 1-5,15,16) 

Rather than carry out liquid-liquid extractions, which are time-consuming and lead to large quantities of waste, solid supports can be constructed that have high affinity for selected types of molecules. Simple filtration of a solution through a column of the solid-phase medium results in efficient extraction of the product. This method is quite widely used and is of particular interest for fluorous chemistry (see below). 

The principal goals of SPE are analyte enrichment, purification, and medium exchange (e.g., from aqueous to organic) [11]. SPE is very similar to liquid chromatography and uses the same physicochemical principles, solvents, and stationary (solid) phases. It has become a very successful and widespread method most biomedical laboratories use it in everyday practice. 

like other sample preparation procedures, requires careful and accmate execution. As it is very common, various steps of the SPE procedure will be 

Development of SPE methods requires a sound knowledge of liquid chromatography. The most important parameters are the SPE cartridge (type of the sorbent) and the type of solvents used. The size of the cartridge (which determines the sample amount) is also important Too small sorbent size is easily overloaded, while too large sorbent size may bind the analyte, thereby decreasing recovery. Like in chromatography, various additives, especially buffers, may be used to improve performance. 

The three most important types of SPE are RP, normal phase (NP), and ion exchange (IE). RP-SPE is best to clean up polar samples from an aqueous phase NP-SPE is best used for apolar compounds dissolved in an organic matrix, while ionic compounds are best retained on lE-SPE cartridges. Fig. 4 provides a useful guide for selecting the SPE method. Retention mechanism on SPE cartridges is essentially the same as that in chromatography and this is discussed in more detail in the next chapter. 

Reverse-phase SPE separations, like RP chromatography, uses a polar (aqueous) sample, an apolar stationary phase, and an organic solvent to elute the analyte. Commonly used stationary phases are alkyl-bonded siUcas such as C18, C8, and C4, 

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In order to one of the most effective separation and preconcentration procedure in trace metal analysis is solid phase extraction (SPE) of analyte. 

Supercritical fluid extraction (SFE) and Solid Phase Extraction (SPE) are excellent alternatives to traditional extraction methods, with both being used independently for clean-up and/or analyte concentration prior to chromatographic analysis. While SFE has been demonstrated to be an excellent method for extracting organic compounds from solid matrices such as soil and food (36, 37), SPE has been mainly used for diluted liquid samples such as water, biological fluids and samples obtained after-liquid-liquid extraction on solid matrices (38, 39). The coupling of these two techniques (SPE-SFE) turns out to be an interesting method for the quantitative transfer... 

When a first column of a very short length (and therefore a low selectivity) is used (this is especially suitable for multiresidue methods), we talk about an on-line precolumn (PC) switching technique coupled to LC (PC-LC or solid-phase extraction (SPE)-LC). This is particulary useful for the enrichment of analytes, and enables a higher sample volume to be injected into the analytical column and a higher sensitivity to be reached. The sample is passed through the precolumn and analytes are retained, while water is eliminated then, by switching the valve, the analytes retained in the precolumn are transferred to the analytical column by the mobile phase, and with not just a fraction, as in the previous cases. 

Although most of the reactions are performed in liquid state - which is the simplest reaction medium - a combination of solid-phase extraction (SPE) and reaction... 

Prepare the solid-phase extraction (SPE) tube (1 ml LC-18 SPE tube) by conditioning with 1 ml of methanol followed by 1 ml of water. 

To determine secondary alkanesulfonates in sewage wastewaters, solid phase extraction (SPE) and a single-step procedure which combines elution and injection port derivatization for analysis with GC-MS were developed [36]. Again a tetrabutylammonium ion pair reagent was employed both to elute the secondary alkanesulfonates as their ion pairs from CI8-bonded silica disks and to derivatize sulfonate ion pairs under GC injection port conditions. Secondary alkanesulfonates were effectively recovered from samples of raw sewage (>92%) and from primary (>98%) and secondary (>85%) effluents. No... 

Kraemer-Schafhalter, A., Fnchs, H., and Pfannhauser, W., Solid-phase extraction (SPE) a comparison of 16 materials for the purification of anthocyanins from Aronw melanocarpa var Nero, J. Sci Food Agr., 78, 435, 1999. 

Fractions are concentrated under vacuum below 35°C. Alternatively, they can be purified and concentrated by solid phase extraction (SPE) before identification and quantification, which is a valuable procedure for samples containing low amounts of pigments. After concentration, fractions can be stored in small flasks, protected from light, sealed under inert atmosphere, and kept below -20°C until analysis. 

Carmine extracted from cochineal insects is one of the most used natural colorings for beverages and other foods. Some representative articles refer to isolation and spectrometric analysis or the use of HPLC or capillary electrophoresis (CE) to separate and characterize all cochineal pigments. Its active ingredient, carminic acid, was quantified by rapid HPLC-DAD or fluorescence spectrometry. Carminic acid, used as an additive in milk beverages, was separated within 9 min using a high-efficiency CE separation at pH 10.0 after a previous polyamide column solid phase extraction (SPE), ... 

Water solubility, dissociation constant(s) and n-octanol/water partition coefficients allow one to predict how an analyte may behave on normal-phase (NP), reversed-phase (RP), or ion-exchange solid-phase extraction (SPE) for sample enrichment and cleanup. 

