Reverse phase SPE

These attractive forces are known as Van der Waals forces and are weak forces of attraction (see Chapter 2 for further explanation), thus allowing the analytes adsorbed to be removed from the packing material by using a nonpolar solvent. As with reverse phase silica columns in chromatography (see Chapter 4), unreacted silanols are also present on the surface of the silica that can act as secondary reaction sites and thus can interact with polar contaminants (or polar analytes) within the mixture. As in solid phase 

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The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Sometimes orthogonal offline SPE steps were used prior to online SPE LC/MS/MS. These preparation steps were used to remove interference and concentrate samples. In an application to measure urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a biomarker for exposure to polyaromatic hydrocarbons (PAHs), a two-step offline SPE was first performed using Sep-Pak C8 (Waters, Milford, Massachusetts) and Strata SCX (Phenomenex, Torrance, California) cartridges to obtain high sensitivity (Chen et al. 2005). The extracts were applied to an online reversed phase SPE LC/MS system. The lower limit of detection was 2.5 fmol/mL when 10 mL of urine was used. 

The first reversed-phase SPE sorbents were based on silica gel particles, similar to the particles used in HPLC. A number of phases are available ranging from C8 to C18 to anion- and cation-exchange functionalities. Recent advances in particle technology have included polymeric materials that combine the benefits of a water-wettable particle to retain polar analytes with a reversed-phase, hydrophobic moiety to... 

Well water Extracted/concentrated using reverse phase SPE columns. Eluted analytes withmethanol. GC (detection not specified) No data 58-67 Hogmire et al. 1990... 

Different reversed phase [195,239,240], mixed mode (ion exchange and reversed phase) SPE cartridges [173,218] and online SPE column [193, 238] have been also reported for samples preparation and extraction. Some of these assays combined both PP and SPE in order to achieve an extensive sample cleanup [193, 195, 238-240], Likewise SPE, LLE provides cleaner plasma extracts than PP. Nevertheless, LLE procedure does not always provide satisfactory results with regard to extraction recovery and selectivity, especially with polar analytes and particularly in the case of multicomponent analysis such as in drug-metabolism studies, where analytes polarity varies widely. This issue was addressed by Zweigenbaum J and Henion J [235] and extraction solvent optimization, using isoamyl alcohol, to achieve acceptable extraction selectivity and recovery for polar analytes has been discussed. 

A previous purification of wine sample can be performed by reverse-phase SPE. A volume of 5 mL of wine is added of 15 mL of water and passed through a C18 cartridge previously activated by passage of methanol and water. After washing the cartridge with 6 mL of 0.3% formic acid aqueous solution and 4mL of water, anthocyanin compounds are recovered with 5 mL of methanol. The solution is dried and the residue re-dissolved in the LC mobile phase (Kosir et al., 2004). 

After the fractions have been collected, the solvent needs to be removed by using a freeze-dryer, rotary evaporator, or high-throughput parallel evaporator. Nonvolatile components can be removed with reversed-phase SPE procedures prior to solvent removal if the aqueous portion of the buffer is sufficiently large. 

Bouvier, E.S.P. Iraneta, P.C. Neue, U.D. McDonald, PD. Phillips, D.J. Capparella, M. Cheng, Y.-F. Polymeric Reversed-Phase SPE Sorbents-Characterization of a Hydrophilic-Lipophilic Balanced SPE Sorbent," LC-GC 16(Supplement), S53-S57 (1998). 

Although the literature chiefly examines bonded-phase SPE sorbents, there are applications of organic polymers as important phases for SPE. The two most common sorbents are styrene-divinylbenzene (SDB) and activated carbon. These two sorbents do not contain silica but are entirely organic polymers (Fig, 2.9). Typically they have large surface areas (600-1200 m /g), greater capacity than the bonded phases because of higher carbon percentage, and a more hydrophobic surface. They have their major application in the area of reversed-phase SPE. 

Figure 2.16. Generic method of elution of reversed-phase SPE sorbents using ethyl acetate and methanol. [After Aga et al. (1994), published with permission.]...

In summarizing reversed-phase SPE, both the and the log of the octanol-water partition coefficient of the compound are related to the aqueous solubility of the analyte, although the relationship is not always straightforward because there are other factors that affect solubility of an analyte but that do not affect sorption, such as crystal lattice energy for solids. In spite of this factor, one could theoretically estimate the from the solubility of the analyte and relate this solubility to the capacity of the solute for the reversed-phase sorbent. The ability to use solubility to predict capacity is also addressed... 

