Filter plate method

Figure 12 Filter plate method for solubility testing. (1) Add compound dissolved in organic solvent to aqueous buffer. (2) Shake for 90 minutes to allow insoluble compound to precipitate. (3) Apply vacuum to filter solution into collection plate. Precipitates remain on membrane. Analyze filtrate in collection plate to quantify the amount of the compound still in the solution. Source Courtesy of Millipore Corporation, Billerica, Massachusetts, U.S.A.

Figure 4.12 Filter plate method for solubility testing.

Recently, Murray and Gellman demonstrated that parallel synthesis in inexpensive 96-well polypropylene filter plates with microwave irradiation in a multimode reactor is a simple and effective method for the rapid preparation of j8-peptide hbraries on sohd support in acceptable purities [156]. 

The majority of commercial LB troughs use the Wilhehny plate method for measurement of surface pressure (II), although some use the alternate Langmuir float method. The plate material most commonly used is cut pieces of filter paper, of negligible cost and completely wetted by water. The other type of plate used is a piece of high-purity platinum metal, which can be cleaned in a flame and gives a reproducible contact angle with water of 60°. 

More information on the comparative evaluation of protein precipitation methods may be obtained from Lei and coworkers.163 An interesting comparison of protein precipitation (PPT) and solid phase extraction (SPE) methods was presented in a technical library publication from Millipore164 that describes use of its Multi-SPE-MPC extraction plate and Multiscreen deep well Solvinert filter plate for SPE and PPT, respectively (Figure 1.45). A Biohit Proline multichannel pipette was used to add 400 /iL of acetonitrile to each well of the filter plate and then, using the pipette s double aspiration program, 100 /iL of spiked serum was aspirated and 100 /iL of acetonitrile from the filter plate was aspirated to initiate protein precipitation in the pipette tip. The mixture was deposited back in the filter plate and shaken vigorously for 2 min. 

The LB film depositions were performed using a Joyce-Loebl Langmuir Trough IV equipped with a microbalance for measurement of the surface pressure by the Wilhelmy plate method. Filtered deionized water with a pH of 7 was used for the subphase. For the electron beam lithography study, PMMA was spread on the water surface from a dilute benzene solution ( 10 mg PMMA in 20 ml benzene). The novolac/PAC mixtures were spread from solutions ( 20 mg solids in 10 ml solvent) of isopropyl acetate. For the fluorescence studies, the PMMA/PDA mixture was spread on fee water surface from a dilute benzene solution (1.75 mg PDA and 8.33 mg PMMA in 20 ml benzene). Prior to compression, a 20 min interval was allowed for solvent evaporation. The Langmuir film was compressed to the desired transfer pressure at a rate of 50 cm2/min, followed by a 20 minute equilibration period. The Cr-coated silicon wafers and quartz wafers were immersed into fee subphase before... 

The Wilhelmy plate method provides an extremely simple approach that, unlike the ring detachment method, permits the measurement of continuously varying or dynamic surface tensions. If a thin plate (e.g., a microscope slide, a strip of platinum foil, or even a slip of filter paper) is attached to a microbalance and suspended so that its lower edge is just immersed in a liquid, the measured apparent weight Wj, is related to the actual weight of the plate Wp and the surface tension y by the following simple equation ... 

Zebrafish Brachydanio/Danio rerio) embryos can be produced in large numbers and carry sufficient nutrients within the egg sack to allow development within micro plate volumes [52, 53]. Mutation frequency has been estimated indirectly using transgenic fish [54[ and UDS, comet, MNT and alkaline filter elution methods have been used effectively [55]. At present, there has been insufficient study of the model to understand predictivity of mammalian genotoxicity, and none of the methods has been demonstrated at throughputs sufficient for hit and lead screening. 

If 30 is accepted as the lowest reliable number to count and a pour plate method uses a 1.0-ml sample, it follows that the procedures described above are unsuitable for any sample that is expected to contain <30 CFU ml-1, e.g. water samples where the count may be 1 CFU ml-1 or less. Here, membrane filter methods are used in which a large, known volume of sample is passed through the membrane which is placed, without inversion, on the agar surface. Nutrients then diffuse up through the membrane and allow the retained cells to grow into colonies on it just as they would on the agar itself. 

The alternative method to turbidimetric detection used for measuring solubility in early discovery is to quantify the aqueous supernatant directly via UV absorbance [13, 20, 21]. Typically, DMSO stock solution is added to aqueous buffer such that the final DMSO composition is kept to a minimum (5% or less) and the resulting precipitate is removed by filtration. A UV plate reader is then used to determine the aqueous solubility by comparing the filtrate absorbance against that of a calibration solution prepared in an identical solvent. It is important to match the sample and calibration solutions to prevent solvochromic effects. Care must also be taken in the selection of the filter plate since nonspecific binding of compound can occur with some filter materials leading to erroneously low solubility values [22], Like nephelometry, the plate-based UV detection approach is amenable to automation. 

The supernatant concentration method uses small volumes of stock solution added to wells containing aqueous buffer in a microtiter filter plate of the type available from Millipore Inc. The solution is incubated for a given amount of time (typically in the range of 1-24 h depending on the requirements of the laboratory) and then filtered or centrifuged. The supernatant is analyzed by UV plate reader or HPLC and the concentration of dissolved species is calculated by reference to a calibration curve. This is often either a three- or a four-point curve prepared from serial dilutions of the stock solution using a solvent such as 80% v/v acetonitrile/water in which the compound is fully soluble. 

Liquid samples can be analysed directly by the spread plate (A) or the pour plate method (B) but they can also be filtered to retain the micro-organism on a filter which is than placed on the culture medium (C). Suspensions need to be filtrated or centrifuged afterwards the filtrate or the filter can be analysed. Solids (e.g. food) must be minced before filtration. Incubation at a given temperature can be aerobic or anaerobic. The result of the counting will depend on the ability to isolate the target organisms with the best recovery rate. 

To the fully grown culture of Aspergillus niger (obtained from soil using dilution plate method), taken in different conical flasks, 150 mg of each of the compounds were added separately and incubated on rotary shaker for a period of 72 hours at room temperatoe. Control was also maintained under the same conditions. At the end of the period, fungal medium was filtered and the mycelium discarded. The filtrate was extracted with chloroform, diethyl ether and ethyl acetate, in the same order. The extracts were dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure conditions to yield a pasty mass for each of the substrates. 

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