Micro-Extraction Methods

Due to phase variation, there are fluctuations in expression levels of certain enzymes in bacteria, therefore, not all colonies or cells make the same structure of lipid A species. A micro-extraction method for extraction of lipid A from a single colony has been developed (Zhou et al., 2009). This method uses microwave-assisted enzymatic digestion and sodium acetate hydrolysis, suitable to analyze lipid A from both cell samples and an individual colony. Because the clean up of SDS is very time-consuming, and the contaminated SDS would seriously interfere with the analysis by mass spectrometry, the proteinase K, instead of SDS, is used to disrupt the cells. Using this method, the entire process for lipid A preparation only takes about 2 h with a detection limit to 1 (xg. 

Method. To 100 Lil of the serum sample add 10 Lil of phosphate buffer (pH 5.6), and extract with 100 Lil of chloroform containing 10 Lig/ml of tetraphenylethylene using the micro-extraction method, above inject 3 to 5 Lil of the chloroform extract on to both columns. 

Method. Extract 100 iLil of plasma with 100 pi of chloroform by the micro-extraction method (described above), and inject 3 to 5 pi of the chloroform extract on to the column, using the following system. 

A Abdollahi, NS Rosenholtz, JL Garwin. Tocopherol micro-extraction method with application to quantitative analysis of lipophflic nutrients. J Food Sci 58 663-666, 1993. 

Some typical industrial applications of HS-SPME are analysis of trace impurities in polymers and solid samples, the determination of solvent residues in raw materials, ppt odour analysis, organics in water, etc. SPME can also be used for quaUtative headspace sampling of fruit volatiles [1000]. Since equilibrium rather than exhaustive extraction occurs in the micro-extraction methods, SPME is suitable for field monitoring. 

E. E. Stashenko, J. R. Martinez, Sampling volatile compounds from natural products with headspace/solid phase micro extraction, J. Biochem. Biophys. Methods, 70, 235 242 (2007). 

The volatile substances were extracted from portions of 0. lg hair using solid-phase micro extraction (SPME). The method uses a fibre coated with an adsorbent that can extract organic compounds from the headspace above the sample. Extracted compounds are desorbed upon exposure of the SPME fibre in the heated injector port of a gas chromatograph (GC). 

In environmental analytical applications where analyte concentrations, e.g. surfactants or their metabolites, are quite low, extraction and concentration steps become essential. Solid phase extraction (SPE) with cartridges, disks or SPME fibres (solid phase micro extraction) because of its good variety of SP materials available has become the method of choice for the analysis of surfactants in water samples in combination with FIA as well as LC—MS analysis. SPE followed by sequential selective elution provides far-reaching pre-separations if eluents with different polarities and their mixtures are applied. The compounds under these conditions are separated in the MS spectrometer by their m/z ratios providing an overview of the ionisable compounds contained in a sample. Identification in the sense it has been mentioned before, however, requires the generation of fragments. 

The mass of sample taken for analysis is primarily dependent on four factors (1) the amount of material available, (2) the concentration of the analyte, (3) the heterogeneity of the sample, and (4) the method of analysis. Most conventional solvent extraction techniques currently start with more sample than is required, use more extraction solvent than is necessary, and ultimately only analyze 0.1% of the material prepared, e.g., 1 pi from 1 ml. Micro-extraction techniques [468] can be used in conjunction with on-line LC-GC or LC-MS to utilize the whole extract in the final determinations. This approach can significantly reduce the size of sample required and the volume of solvent used. Many workers have reported the use of solid phase microextraction (SPME) in different environmental matrices for various pollutants [288,342,345,469 - 477]. 

Classical sample preparation methods such as distillation, soxhlet extraction are still used [839, 840], but specific techniques such as supercritical fluid extraction (SFE) [841], and increasingly in recent years, adsorption techniques such as solid phase micro-extraction (SPME) [841a] are also being used for isolation, separation, and identification of flavor and fragrance materials. 

