Solid—phase extraction

Liquid-liquid extractions are very useful but have certain limitations. The extracting solvents are limited to those that are water immiscible (for aqueous samples). Emulsions tend to form when the solvents are shaken, and relatively large volumes of solvents are used that generate a substantial waste disposal problem. The operations are often manually performed and may require a back extraction. 

When silica particles are bonded with a hydrophobic phase, they become waterproof and must be conditioned in order to interact with aqueous samples. This is accomplished by passing methanol or a similar solvent through the sorbent bed. This penetrates into the bonded layer and permits water molecules and analyte to diffuse into the bonded phase. After conditioning, water is passed to remove the excess solvent prior to adding the sample. 

In solid-phase extraction, the bonded Cis chains take the place of the organic solvent. 

Solid-phase extractants utilizing nonpolar, polar, and electrostatic interactions. (Adapted from N. Simpson, Am. Lab., August, 1992, p. 37. Reproduced by permission of American Laboratory, Inc.) 

SAMPLE PREPARATION SOLVENT AND SOLID-PHASE EXTRACTION 

Solid-phase extraction is based on differences in the physico-chemical properties (predominantly basic or acidic properties) or chemical reactivity between starting materials, products, and auxiliaries of a chemical reaction.The reaction solution is brought into contact with a solid phase having chemical groups whose properties are complementary to the reagent that is to be extracted. After the excess starting materials or auxiliaries have been removed, the products are obtained by simply evaporating the solvent. If instead the products are extracted, an additional elution step is required. 

Examples in which solid-phase extraction has been applied to solution-phase combinatorial synthesis are listed in Table 3.12 for extraction by ionic interactions, and in Table 3.13 for extraction by covalent bond formation. 

Hydrophobic interactions may also serve to purify products of combinatorial syntheses. As an example, penta-O-lauroyl-l-thio-p-galactose was subjected to a Michael addition to a,p-unsaturated ketones or to alkylation by a-chloro ketones followed by reduction of the keto groups or by reductive alkylation with amino acid esters. The reaction mixtures were passed through Cl8 silica gel, whereby the desired products were adsorbed while unwanted products were washed away. Desorption was carried out with pentane, and the hydrophobic labels were removed with methanolic sodium methoxide.The methyl laurates were finally separated from the desired products by chromatography on silica 

For further examples concerning the synthesis and the application of functionalized resins in solid-phase extraction, see also [63]. 

Reaction Extracted material Solid phase Reference 

Solid phase extraction has several advantages over conventional liquid-liquid extraction when trace components are of interest. It is faster, requires less solvent, reduces the need for large concentration steps, and is easily automatable. Several liters, if necessary, can be poured through the column, and the trace components collected. These can be eluted with only a few mL of solvent, so the solvent removal for further concentration is nearly or completely eliminated. However, an HPLC column of 30 cm can provide 15,000 plates, whereas a solid phase disc can provide only 10 to 50 plates. What this means is that a solid phase disc can separate classes of compounds, but HPLC can then separate the compounds within that class. In fact, one of the major uses for SPE is as a clean-up for HPLC. The sequence is (1) select the proper size SPE tube, (2) condition the tube, (3) add the sample, (4) wash the packing, and (5) elute the compounds of interest. 

An excellent summary of the technique and several representative methods are presented in the Supelco Guide to Solid Phase Extraction which can be obtained from Supelco, Inc. Bellefonte, PA 16823-0048 (Telephone 1-800-247-6628). 

Retain Compounds of Interest and Let Impurities Pass through Tube 

Retain Impurities and Let Compounds of Interest Pass through Tube 

The following material is abstracted from an article by Edouard Bouvier of Waters Corp. with modifications to fit this chapter. Can a Sep-Pak cartridge be used as an inexpensive analytical column The answer can be found by examining the fundamental HPLC equation (see Chapter 19, p. 190, for more details) for the resolution between two components  

In solid-phase extraction (SPE) a solution of the sample is passed through a column of a sorbent, which retains the desired substance and possibly others. Unwanted materials can then be eluted and the desired substance selectively recovered. The available sorbents are mostly chemically modified silicas and include the following types. 

