Mobile phases solid phase extraction

Figure 6 HILIC-ESI-MS separation of the polar components (unretained by reversed-phase solid-phase extraction pretreatment) of a fermentation extract, represented as a total ion chromatogram in positive ion ESI. The chromatogram was obtained using a TSKGel Amide 80 packing, 6.5 mmol r1 ammonium acetate pH 5.5-buffered mobile phases, and a 90 min 10-40% aqueous gradient. Reproduced from M. A. Strege, Anal. Chem. 1998, 70, 2439-2445.

Reversed-phase solid-phase extraction (SPE) involves the partitioning of organic solutes from a polar mobile phase, such as water, into a nonpolar solid phase, such as the C-18 sorbent (Fig. 4.1). Partitioning involves the interaction of the solute within the chains of the stationary phase, which may be a C-18 hydrocarbon, C-8 hydrocarbon, or the polymeric sorbents (such as styrene-divinylbenzene). The word hydrophobic mechanism is commonly... 

The first bioanalytical application of LC-GC was presented by Grob et al. (119). These authors proposed this coupled system for the determination of diethylstilbe-strol in urine as a replacement for GC-MS. After hydrolysis, clean-up by solid-phase extraction and derivatization by pentafluorobenzyl bromide, the extract was separated with normal-phase LC by using cyclohexane/1 % tetrahydrofuran (THE) at a flow-rate of 260 p.l/min as the mobile phase. The result of LC-UV analysis of a urine sample and GC with electron-capture detection (ECD) of the LC fraction are shown in Ligures 11.8(a) and (b), respectively. The practical detection limits varied between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of... 

O. V. Olesen and B. Poulsen, On-line fully automated deteimination of clozapine and desmethylclozapine in human semm by solid-phase extraction on exchangeable car-ti idges and liquid cliromatography using a methanol buffer mobile phase on unmodified silica , 7. Chromatogr. 622 39-46 (1993). 

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. 

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... 

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

T.L. Buxton and P. de B. Harrington, Trace explosive detection in aqueous samples by solid-phase extraction ion mobility spectrometry (SPE-IMS), Applied Spectroscopy 57(2) (2003) 223—232. 

Despite the distinct advantages of pneumatic nebulizers, ultrasonic nebulizers may alternatively be used, in some instances, with success. In a recent application, a variation of ultrasonic nebulizer called spray nozzle-rotating disk FTIR interface was successfully applied to confirm the presence of methyltestosterone, testosterone, fluoxymesterone, epitestosterone, and estradiol and testosterone cyp-ionate in urine, after solid-phase extraction and reversed-phase LC separation (151). Using a commercial infrared microscopy spectrometer, usable spectra from 5 ng steroid deposits could be readily obtained. To achieve success with this interface, phosphate buffers in the mobile phase were not used because these nonvolatile salts accumulate on the collection disk and their spectra tend to swamp out small mass deposits. Another limitation of the method was that only nonvolatile analytes could be analyzed because volatile compounds simply evaporated off the collection-disk surface prior to scanning. 

Actually, solid-phase extraction is used not only as a rough preliminary fractionation procedure. Prieto et al. described the complete fractionation of the total lipids from wheat into eight neutral lipid, two glycolipid, and four phospholipid classes in addition to PC and LPC, TV-acyl PE and A-acyl LPE were detected (37). However, two separate stationary phases (silica and aminopropyl) as well as seven different mobile phases were needed. Moreover, 14% crosscontamination of PC and LPC was observed, and the recovery of the phospholipids was limited to about 85%. Hence, SPE is a rapid and efficient technique for preliminary fractionation, but loses its advantages if more complex separations are tried. 

Motevalian et al. [62] developed a rapid, simple, and sensitive HPLC assay method for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 /d) was injected on a /rBondapak Ci8 reversed-phase column (4.6 mm x 250 mm, 10 /un). The mobile phase consisted of 0.05 M phosphate buffer (pH 7.5) and acetonitrile (75 25) at a flow-rate of 0.8 ml/min. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50-2000 ng/ml. The minimum detection limits were 10, 10, and 15 ng/ml for omeprazole, omeprazole sulfone, and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg of omeprazole. 

Orlando and Bonato [73] presented a practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma. Ci8 solid-phase extraction cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol-hexane (70 30) as the mobile phase, at a flow-rate of 0.5 ml/min. The detection was carried out at 302 nm. The method is linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%. 

Another popular and selective extraction technique widely used in bioanalysis is solid phase extraction (SPE). SPE is a separation process utilizing the affinity of the analytes to a solid stationary phase. By manipulating the polarity and pH of the mobile phase, the analytes of interest or undesired impurities pass through stationary phase sequentially according to their physical and chemical properties. For a SPE procedure, a wash step refers to the elution of the unwanted impurities which are discarded and the elution step refers to the elution of the analytes of interest which are collected. While the fundamental remains the same in decades, the continuing invention and introduction of new commercial stationary phases and accessory devices have boosted the application of SPE in bioanalysis and many other fields. 

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