Quantification Quantitative analysis

Quantification Quantitative analysis with TOF-SIMS requires careful handling, as is the case with other SIMS applications, because the secondary ion yield depends on several sensitive factors, such as the sample matrix and elements involved, which are not directly proportional to the concentration of the components in the samples [2,38]. 

As mentioned above, the interpretation of CL cannot be unified under a simple law, and one of the fundamental difficulties involved in luminescence analysis is the lack of information on the competing nonradiative processes present in the material. In addition, the influence of defects, the surface, and various external perturbations (such as temperature, electric field, and stress) have to be taken into account in quantitative CL analysis. All these make the quantification of CL intensities difficult. Correlations between dopant concentrations and such band-shape parameters as the peak energy and the half-width of the CL emission currently are more reliable as means for the quantitative analysis of the carrier concentration. 

The accuracy and precision of carotenoid quantification by HPLC depend on the standard purity and measurement of the peak areas thus quantification of overlapping peaks can cause high variation of peak areas. In addition, preparation and dilution of standard and sample solutions are among the main causes of error in quantitative analysis. For example, the absorbance levels at of lutein in concentrations up to 10 mM have a linear relationship between concentration and absorbance in hexane and MeOH on the other hand, the absorbance of P-carotene in hexane increased linearly with increasing concentration, whereas in MeOH, its absorbance increased linearly up to 5 mM but non-linearly at increasingly higher concentrations. In other words, when a stock solution of carotenoids is prepared, care should be taken to ensure that the compounds are fuUy soluble at the desired concentrations in a particular solvent. 

Unfortunately these and other existing quality control procedures do not answer aU problems. There remains a clear need for development of PCR reference materials that win provide information both on quality and quantity levels. For quality the reference materials should be host-specific and PCR primers, for positive control, may correspond to host specific house keeping genes e.g. b-actin. For quantitative analysis, fluorescence dyes in specific primers might be used in order to measure accurately the amount of DNA present. Such practices, and other as yet un-realized procedures, will be needed to achieve reliable results in the quantification of DNA analysis. 

FRET applications employing CFP and YFP are complicated due to considerable bleed-through between CFP and YFP fluorescence (Figs. 5.5B and 5.6B). Direct excitation of YFP and bleed-through of CFP fluorescence into the YFP detection channel have to be corrected for as shown in Chapters 7 and 8. The multiexponential fluorescence decay of all CFP variants complicates the quantification of FRET by donor lifetime methods. Altogether these factors make quantitative analysis of the FRET efficiency relatively difficult. 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

GC and HPLC allow for ready quantification of the components exiting the column in that the area under the peak in the chromatogram is proportional to the amount of component present. However, to make a quantitative analysis, it is essential to have a calibration curve (see Chapter 14) for each of the components of interest. This means making solutions of differing but known concentrations, injecting them, and finding the relationship between peak area and amount of material present in a manner similar to that described in Section 14.9, for colorimetric analysis. In many cases, the software that controls the chromatograph can be set up to automatically do this analysis (see also Chapter 15). 

Quantitative analysis of AP/APEO by HPLC-FL can be performed with external standard solutions of mixtures of AP or APEO. Initially quantification of oligomeric mixtures was based on the elaborate procedure of normal-phase analysis with subsequent quantification of all oligomeric peaks [27]. Kiewiet et al. [28] have described the general principle of quantification of ethoxymers in reversed-phase LC with spectroscopic detection in detail using the example of derivatised alcohol ethoxylates. Based on this method the quantitative analysis of... 

There is a recent trend towards simultaneous CE separations of several classes of food additives. This has so far been applied to soft drinks and preserved fruits, but could also be used for other food products. An MEKC method was published (Lin et al., 2000) for simultaneous separation of intense sweeteners (dulcin, aspartame, saccharin and acesulfame K) and some preservatives (sorbic and benzoic acids, sodium dehydroacetate, methyl-, ethyl-, propyl- and isopropyl- p-hydroxybenzoates) in preserved fruits. Ion pair extraction and SPE cleanup were used prior to CE analysis. The average recovery of these various additives was 90% with good within-laboratory reproducibility of results. Another procedure was described by Frazier et al. (2000b) for separation of intense sweeteners, preservatives and colours as well as caffeine and caramel in soft drinks. Using the MEKC mode, separation was obtained in 15 min. The aqueous phase was 20 mM carbonate buffer at pH 9.5 and the micellar phase was 62 mM sodium dodecyl sulphate. A diode array detector was used for quantification in the range 190-600 nm, and limits of quantification of 0.01 mg/1 per analyte were reported. The authors observed that their procedure requires further validation for quantitative analysis. 

Methods used for the detection of PAs in cmde or partially purified extracts can also be adapted for post-column analysis after fractionation (see below). Direct quantitative analysis of PAs in crude grape phenolic extracts is often impossible due to the complex sample matrix. Thus, fractionation or purification is often necessary before analysis. The Folin-Ciocalteu and Pmssian Blue assays are widely used for the quantification of total polyphenols in plants [27,28]. These methods are not specific for PAs due to the reaction of other phenolic compounds with these reagents. 

Quantitative determination of the major and minor minerals In geological materials Is commonly attempted by x-ray diffraction (XRD) techniques. Mineralogists use a variety of sophisticated and often tedious procedures to obtain semlquantltatlve estimates of the minerals In a solid sample. The mineralogist knows that XRD Intensities depend on the quantity of each mineral component In the sample even through expressions for conversion of signal Intensity to quantitative analysis often are unknown or difficult to determine. Serious difficulties caused by variables such as particle size, crystallinity, and orientation make quantification of many sample types Impractical. Because of a lack of suitable standards, these difficulties are particularly manifest for clay minerals. Nevertheless, XRD remains the most generally used method for quan-... 

Quantitative analysis of multiple components in a sample and in pack quantification of drugs in formulations... 

Instruments based on GD-MS coupling have been employed most commonly for the quantitative analysis of trace and ultratrace amounts in high-purity materials. However, it has been demonstrated that, as in GD-OES, quantitative depth profile analysis by GD-MS is possible [33]. At present, a GD-MS prototype which allows the depth quantification of thin layers on conducting or insulating materials is being developed for commercial purposes [34]. 

Several gas chromatographic detectors are sensitive to changes in the flowrate of the carrier gas. Any changes in flow rate cause the baseline to be displaced. These displacements make quantification quite difficult especially since the response of certain detectors such as thermal conductivity also changes with changes in flowrate. When an accuracy of 1% in quantitative analysis is required, the flowrate should not fluctuate more than 0.2 percent (see Chapter 4). 

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