Protein solubilities of soy flour and extrudates in the following solvent systems were determined by the micro-Kjeldahl method ( 9). A portion (0.1 g) of finely-ground sample (No. 60 sieve) was extracted with 9.9 ml of solvent for 1 hr at room temperature followed by centrifugation and filtration. An aliquot of the supernatant was used for nitrogen determination. 

Pasztor, L., J. D. Bode, and Q. Fernando Determination of Micro Quantities of Boron in Steel by a Solvent Extraction Method. Anal. Chem. 32, 277 (1960). 

Nguyen, R.T., and Harvey, H.R. (1994) A rapid micro-scale method for the extraction and analysis of protein in marine samples. Mar. Chem. 45, 1-14. 

Because LPS molecules in different bacteria have different structures and amphi-pathic properties, there is no universal method for extraction of LPS. Several methods have been developed each favors some specific groups of LPS. For example, the phenol-water extraction method favors S-LPS extraction, but not R-LPS (Hickman and Ashwell, 1966 Kasai and Nowotny, 1967) the ether extraction method favors the extraction of R-LPS (Galanos et al., 1969). Both methods for large scale or micro-scale extractions have been developed. Agents used by different extraction methods are listed in Table 2.1. 

As the typical molecule in Gram-negative bacteria, lipid A can be used to detect the bacteria. Detection methods need to be rapid and sensitive therefore, a few rapid methods for lipid A micro-extraction from whole bacteria have been developed. 

CWC-related chemicals in aqueous liquid samples (water samples) are usually recovered by extraction with an organic solvent. Modem methods such as SPE and solid phase micro extraction (SPME) have also been presented 04 24). Organic extractions and these modem methods mainly recover nonpolar CWC-related chemicals, but leave behind the water-soluble and nonvolatile chemicals. These must also be recovered, however, because the agents tend to decompose (hydrolyze) rapidly under conditions in the environment. In the past few years, techniques such as CE and LC, relying on element specific or mass spectrometric detection, have been intensively developed to provide easy and effective ways of recovering these chemicals from water samples with only minor sample preparation (2S 44,1. For GC/MS analysis, the water must be displaced and the analytes derivatized. 

Solid phase micro-extraction (SPME) allows isolation and concentration of volatile components rapidly and easily without the use of a solvent. These techniques are independent of the form of the matrix liquids, solids and gases can be sampled quite readily. SPME is an equilibrium technique and accurate quantification requires that the extraction conditions be controlled carefully. Each chemical component will behave differently depending on its polarity, volatility, organic/water partition coefficient, volume of the sample and headspace, speed of agitation, pH of the solution and temperature of the sample (Harmon, 2002). The techniques involve the use of an inert fiber coated with an absorbant, which govern its properties. Volatile components are adsorbed onto a suitable SPME fiber (which are usually discriminative for a range of volatile components), desorbed in the injection chamber and separated by a suitable GC column. To use this method effectively, it is important to be familiar with the factors that influence recovery of the volatiles (Reineccius, 2002). 

Reference Solutions. Prepare chloroform solutions containing standard mixtures of drugs, all at concentrations of 5 iLig/ml, and inject 3 to 5 Lil on to both columns to obtain the standard chromatogram. Method. To 100 Ll1 of stomach contents or 500 Lil of urine add 100 Ll1 of M sodium hydroxide, and extract with 100 Lil of chloroform by the micro-exfraction method (above) inject 3 to 5 ll1 of the chloroform extract on to both columns. Identify any peaks which appear on the chromatograms by reference to the standard chromatogram and to the retention data given on p. 193. Corroborative evidence must be obtained from both systems. 

Murray, D.A.J. Rapid micro extraction procedure for analyses of trace amounts of organic compounds in water by gas chromatography and comparisons with macro extraction method. J. Chromatogr. A 177, 135-140 (1979)... 

This cold extraction method is successfully performed in our and other laboratories for more than 20 years. Because hexane is neurotoxic, isohexane or heptane can be used as a hydrophobic solvent. A modified method can also be used as a semi-micro method for the lipid determination in fish, mussels, oysters, Daphnia, and other aquatic organisms or tissues. 

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