Sorption type Non-polar (reverse phase) Polar 

Sorbent functional groups Alkyl—octadecyl, octyl, ethyl cyclic— cyclohexyl aromatic— phenyl Cyanopropyl, diol, silica, amine 

Anion exchange—strongly basic (quaternary) weakly basic (primary, tertiary and mixed amine) Cation exchange—sulphonic acids carboxylic acids 

These and other sorbents are supplied in prepacked columns. Analy-tichem (Varian Sample Preparation Products) Bond Elut, International Sorbent Technology (Jones Chromatography) Isolute and Waters Sep-Pak are all well-known names. The sorbents are also available in bulk, and a wide range of accessories is available, such as Luer stop-cocks, vacuum pumps, manifolds and regulators. The sorbents supplied by Varian and International Sorbent Technology appear to be identical, but 

Classical LLEs have also been replaced by membrane extractions such as SLM (supported liquid membrane extraction), MMLLE (microporous membrane liquid-liquid extraction) and MESI (membrane extraction with a sorbent interface). All of these techniques use a nonporous membrane, involving partitioning of the analytes [499]. SLM is a sample handling technique which can be used for selective extraction of a particular class of compounds from complex (aqueous) matrices [500]. Membrane extraction with a sorbent interface (MESI) is suitable for VOC analysis (e.g. in a MESI- xGC-TCD configuration) [501,502]. 

Principles and Characteristics Solid-phase extraction (SPE) is a very popular sample preparation and clean-up technique. In SPE solutes are extracted from a liquid (or gaseous) phase into a solid phase. Substances that have been extracted by the solid particles can be removed by washing with an appropriate liquid eluent. Usually, the volume of solvent needed for complete elution of the analytes is much smaller (typically 1 mL) than the original sample volume. A concentration of the analytes is thus achieved. 

Although SPE can be done in a batch equilibration similar to that used in LLE, it is much more common to use a small tube (minicolumn) or cartridge packed with the solid particles. SPE is often referred to as LSE, bonded phase or sorbent extraction SPE is a refinement of open-column chromatography. The mechanisms of retention include reversed phase, normal phase, and ion exchange. 

Packed-bed SPE was introduced as a sample preparation technique in the early 1970s but did not start 

The sample volume initially introduced onto the sorbent, the choice of sorbent and solvent system and careful control of the amount of solvent used are of paramount importance for effective pre-concentration and/or clean-up of the analyte in the sample. The number of theoretical plates in an SPE column is low (/V = 10-25). SPE is a multistage separation method and as such requires only a reasonable difference in extractability to separate two solutes. In SPE concentration factors of 1000 or more are possible, as compared to up to 100 for LLE with vortex mixing. 

The types of organic compounds that can be bonded to a solid substrate vary widely. They cau be hydrophobic nonpolar molecules such as Cg aud Q hydrocarbou chains, chains with highly polar functional groups such as cyauo (—C N), amine ( NH2), and hydroxyl (—OH) groups aud with 

SPE is used widely for the cleanup aud couceutration of aualytes for analysis using LC, HPLC, and LC-MS, discussed in Chapter 13. As you will see, the phases used in HPLC for the separation of compounds are in many cases identical to the bonded solid-phase extractants described here. Detailed examples and application notes are available from a number of SPE equipmeut suppliers Avantor Performance Materials, Inc. (www.avantormaterials.com), Supelco (www.sigma-aldrich.com/supelco), Phenomenex (www.phenomenex.com), and Biichi Corporation (www.mybuchi.com) are a few of the companies that supply these products. 

Klejdus et al. (1999) used different solid-phase extraction (SPE) methods for isolation of daidzein, genistein, formononetin, and biochanin A to compare two red clover varieties. Eiltered ethanoUc (80%) extract of isoflavone is diluted with water (1 3, extract to water) before SPE. The cartridges are conditioned with either water or methanol, samples are then passed through, washed with methanol (5% or 10%) to remove impurities, and the retained isoflavones are eluted with 80% methanol. The extract is evaporated to dryness, dissolved in mobile phase, and directly injected onto an RP-HPLC column. 

The application of MIPs as the stationary phase in solid-phase extraction (SPE), often referred to as molecularly imprinted polymer solid-phase extraction (MIS P E), is a rapidly growing area [197-199]. With MISPE, highly specific enrichment of substances present at trace levels is possible. The technique has been applied to the analysis of drugs, for example, caffeine [200], scopolamine [201], naproxen [202], tetracycline [203], cholesterol [204] and local anesthetics [205], as well as environmental pollutants, exemplified by organophosphate flame retardants [206-208], triazines in soil and vegetable samples [71] and naphthalene sulfonates in river water [209]. 

Rinse the SPE cartridge with a strong solvent to remove adsorbed impurities. 

Wash the cartridge with a solvent suitable for initial conditions. 

