Internal markers

The pumps now available for liquid chromatography applications are very consistent in their flow delivery, but a very small error in the flow rate can lead to a much more significant error in the calculated average molecular masses. Consequently, the use of an internal marker for a flow rate correction plays an essential role in calibrating an SEC system and in the subsequent calculation of results. 

Ideally, an internal marker should be a polymeric species under the same influences as the samples. In practice, since the elution volume range available in SEC is very limited, the internal markers used normally have low molecular mass, either added solvents or system peaks, for instance those due to antioxidant [11]. However, some care is required, as these solvent or system peaks can shift as a consequence of non-size exclusion effects, for instance moisture content of solvent encouraging selective adsorption. An internal marker is run at the same time as the calibrants, and when samples are run, the elution times of the internal marker are compared and the elution times of the sample are adjusted to correspond to the calibration. 

The use of an internal marker can make allowance for quite large variations in flow rate. However, the flow rate over the time of the run must remain consistent. 

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In situations where conserved internal markers caimot be used, such as in spills of essentially pure compounds, the evidence for enhanced biodegradation may have to be more indirect. Oxygen consumption, increases in microbial activity or population, and carbon dioxide evolution have all been used with success. 

Push-down Automata push-down automata generalize finite automata by introducing an internal memory. Just as for finite automata, push-down automata have a finite input alphabet and a finite set of intermediate states, a subset of which constitutes the set of its output (or accepting) states. The difference is that push-down automata have an additional stack-space, consisting of some or all of the symbols of the machine s alphabet (along with perhaps some additional symbols to be used as internal markers) which they can use to store information for later use. We can therefore generalize our definition for finite automata (equation 6.4) to ... 

Host marking pheromones are important in many species of parasitic hymen-optera, because they ensure that a female parasitoid focuses on non-parasitized hosts. This, in turn, ensures a more effective use of limited host resources. Marking pheromones can be internal (injected into the host at the time of oviposition) or external (applied to the host during inspection and/or ovipo-sition). The internal markers can be detected by sensory hairs on the parasitoid ovipositor [11]. The internal markers often also delay the development of the host. 

Additionally, all samples contained 6.25 nM fluorescein-labeled proline as internal marker. (Reprinted with permission from Ref. 32. Copyright 2000, Academic Press.)... 

Each compound in the mixture is characterised by its relative mobility Rf, but the absence of a solvent front, compared to TLC, requires that an internal marker is used in order to measure the relative distance of migration. 

Capillary isoelectric focusing separates analytes based on differences in their isoelectric points (pi) using the same principles as in preparative solution IEF. After a focusing step, that builds up a linear pH gradient in the capillary (controlled with zwitterionic internal markers), the analytes move as a function of their respective charge until they reach a position of zero charge (isoelectric point). The solution is then mobilized in CIEF to the detector hydrodynamically. 

Fig. 3.10. Separation of neutral parahydroxybenzoates and internal marker compounds using a standard MEEKC method. Separation conditions as Fig. 3.9. Reprixluced with permission from [17],...

England), the on-line detectors were in sequence light scattering (model KMX-6, Chromatix Thermo Separation Products, Riviera Beach, FL), infrared (model lA, Wilks-Miran), and RI (model R401, Waters Associates). Separations were performed with tetrachloroethylene as eluent at 353 K. Solution concentrations were 5 mg/cm and toluene (0.1%) was added as internal marker. 

However, creatinine and purine derivatives are very important in the field of animal nutrition because measurements of urinary excretion of these compounds have been proposed as an internal marker for microbial protein synthesis. 

A variant of this method was described by Malvano et al. (1982) standard unit response curves are constructed with aliquots of a highly positive serum diluted with a negative sample. This approach is based on the assumption that dose-response curve parallellism eliminates the necessity to employ many different sera for the calibration curve. This positive serum is then calibrated against a WHO standard serum and the antibody content of the positive serum is expressed in international units (lU). Calibration samples of the positive serum diluted with a negative serum are included as internal markers for between-run, between-laboratory and between-method normalizations to provide analytical consistency to the measurements. 

With the HDC method, no special instrumentation or experience is needed for the simple peak-position method, and elution of the sol is directly correlated to particle size. Good repeatability for the peak-position method is experienced if an internal marker is used. HDC can be used with silica sols in the range of about 6-600 nm. The upper limit of sol size has not been clearly defined. However, experiments have suggested that sols >600 nm do not completely elute from a packed bed of 20-pm glass beads. 

The position of the internal markers (proteins with no affinity for the ligand) are revealed by staining the gets with Coomassie brilliant blue R 250. When serum albumin is used as a standard, its position is revealed during the migration by a blue band because it binds bromophenol blue. 

Internal markers or standard proteins have most often been used by researchers to assure themselves that separations by electrophoresis are consistent and reproducible (21). In addition, charge markers In Isoelectric focusing are also used, although standard preparations of these materials are less reliable, especially under denaturing conditions. Other Internal standards that are often reported are materials for assuring radlolabel activity, enzyme activity, or other materials to monitor repeatability of measurements. In all cases, the stability of the standard material Is the key to long-term quality control. Large, batches of standards that are reproducible from lot to lot would also be useful. 

Fig, 5 Isokinetic binding sedimentation of T3 receptors from oligodendrocytes and astrocytes. The receptors were extracted from nuclei of rat oligodendroglial and astroglial cells after 30 and 24 days in culture, respectively, with 0.4 M KCl and labelled in vitro with L-[ I]T3 in the absence ( -— ) or presence (O—O) of an excess of non-radioactive T3. The arrows represent the position of hemoglobin which has a sedimentation coefficient 4 3 S and was used as an internal marker (from Yusta et al.. Endocrinology 122 in press (1988) (71). With permission). 

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