Methods development interferences removal

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

In a polyphenolic extract, anthocyanins can interfere with other polyphenolics such as pro-cyanidins during HPLC analysis and hence should be removed prior to analysis. Anthocyanins from crude polyphenolic extracts can be removed as described in Basic Protocol 2. The ethyl acetate used for elution of phenolic compounds other than anthocyanins is removed using a rotary evaporator at 20°C. The non-anthocyanin polyphenolics are dissolved in deionized distilled water and the pH is adjusted to 7.0 with NaOH as described in Alternate Protocol 2 or the method developed by Oszmianski and Lee (1990a). In the latter method, polyphenolics were fractionated into three groups neutral fraction A (flavanols and other polar phenolics), neutral fraction B (flavonols), and acidic phenolics. Polyphenolic extracts were adjusted to pH 7.0 with NaOH... 

It has become a standard procedure to test how much the washing step removes the interferents and how little of the analyte is removed during washing. This test is useful to support the method development, but often it is based merely on a trial and error approach, so that any changes in the method or the sample matrix would require a reoptimization. 

Usually, separations are required for only one of two reasons, to remove a major cause of interference or to concentrate the elements to be determined should they be present in amounts less than their detection limit. While separation procedures must therefore be quantitative for the elements concerned, they do not necessarily have to be specific as it is possible to determine a number of elements together in one solution. This concept leads to the separation of groups of elements rather than individuals, and indeed to a general philosophy of chemical preparation and samples for atomic absorption in which as many elements as possible are brought together for determination in the final analysis solution. This should always be the aim in method development. 

The cloning of functional genes from natural microbial consortia is dependent on the high quality of the extracted and purified environmental DNA since the enzymatic modifications required during the cloning steps are sensitive to contamination by various biotic and abiotic components that are present in environmental ecosystems. For example, extraction of DNA from soils always results in coextraction of humic substances, which interfere with restriction enzyme digestion and PCR amplification and reduce transformation efficiency and DNA hybridization specificity [19 -22], Therefore, extraction methods have been developed to remove or minimize contamination of the purified DNA by humic or other interfering substances. Several protocols for the isolation of bacterial community DNA from various environmental samples have been reported in recent years. These methods are based either on re-... 

Based on the most widely accepted meanings of leaching and digestion, the former is always desirable when possible. Partial dissolution of the sample with complete removal of the target analytes provides a less complex liquid and the possibility to avoid interferences. It should be noted, however, that complete dissolution of the analytes must be ensured if the target analytes are to be quantified with adequate precision. Special care must be exercised in applying a method developed with spiked matrices to natural samples — rather than with real samples as opposed to prepared or artificial samples since the opposite of real samples would be imaginary samples . 

An alternative interference removal step was developed by Ledo de Medina et al. who developed an IC method for phosphate in natural waters in the presence of high concentration of sulphates. This interference was avoided by first precipitating sulphate as lead sulphate prior to 1C analysis. Samples with high iron content were investigated by Simon. Interferences caused by the precipitation of iron hydroxides from air oxidation of ferrous iron in anoxic water samples and from the alkaline eluent used in IC, were found to affect the determination of phosphate and other inorganic anions in riverine sediment interstitial water samples with high concentrations of dissolved iron (0.5 to 2.0 mmol/1). To eliminate this interference the complexation of iron with cyanide was used prior to IC analysis. ... 

A method development and testing function is a prerequisite to initiating laboratory operation. Each selected method must be tested for reliability in recovering the radionuclide of interest and removing impurities that interfere with measurement. Method modifications are inevitable if the sample matrices in the laboratory differ from those for which the selected method was designed. 

Welz et al. [151] investigated in detail the determination of thallium in marine sediment reference materials, a particularly complex topic, because of the volatility of TlCl, and the various spectral interferences that could be observed in the vicinity of the T1 line (refer to Section 8.2.1). The authors had previously investigated this using direct analysis of solid samples and Zeeman-effect BC [142], however they only succeeded in obtaining reliable results when ruthenium was used as permanent modifier, and a solution of ammonium nitrate was pipetted on top of the solid sample as an additional modifier. Method development turned out to be much easier in HR-CS AAS, and the spectral interference due to the sulfur content of the sediments could be completely removed using least-squares BC (refer to Section 8.2.1). 

Two methods are used to measure pH electrometric and chemical indicator (1 7). The most common is electrometric and uses the commercial pH meter with a glass electrode. This procedure is based on the measurement of the difference between the pH of an unknown or test solution and that of a standard solution. The instmment measures the emf developed between the glass electrode and a reference electrode of constant potential. The difference in emf when the electrodes are removed from the standard solution and placed in the test solution is converted to a difference in pH. Electrodes based on metal—metal oxides, eg, antimony—antimony oxide (see Antimony AND ANTIMONY ALLOYS Antimony COMPOUNDS), have also found use as pH sensors (8), especially for industrial appHcations where superior mechanical stabiUty is needed (see Sensors). However, because of the presence of the metallic element, these electrodes suffer from interferences by oxidation—reduction systems in the test solution. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

In TLC the detection process is static (sepaurations achieved in space rather than time) and free from time constraints, or from interference by the mobile phase, which is removed between the development and detection process. Freedom from time constraints permits the utilization of any variety of techniques to enhance detection sensitivity, which if the methods are nondestructive, nay be applied sequentially. Thus, the detection process in TLC is nore flexible and variable than for HPLC. For optical detection the minimum detectable quantities are similar for both technlqpies with, perhaps, a slight advantage for HPLC. Direct comparisons are difficult because of the differences in detection variables and how these are optimized. Detection in TLC, however, is generally limited to optical detection without the equivalent of refractive... 

An analytical procedure that quantifies the total AE concentration resolved by alkyl chain length for various environmental matrices (influent, effluent, and river water) was developed by Di Corcia et al. [41]. The method utilises a reverse-phase column to extract and concentrate AE from surface waters and wastewaters and utilises strong anionic and cationic exchange columns to remove potential interferences. Samples are passed through the RP extraction column (Ci). AE and potential anionic and cationic interferences are eluted from the Ci column and passed directly through the SAX and SCX. The SAX and SCX columns retain anionic and cationic materials while non-ionic AE are not retained. Recovery of AE from influent, treatment plant effluent, and river water is quantitative (65—102%) over a range of concentrations for all matrices. 

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