Analysis without external calibration

Analysis of binary mixtures without external calibration... 

Each detection system has its own limitations. Compounds with no chromophor are not UV active, for instance, and may be quantified with ELS. A short analysis of the compound class is therefore needed to prepare and interpret the solubility experiments. Direct quantification of the sample without any external calibration is possible if a nitrogen detector is used. Throughput is higher and a standard injection is not necessary. 

A general spectroscopic method of quantitatively analyzing binary mixtures, which does not require external calibration, has been developed by Diem and Krimm (1981). It is based on a technique introduced by Hirschfeld (1976), which makes it possible to resolve a spectrum of a mixture of a small number of components whose proportions are unknown into the spectra of the constituents without isolating them. If the components are recognized, traditional methods of quantitative analysis can be applied. If the components are not known, a spectrum of the original mixture and a spectrum obtained after partial... 

SEC-ESIMS is a valuable tool for polymer characterization. Compounds are separated based on their hydrodynamic size in solution, but upon detection, an absolute molecular weight is also furnished. Only 1% of the SEC effluent is required for ESIMS analysis, thereby accommodating the popular SEC detectors. SEC-ESIMS provides an attractive solution to the calibration of SEC without the use of external calibrants. Chemical composition distribution information on copolymers is easily afforded provided the individual monomers differ in molecular weight. The successively acquired mass spectra contain narrow fractions of the overall distribution that simplifies the analysis of complex formulations. Unfortunately, we have not been able to provide structured details on materials beyond 5000 Da due to the low resolution of the quadrupole mass spectrometer. Nevertheless, SEC-ESIMS is an exciting hyphenated techniques for polymer characterization. 

The MSA is often used if no suitable external calibration curve has been prepared. There may be no time to prepare calibration standards— for example, in an emergency situation in a hospital it may be necessary to measure sodium rapidly in a patient s serum. It may not be possible to prepare a valid set of calibration standards because of the complexity of the sample matrix or due to lack of sufficient information about the sample—for example, industries often require the analysis of mystery samples when something goes wrong in a process. MSA calibration is very useful when certain types of interferences are present in the sample matrix. MSA permits us to obtain accurate results without removing the interferences by performing the calibration in the presence of the interferences. It is often used when only one sample must be analyzed, and the preparation of external standards would be inefficient. 

The quality assurance sample used is an aliquot of a urine pool prepared by mixing equal proportions of 24-h samples from 20 men and women. Thus, the values obtained on GC-MS analysis should approximate true average steroid excretions. We use these values to adjust the calibration of steroids without authentic materials in the external standard. We have determined that the excretion of 18-OH-THA in the quality assurance sample should be 90 pg/24 h THAldo, 30 pg/24 h, and 18-oxo-THF, 5 pg/24 h. The amount of these steroids in clinical samples is thus calibrated against a normal excretion. 

The methyl esters can be also determined by GC-FID. Using a 30 m x 0.32 mm ID x 0.25 pm (film thickness) capillary column, such as DB-1701 or equivalent, the compounds can be adequately separated and detected by FID. The recommended carrier gas (helium) flow rate is 35 cm/s, while that of the makeup gas (nitrogen) is 30 cm/min. All of the listed herbicides may be analyzed within 25 min. The oven temperature is programmed between 50 and 260°C, while the detector and injector temperatures should be 300 and 250°C, respectively. The herbicides may alternatively converted into their trimethylsilyl esters and analyzed by GC-FID under the same conditions. FID, however, gives a lower response as compared with ECD. The detection level ranges from 50 to 100 ng. For quantitation, either the external standard or the internal standard method may be applied. Any chlorinated compound stable under the above analytical conditions, which produces a sharp peak in the same RT range without coeluting with any analyte, may be used as an internal standard for GC-ECD analysis. U.S. EPA Method 8151 refers the use of 4,4,-dibromooctafluorobiphenyl and 1,4-dichlorobenzene as internal standards. The quantitation results are expressed as acid equivalent of esters. If pure chlorophenoxy acid neat compounds are esterified and used for calibration, the results would determine the actual concentrations of herbicides in the sample. Alternatively, if required, the herbicide acids can be stoichiometrically calculated as follows from the concentration of their methyl esters determined in the analysis ... 

The third concept is to generate as much useful information as possible from a single analysis (e.g., to obtain both structural and quantitative information from a single run without tedious external or internal calibrations) [39]. 

The colloidal state inevitably brings about difficulties for the experimentalist when separation of the disperse phase from the dispersion medium is needed. This is the case when the speciation and concentration of only the free soluble species have to be determined. Separation of the ionic solution from the small colloidal particles for conventional chemical analysis is nontrivial, although separation techniques such as ultracentrifugation, dialysis, and field-flow fractionation have been successfully used. If the soluble species of interest have an active nuclear spin, the liquid NMR technique wiU constitute an alternative and simpler way to characterize and quantify those species without being affected by the disperse phase. An exception is the case where the colloidal species gives a signal that fully overlaps the sharp resonance of the solution entity. As NMR is quantitative, the absolute concentration of the species can be estimated based on an internal reference of known concentration but different chemical shift relative to the sample signals. Alternatively, a calibration curve can be established from a set of external standard solutions (preferably the same substance found in the sample) measured under the same experimental NMR conditions as those applied to the sample. 

The on-line system is the most commonly used system for compositional analyzers. In this approach, the sampling system is automated and directly interfaced to the analyzer. With the analyzer system external to the process, it is fairly easy to repair and/or calibrate the system without the need to shut the process down. As this is the most common real-time analyzer approach, it will be discussed in more detail later in the chapter. The at-line process is similar to the old laboratory analysis in that a manual sample must be taken out of the process but in this case the analyzer is rugged enough to be located in the process area to do an immediate analysis once the sample is obtained. This method is labor intensive to do but does solve the sampling... 

Instead of using an IS, calibration can also be obtained by using a hop extract with known composition. The peak area of the reference extract is then directly compared with that of the sample to be analysed. The unknown and reference extract should be analysed consecutively, which means double analysis time. The choice of internal or external reference was discussed in more detail earlier in this chapter. In some cases oxidation during the sample preparation and during the chromatography may be feared or is a reality. Therefore the addition of 1 % BHT to all solvents is sometimes needed. Whether such oxidation occurs or not can be evaluated by running the analysis with and without BHT addition. If the results are similar, the system is not oxidizing the hop acids and BHT can be ommitted. 

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