Freeze drying sample preparation

Starch may be extracted simply with hot water, but the mildness of this exercise requires separate inactivation of depolymerases (by heat or ethanol) if this biopolymer is to be used for food, or mercuric chloride (0.01 M) if it is to be used other than for food. The crude water extracts are centrifuged, filtered, and spray- or freeze-dried in preparation for storage. Where there is protein, in small samples, most of it can be removed by shaking the crude extract with one-tenth its volume of toluene and discarding the toluene layer. Lipid matter is dissolved by refluxing with aqueous 80% methanol. 

The method of sample preparation to be used for a given analysis is governed by the nature and concentration of the analyte, the nature (solid or liquid) and type of matrix, the available sample amount, and also by the instrumental technique employed. Freeze-dried samples will require some form of digestion or dissolution in order to be analyzed by a classic atomic technique (i.e., using nebuliza-tion). Liquids might be analyzed by direct nebulization, but this is not always possible due to matrix interferences. Milk pretreatment may be necessary under such circumstances. 

Micrographs of the freeze-dried RDP preparations are shown in Figure 6. The unheated and microwave treated samples are clearly differentiated from those treated with hot water. The former consist of ragged fragments containing numerous but small pores while the latter appeared more aggregated and exhibits larger orifices. A consideration of bulk density (Table IV) and microstructure may help to explain some aspects of protein/water interaction properties. Porosity and particle size could be important parameters, however they are difficult to control and are rarely measured in studies of functional properties. 

Steroid Analysis. Steroid analysis was carried out on selected humic and fulvic freeze-dried samples of stream, foam, and foam extract from the Suwannee River by Microbial Insights, Inc., (Knoxville, TN). Tri-methyl silyl derivatives of 3 P ol sterols were prepared from either the neutral lipid or total lipid fraction by alkaline saponification as described by Nichols et al. (23). 

The LDH activity was assayed according to the method described by Suzuki et al. (1998) at the three stages before freeze-drying, after preparation of the freeze-dried sample, and after storage for several days at either 60 or 40°C. The remaining LDH activity was expressed as activity relative to that determined before freeze-drying. 

The NEB extent in the trehalose model is highest at 33.2 and 44.1% RVP probably because of local heterogeneities. In fact, during preparation of the freeze-dried samples in Petri dishes, we found that the trehalose model showed different extents of browning between the top and bottom of the sample. Of course, the uptake of water at 33.2 and 44.1% RVP environments enhanced the NEB reaction. NEB was observed at a temperature lower than the glass-transition temperature in the present study, which might be related to the heterogeneities and porosity of the matrix structure. 

The action of the enzyme is represented in Figure 15.24. All these spectra were run on freeze-dried samples from which KBr pellets were prepared. Total inactivation of an antibiotic with abolition of the so-called c band ( 1760cm ) is characteristic of the action of this enzyme. Partial inactivation with preservation of this band implies deacylation or other degradation leaving the /S-lactam ring intact. 

In order to increase the accuracy and reliability or results, Karl Fischer results are usually compared to TG results. The moisture results should be nearly equivalent from these two methods since they are both measures of total bound and surface moisture in the sample cake [17]. Vial-to-vial moisture variability in the samples is usually inherent in freeze-dried samples since each vial is unique with respect to freeze-drier shelf and position on the shelf. Both the Karl Fischer and TG methods are frequently capable of measuring the moisture content of one vial and therefore vial-to-vial variability for one lot since one test requires either 20 or 5 mg of sample, respectively. For these two methods also the relative standard deviation is near 10%. Low vial-to-vial variability is produced by a well-controlled sample lyophilization process. After the freeze-drying of U.S. National Reference Preparation for a-Fetoprotein in Mid-Pregnancy Maternal Serum [51], 85 samples were chosen at random. The mean residual moisture content of the 85 samples was 0.55% with a standard deviation of 0.19%. 

Figure 9.4. Microstructure of a cellulose aerogel as prepared by Jin and co-workers [10] (with permission of Elsevier). The samples shown have a cellulose concentration of 2 wt%. The figure in the left panel shows a regularly freeze dried sample, in the right a sample rapidly freeze dried as described above with gel casting onto a copper plate kept at liquid nitrogen temperature.

