Filter probe method

Where the compounds are poorly soluble or where the quantity of compotmd available is small, pK values are calculated from ultraviolet (UV) spectrophoto-metric measurements. The method simply relies on the change in UV spectra at different pH values. An adaptation of the filter probe method (described next) is used. Sample concentrations down to 4 mg/400 mL may be determined (approx. 0.000025 M). Sirius Analytical Instruments supplies the D-PAS probe, which enables spectrophotometric determinations using the GLpfCa instrument (Table 6). 

To sum up, a lot of experience is needed to determine log P values by the classical shake flask method. Alternatives have been developed and compared with each other, e.g. filter probe methods [219, 220], the AKUFVE method [221], and different centrifugal partition chromatographic techniques (which correspond to true partitioning because only two immiscible liquid phases and no solid support are involved) [222-225]. As the scope and limitations of most of these techniques have been reviewed [173, 217, 218, 225], they shall not be discussed here in detail. 

Other manual methods include a solid-liquid extraction, which consists of placing the solvent in a tube with sample and shaking the sample and filtering. This method is also called the shake-filter method. The solvent may be heated to improve extraction efficiency. Several other simple methods include sonica-tion of the sample with an ultrasonic probe and heated solvent, and dissolution of the sample directly with solvent (Majors, 1996). 

Log D profiles may be obtained by performing the filter probe experiment over a range of pH values. The critical part of the experiment is to discover whether the compound of interest has an isobestic point in its UV spectrum. If it does not, the experiment cannot be performed if it does, both log P and pKa values may be determined. This method is similar to the potentiometric method described earlier except that the amount of un-partitioned substance is determined by spectroscopy rather than by potentiometry. The advantage of this method is that only one experiment needs to be performed to yield log P, log P pp, and pKaS, but not all compounds have an isobestic point. Sample concentrations down to 4 mg/400 mL may be determined (approx. 0.000025 M). 

Barnett et al. (1992) have described a method for estimating partition coefficients from data collected using a filter probe extractor. They developed the method to analyze data from two component mixtures (e.g., in the presence of an impurity) for which there existed no suitable method. The technique is based on model fitting and may also be used for pure compounds. 

Total RNA was isolated as Wallace (19), and poly(A )-RNA purified using oligo-dT-cellulose. Northern blot analysis were performed by e-lectrophoresis of the RNA in formaldehyde-MOPS agarose and blotted onto Gene Screen membranes. trxA gene labelled with P-dCTP by nick-translation was used as a probe. Methods used for hybridization and washing of filters were those recommended by the manufacturers. 

Northern blot A method for transferring RNA from an agarose gel to a nitrocellulose filter, on which the RNA can be detected by a suitable probe. 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

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