Another equally important consideration before development of a determinative or confirmatory method is an understanding of the chemical properties of the analyte. Such an understanding becomes the cornerstone of a successful method since the unique chemical properties of each analyte provide the basis for isolation and detection schemes. Table 1 lists some of the important chemical properties that could be considered. For example, knowing the or p/fb of an analyte could influence the choice of a liquid-liquid extraction scheme, solid-phase extraction (SPE) cartridge, mobile phase pH, or mass spectrometric ionization. Knowing the overall polarity of the analyte can be very helpful in the evaluation of an extraction or separation. Currently, computational methods are available to obtain an estimate of the logP... 

The extent of cleanup needed depends on the target analyte, the quality of the sample extract, the method of detection and sensitivity. Liquid-liquid partition (LLP) and solid-phase extraction (SPE) columns such as the Cig cartridge and macroporous diatomaceous column are the cleanup method of choice. 

Analytical methods for parent chloroacetanilide herbicides in soil typically involve extraction of the soil with solvent, followed by solid-phase extraction (SPE), and analysis by gas chromatography/electron capture detection (GC/ECD) or gas chromatog-raphy/mass spectrometry (GC/MS). Analytical methods for parent chloroacetanilides in water are similarly based on extraction followed by GC with various detection techniques. Many of the water methods, such as the Environmental Protection Agency (EPA) official methods, are multi-residue methods that include other compound classes in addition to chloroacetanilides. While liquid-liquid partitioning was used initially to extract acetanilides from water samples, SPE using... 

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. 

A soil sample (10 g) was extracted by mechanically shaking with methanol-deionized water. This mixture was filtered and a portion was removed for partitioning into toluene-hexane. A phenyl solid-phase extraction (SPE) cartridge was employed... 

Residues of isoxaflutole, RPA 202248 and RPA 203328 are extracted from surface water or groundwater on to an RP-102 resin solid-phase extraction (SPE) cartridge, then eluted with an acetonitrile-methanol solvent mixture. Residues are determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Cg column. Quantitation of results is based on a comparison of the ratio of analyte response to isotopically labeled internal standard response versus analyte response to internal standard response for calibration standards. 

The need to understand the fate of pesticides in the environment has necessitated the development of analytical methods for the determination of residues in environmental media. Adoption of methods utilizing instrumentation such as gas chro-matography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), liquid chromatography/tandem mass spectrometry (LC/MS/MS), or enzyme-linked immunosorbent assay (ELISA) has allowed the detection of minute amounts of pesticides and their degradation products in environmental samples. Sample preparation techniques such as solid-phase extraction (SPE), accelerated solvent extraction (ASE), or solid-phase microextraction (SPME) have also been important in the development of more reliable and sensitive analytical methods. 

Solid phase extraction (SPE) is a very simple, rapid and reproducible cleanup technique that is now widely accepted as an alternative to the time-consuming liquid-liquid extractions. Additionally, SPE uses relatively small volumes of solvents, and is easy to automate. It is available in a number of different formats, including cartridges, disks, loose material, well plates or SPME using film-coated capillaries. SPE can be considered as an extraction technique when used for isolation and concentration or a cleanup technique when used to remove co-extractives from solvent extracts. The use of SPE for cleanup is discussed later. 

The development of solid-phase extraction (SPE) absorbents such as silica gel, alumina and Florisil tremendously aided in the purification or cleanup of pesticide residues from water. 

The extent of the cleanup depends on the sample matrix to be analyzed, the extraction procedure, the method of detection and the desired sensitivity. Generally, the cleanup method is liquid-liquid partitioning (LLP), but recently it has become simpler and more reliable to use solid-phase extraction (SPE) columns. 

A cleanup procedure is usually carried out to remove co-extracted matrix components that may interfere in the chromatographic analysis or be detrimental to the analytical instrument. The cleanup procedure is dependent on the nature of the analyte, the type of sample to be analyzed, and the selectivity and sensitivity of the analytical instrument used in the analysis. Preliminary purification of the sample extracts prior to chromatographic separation involves liquid-liquid partitioning and/or solid-phase extraction (SPE) using charcoal/Celite, Elorisil, carbon black, silica, or aminopropyl-silica based adsorbents or gel permeation chromatography (GPC). 

Crop material is homogenized with acetonitrile-water (9 1, v/v). The crop extract is centrifuged and an aliquot is rotary evaporated to a small volume. The sample is subjected to a Cig solid-phase extraction (SPE) cleanup procedure. The concentrated eluate is subjected to liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. 

Diolsilane solid-phase extraction (SPE) cartridges, 3-mL (J.T. Baker Inc.) Aminopropyl SPE cartridges, 3-mL (J.T. Baker Inc.)... 

The water samples are extracted by solid-phase extraction (SPE) and analyzed by GC/NPD. 

Solid-phase extraction (SPE) cartridge, LC Si 2-g, 12-mL (Supelco), or equivalent ABC Laboratories Model SP 1000 gel permeation chromatograph system equipped with a 2.5 x 32.0 cm glass column of Bio-Beads S-X3 Select 200-400 mesh (ca 50 g, Bio-Rad Laboratories) preconditioned with ethyl acetate-cyclohexane (1 1, v/v), or equivalent... 

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

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