The selection and development of a reversed-phase method can be a straightforward process, and for this reason, many methods have been developed on reversed-phase SPE, especially the C-18 sorbent due to its reliable nature. The following examples describe the reversed-phase extraction of compounds of differing polarities from various matrices, as an indication of the broad extent to which reversed-phase SPE can be applied. 

The trace enrichment of pesticides from groundwater is a straightforward problem for reversed-phase SPE. The following example uses the EMPORE disk (C-18) to isolate organochlorine pesticides including aldrin, chlordane, endrin, heptachlor, lindane, methoxychlor, pentachlorophenol, and toxaphene from groundwater (Fig. 4.15). 

Next, condition either the silica-based bonded phases or the styrene-divinylbenzene copolymers with methanol to wet and activate the sorbent for good mass transfer when the sample is passed through the sorbent. Rinse the methanol from the sorbent with deionized water. Several bed volumes should be sufficient to displace the majority of methanol and is comparable to that used in reversed-phase SPE. 

These compounds are permanently anionic and are moderately polar (surfactants are organic molecules that are surface active). This means that they concentrate on the surface of a liquid in which they are dissolved. Generally, these types of analytes contain both a hydrophobic and a hydrophilic segment. There are anionic, cationic, neutral, and amphoteric surfactants. They may be readily sorbed from water by reversed-phase SPE. Elution requires methanol or acetonitrile rather than ethyl acetate because of their polar, ionic functional groups, which are typically sulfate esters or sulfonic acids (Fig. 7.18). 

Aliquots of 50 ml of raw and settled influent of the treatment plants were evaporated to dryness on a steambath. The residue was extracted several times with methanol and the combined centrifuged extracts were brought to a volume of 100 ml with methanol an aliquot of 20 ml of the extract was then evaporated to a few milliliters on a steambath and finally diluted with water to a volume of 50 ml. This solution was then passed through an octadecyl reversed phase SPE column where the LAS was retained. After rinsing the column with 3 ml of a methanol/water 30/70 solution, the LAS was quantitatively recovered by elution with 4 ml methanol. 

Samples of 1 gram of dried primary and dried wasted sludge were extracted in a Soxhlet extraction apparatus with 150 ml methanol for 4 hours. An aliquot of the methanol extract (20 ml out of a total volume of 200 ml) was passed over a small anion exchange column. The column was washed with 10 ml methanol before elution of the LAS with 3 ml of acidic methanol. The effluent was diluted to about 70 ml with water and the pH was adjusted to a value of 7. The solution was then brought to a value of 100 ml and an aliquot of 25 ml was passed over an octadecyl reversed phase SPE column. The sample was further treated as described above. The samples were kept in a dry state until analysis by HPLC. Prior to HPLC, the residues were dissolved in an acetonitrile/water 70/30 solvent mixture. 

Several steps are required in a typical reversed-phase SPE ... 

SPE has been used to estimate the amount of freely dissolved PAHs versus DOM-associated and particulate-associated PAHs. Glass fiber filters used to remove particulate matter from water samples before extraction can also retain some DOM-associated PAHs. It is largely thought that DOM-associated PAHs will pass through reverse-phase SPE cartridges allowing for the measurement of the freely-dissolved fraction, however, some DOM-associated PAHs are retained... 

A new selective enrichment technique was investigated for the sample preparation for GC-MS analysis of 16 acidic herbicides in water. By using a dynamic ion-exchange solid-phase extraction (DIE-SPE) combined with reverse-phase SPE, interference by humic substances could be reduced and most of the acidic herbicides were extracted with recoveries above 70%. ... 

Reversed-phase SPE is applied principally in two areas of TEQA. The first application is in the determination of organochlorine pesticides (OCs) in connection with the determinative technique of GC using a chlorine-selective detector. The second application is in the determination of priority pollutant semivolatile organics that are adequately recovered from drinking water. Off-line RP-SPE coupled to a determinative technique such as GC using an element-selective detector is a very powerful combination with which to achieve the objectives of TEQA. To illustrate, this author s study of various organophosphorous pesticides (OPs) from spiked water using RP-SPE will now be discussed. 

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