Depending upon the application, steps 4 and/or 5 may not be required. By careful selection of eluting solvents the compound to be measured may not only be enriched relative to the drug, but may also be concentrated relative to the original loading solution, thus enhancing sensitivity. 

Prefiltering samples prior to SPE in a standardized manner using glass-fiber filters having no organic binders and testing the analytes of interest to establish that they are not adsorbed on the filter selected is recommended [43], Alternatively, Simpson and Wynne [76] present the counter viewpoint 

The aim with SPE is to selectively isolate solutes from a liquid sample. The matrix is applied at the top of the column and pulled through the 

F ureS.] A typical solid phase extraction cartridge. The sorbent is usually contained in plastic syringe shaped column. 

The polypropylene columns can be obtained packed with 100-1000 mg of 40-(xm sorbent sandwiched between two 20-(xm polyethylene frits. The columns are typically 5-6 cm long. Sample volumes are generally 1-6 mL. 

The adsorbent (stationary phase) used in these colromns is a nonpolar adsorbent chemically bonded to siFca gel. In fact, they are the same nonpolar adsorbents used in the reversed-phase higfi-performance liquid chromatography (HPLC). More specifically, the adsorbents are derivatized ca gel where the — OH groups of the siFca gel have been replaced with siloxane groups by treating sibca gel with the appropriate organochlorosilanes. 

Silica surface Chemically bonded silica surface 

An example of a typical solid-phase extraction is the determination of the amount of caffeine in coffee using a 1-mL column containing 100 mg of oc-tadecyl-bonded silica. This efficient method isolates about 95% of the available caffeine. The column is conditioned by flushing 2 mL of methanol followed by 2 mL of water through the column. One milliliter of a coffee solution ( 0.75 mg of caffeine/mL) is then drawn through the column at a flow rate of 1 mL/min. The column is washed with 1 mL of water and air dried (vacuum) for 10 min. The adsorbed caffeine is then eluted with two 500- xL portions of chloroform. 

You are presented a two-phase system. The two liquids are immiscible. The top phase is blue and the bottom, orange. One phase is water. Please devise an experiment to definitively differentiate which phase is water. 

Euture developments in sample preparation are expected from the application of nanomaterials. Due to their specific properties and the flexibility in tailored surface modifications carbon-based nanomaterials have already found a wide range of applications in different sample preparation procedures. They can be used as selective adsorbents by direct interaction between the analyte and the nanoparticles (Zhang, 2013) (Table 2.1). 

Materials Special material characteristics Derivatizatlon methods Sample preparation potential Current status Perspective  

Graphene 1. Both available sides for adsorption 2. Easily synthesized in lab 3. Easily modified with functional groups Modified via graphene oxide SPE, MSPE, SPME, LDI substrate, p-SPE, PMME, MSDP, SRSE  

Carbon nanofibers 1. Easily available on a large scale 2. Larger dimensions without coating or functionalization Polar groups can be introduced by treating with the concentrated nitric acid SPE, SPME, on-line p-SPE  

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Solid-Phase Extractions In a solid-phase extraction the sample is passed through a cartridge containing solid particulates that serve as the adsorbent material. For liquid samples the solid adsorbent is isolated in either a disk cartridge or a column (Figure 7.17). The choice of adsorbent is determined by the properties of the species being retained and the matrix in which it is found. Representative solid adsorbents... 

Solid-phase extraction cartridges (a) disk cartridge (b) column cartridge. 

Selected Adsorbents for Solid-Phase Extraction of Liquid Samples... 

Two examples from the analysis of water samples illustrate how a separation and preconcentration can be accomplished simultaneously. In the gas chromatographic analysis for organophosphorous pesticides in environmental waters, the analytes in a 1000-mL sample may be separated from their aqueous matrix by a solid-phase extraction using 15 mb of ethyl acetate. After the extraction, the analytes are present in the ethyl acetate at a concentration that is 67 times greater than that in... 

In a solid-phase extraction the analytes are first extracted from their solution matrix into a solid adsorbent. After washing to remove impurities, the analytes are removed from the adsorbent with a suitable solvent. Alternatively, the extraction can be carried out using a Soxhlet extractor. 

This publication provides several examples of the use of solid-phase extractions for separating analytes from their matrices. Some of the examples included are caffeine from coffee, polyaromatic hydrocarbons from water, parabens from cosmetics, chlorinated pesticides from water, and steroids from hydrocortisone creams. Extracted analytes maybe determined quantitatively by gas (GC) or liquid chromatography (LG). 