Freeze-dry sample for storage and measurement Immediate data collection and analysis following sample preparation... 

Silylation procedure. Add 1.0 ml of the internal standard soln. and 80 pi of N-trimethylsilyldi-ethylamine (Pierce) to each vial containing the freeze-dried sample. Heat the vial in a 75°C oil bath for 40 min. Silylated neomycins thus prepared are extremely sensitive to moisture and better stability may be obtained when the sample is prepared in a l.S-ml serum vial with a I3-mm natural red rubber closure Calculations... 

Bacillus licbeniformis (ATCC 9945a) was obtained from Dr. L- K. Nakamura, of the U. S. Department of Agriculture, Peoria, IL. This freeze dried sample was suspended using 0.5 mL Tryptic Soy Broth (Difco Labs). This solution was transferred to a larger volume of Tryptic Soy Broth (100 mL) and incubated on a rotary-shaker (250 RPM, 37 C) for 18 h. From this inoculum four stock culture plates were prepared using Tryptic Soy Broth supplemented with 1.5% agar. These culture plates were then incubated at 37 C for 18 h. 

For the polymerization studies, multilamellar vesicular dispersions were prepared by vortexing the hydrated lipid dispersions. These dispersions were gel filtered on a Sephadex G 50-150 column and were polymerized at 5 C by 254 nm irradiation in a Rayonette Photoreactor. The course of the polymerization was monitored by thin layer chromatography on silica gel using a chloroformrmethanol water (65 25 4) solvent system. The monomer participation was measured in polymerized and freeze dried samples by dissolving the monomers in chloroform and spotting on the TLC plate, developing in lipid solvent and measuring the phosphorus content under monomer and polymer spots. 

Frozen reference materials have been produced by NIST (Wise et al. 1993). These materials do not have the disadvantages of the oils or freeze-dried materials, but are more difficult to transport. Obviously they have to be kept deep-frozen during transport, which makes their use rather expensive. Since the early 1990 s a new approach in this field has been introduced. This concerned the use of wet, sterilized fish and shellfish samples. These samples, packed in glass jars or in tins, were firstly used in the QUASIMEME program as reference materials for inter-laboratory studies (de Boer 1997). Later, when it appeared that the stability was maintained for longer periods, tests for organic contaminants based on this principle were also prepared. 

Supercritical fluid extraction (SFE) is generally used for the extraction of selected analytes from solid sample matrices, but applications have been reported for aqueous samples. In one study, recoveries of 87-100% were obtained for simazine, propazine, and trietazine at the 0.05 ug mL concentration level using methanol-modified CO2 (10%, v/v) to extract the analytes, previously preconcentrated on a C-18 Empore extraction disk. The analysis was performed using LC/UV detection. Freeze-dried water samples were subjected to SFE for atrazine and simazine, and the optimum recoveries were obtained using the mildest conditions studied (50 °C, 20 MPa, and 30 mL of CO2). In some cases when using LEE and LC analysis, co-extracted humic substances created interference for the more polar metabolites when compared with SFE for the preparation of the same water sample. ... 

Polystyrene was prepared by the anionic polymerisation of styrene in toluene plus THF mixtures (4 1 volume ratio) using n-butyl lithium as initiator. After removing a sample for analysis at this stage, the remainder of the living polystyrene was reacted with a five molar excess of trichloromethylsilane for 15 min and then excess methanol introduced. The methoxy-terminated polystyrene was freeze-dried from dioxan. The method described here essentially follows the route proposed by Laible and Hamann (6). 

Materials. Na-Kaolinite A homoionic sample of kaolinite was prepared from a well-crystallized sample purchased from Source Clays, University of Missouri, using a standardized technique (14) which involved repeated washing with distilled water and by treatment with NaCl solutions to remove exchangeable ions such as Ca, and freeze-drying of the final product. Nitrogen specific surface area of this kaolinite was estimated to be 9.4nr/g and X-ray analysis showed the characteristic pattern of kaolinite. 

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