Thurman, E. M. Mills, M. S. Solid-Phase Extraction Principles an Practice, Wiley NewYork, 1998. 

Two approaches have been used to separate the analyte from its matrix in particulate gravimetry. The most common approach is filtration, in which solid particulates are separated from their gas, liquid, or solid matrix. A second approach uses a liquid-phase or solid-phase extraction. 

Extraction Eiltering limits particulate gravimetry to solid particulate analytes that are easily separated from their matrix. Particulate gravimetry can be extended to the analysis of gas-phase analytes, solutes, and poorly filterable solids if the analyte can be extracted from its matrix with a suitable solvent. After extraction, the solvent can be evaporated and the mass of the extracted analyte determined. Alternatively, the analyte can be determined indirectly by measuring the change in a sample s mass after extracting the analyte. Solid-phase extractions, such as those described in Ghapter 7, also may be used. 

Saito described a quantitative spectrophotometric procedure for iron based on a solid-phase extraction using bathophenanthroline in a poly(vinyl chloride) membrane. ... 

Volatile analytes can be separated from a nonvolatile matrix using any of the extraction techniques described in Ghapter 7. Fiquid-liquid extractions, in which analytes are extracted from an aqueous matrix into methylene chloride or other organic solvent, are commonly used. Solid-phase extractions also are used to remove unwanted matrix constituents. 

A solid-phase extraction in which the solid adsorbent is coated on a fused-silica fiber held within a syringe needle. 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

What is the purpose of including an initial solid-phase extraction ... 

Table 1. Samples Analyzed by Solid-Phase Extraction...

SOLID-PHASE EXTRACTION ON SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION TRACE IONS IN WATER WITH l-(2-PYRIDILAZO)-2-NAPHTOL... 

ATOMIC ABSORPTION DETERMINATION OF Cu(II), Cd(II), Zn(II), Pb(II) USING PRECONCENTRATION BY SOLID-PHASE EXTRACTION ON PROPYLTHIOETHYLEAMINE MODIFIED... 

SOLID PHASE EXTRACTION AND DETERMINATION OF TRACE AMOUNT OF Co + BY MODIFIED ANALCIME ZEOLITE WITH A NEW SCHIFF-BASE LIGAND... 

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

The liquid chromatography - tandem mass spectrometry (LC/MS/MS) technique was proposed for the determination of corticosteroids in plasma and cerebrospinal fluid (CSF, liquor) of children with leucosis. Preliminai y sample prepai ation included the sedimentation of proteins, spinning and solid-phase extraction. MS detection was performed by scanning selected ions, with three chai acteristic ions for every corticosteroids. The limit of detection was found 80 pg/ml of plasma. 

SOLID-PHASE EXTRACTION IN DETERMINATION OF ORGANIC COMPOUNDS IN WATER BY GC-MS Milyukin M.V. 

Prepai ative isolation of nonvolatile and semivolatile organic compounds fractions (hydrophobic weak acids, hydrophobic weak bases, hydrophobic neutrals, humic and fulvic acids) from natural and drinking waters in optimal conditions was systematically investigated by solid-phase extraction method with porous polymer sorbents followed by isolation from general concentrate of antropogenic and/or toxic semivolatile compounds produced in chlorination and ozonation processes. 

In recent decades the development of preconcentration steps to be implemented prior to analytical determinations of trace level compounds has been explored in considerable depth. With a view to eliminating or at least minimising the use of organic solvents used in conventional liquid-liquid extraction, other methodologies have been developed, such as membrane extraction, solid-phase extraction, solid-phase microextraction, etc. 

E.M. Thurman and M.S. Mills, Solid-phase Extraction Principles and Practice, Chemical Analysis Series Vol. 147, J. Wiley Sons, New York, 1998. ISBN 047161422X. 

Figure 2.12 Schematic representation of an on-line SPE-GC system consisting of three switching valves (VI-V3), two pumps (a solvent-delivery unit (SDU) pump and a syringe pump) and a GC system equipped with a solvent-vapour exit (SVE), an MS instrument detector, a retention gap, a retaining precolumn and an analytical column. Reprinted from Journal of Chromatography, AIIS, A. J. H. Eouter et al, Analysis of microcontaminants in aqueous samples hy fully automated on-line solid-phase extraction-gas chromatography-mass selective detection , pp. 67-83, copyright 1996, with permission from Elsevier Science.

S. Nelieu, M. Stobiecki and J. Einhom, Tandem solid-phase extraction of ati azine ozonation products in water , / Chromatogr. 866 195 - 201 (2000). 

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... 

A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

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