Quantitation and Standardization

In order to determine the substance concentration in an unknown analysis sample, the peak areas of the sample are calculated using the calibration function and the results are given in terms of quantity or concentration. Many data systems also take into accormt the sample amount as weighed out and dilution or concentration steps in order to be able to give the concentration in the original sample. 

For GC-MS, the methods of external or internal standard calibration or standard addition are used as standardization procedures (Funket /., 2008). 

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Presently, immunohistochemistry requires improvements in quality, reproducibility, speed, quantitation, and standardization. Some of these goals can be achieved by using computerized bar code-driven automatic immunostainers that automatically dispense reagents, control washing, mixing, and heating to optimize immunohistochemical reaction... 

Quantitation and standardization of the amounts of total DNA isolated from each tissue sample must also be performed prior to analysis by Southern blot hybridization so that the amounts of total DNA loaded into each lane are equivalent and so that the autoradiographic signals obtained after Southern blot hybridization are directly comparable. This is not required for the analysis of DNA isolated from tissue culture as the number of PDH extracted are standardized at the time of seeding. By recording the exact weight of the tissue extracted, it is possible to calculate the viral load per cell in each sample. 

Some of the answers have already been provided in the previous sections. All of these properties, however, have to be measured in quantitative and standard tests. This testing has done much to reassure the general public of the value and suitability of polymers. Many of these tests are carried out under conditions stipulated by ASTM (American Society for Testing and Materials). Not only new polymers are analysed by these techniques but also many new grades of old polymers. For example there are already dozens of grades of PE and nylon, new grades appear yearly, and data for all these grades are necessary. 

Connolly surfaces are standard in Molecular Modeling tools, and permit the quantitative and qualitative comparison of different molecules. 

Although many quantitative applications of acid-base titrimetry have been replaced by other analytical methods, there are several important applications that continue to be listed as standard methods. In this section we review the general application of acid-base titrimetry to the analysis of inorganic and organic compounds, with an emphasis on selected applications in environmental and clinical analysis. First, however, we discuss the selection and standardization of acidic and basic titrants. 

Selecting and Standardizing a Titrant Most common acid-base titrants are not readily available as primary standards and must be standardized before they can be used in a quantitative analysis. Standardization is accomplished by titrating a known amount of an appropriate acidic or basic primary standard. 

Quantitative Analysis Using the Method of Standard Additions Because of the difficulty of maintaining a constant matrix for samples and standards, many quantitative potentiometric methods use the method of standard additions. A sample of volume, Vx) and analyte concentration, Cx, is transferred to a sample cell, and the potential, (ficell)x) measured. A standard addition is made by adding a small volume, Vs) of a standard containing a known concentration of analyte, Cs, to the sample, and the potential, (ficell)s) measured. Provided that Vs is significantly smaller than Vx, the change in sample matrix is ignored, and the analyte s activity coefficient remains constant. Example 11.7 shows how a one-point standard addition can be used to determine the concentration of an analyte. 

Earlier we described a voltammogram as the electrochemical equivalent of a spectrum in spectroscopy. In this section we consider how quantitative and qualitative information may be extracted from a voltammogram. Quantitative information is obtained by relating current to the concentration of analyte in the bulk solution. Qualitative information is obtained from the voltammogram by extracting the standard-state potential for the redox reaction. For simplicity we only consider voltammograms similar to that shown in Figure 11.33a. 

The pH must be kept at 7.0—7.2 for this method to be quantitative and to give a stable end poiut. This condition is easily met by addition of soHd sodium bicarbonate to neutralize the HI formed. With starch as iudicator and an appropriate standardized iodine solution, this method is appHcable to both concentrated and dilute (to ca 50 ppm) hydraziue solutious. The iodiue solutiou is best standardized usiug mouohydraziuium sulfate or sodium thiosulfate. Using an iodide-selective electrode, low levels down to the ppb range are detectable (see Electro analytical techniques) (141,142). Potassium iodate (143,144), bromate (145), and permanganate (146) have also been employed as oxidants. 

Quantitative Phase Analysis. Once the identity of the components in a sample are known, it is possible to determine the quantitative composition of the sample. There are several different methods for doing a quantitative analysis, but the most rehable method is to use mixtures of known composition as standards. The computer can determine quantitatively the relative amounts of each component in the unknown sample. For accurate calculations of relative amounts in the unknown sample, it is necessary that the sample and standards have uniform distributions of crystaUites. Often the sample and standards are rotated during data collection to provide a more even distribution of crystaUites which diffract. 

Bromine is used as an analytical reagent to determine the amount of unsaturation in organic compounds because carbon—carbon double bonds add bromine quantitatively, and for phenols which add bromine in the ortho and para positions. Standard bromine is added in excess and the amount unreacted is deterrnined by an indirect iodine titration. Bromine is also used to oxidize several elements, such as T1(I) to T1(III). Excess bromine is removed by adding phenol. Bromine plus an acid, such as nitric and/or hydrochloric, provides an oxidizing acid mixture usefiil in dissolving metal or mineral samples prior to analysis for sulfur. 

GDMS is slowly replacing SSMS because of its increased quantitative accuracy and improved detection limits. Like SNMS and SALI, GDMS is semiquantitative without standards ( a factor of 3) and quantitative with standards ( 20%) because sputtering and ionizadon are decoupled. GDMS is often used to measure impuri-des in metals and other materials which are eventually used to form thin films in other materials applications. 

Because of the complex nature of the discharge conditions, GD-OES is a comparative analytical method and standard reference materials must be used to establish a unique relationship between the measured line intensities and the elemental concentration. In quantitative bulk analysis, which has been developed to very high standards, calibration is performed with a set of calibration samples of composition similar to the unknown samples. Normally, a major element is used as reference and the internal standard method is applied. This approach is not generally applicable in depth-profile analysis, because the different layers encountered in a depth profile of ten comprise widely different types of material which means that a common reference element is not available. 

Equation (1) can be viewed in an over-simplistic manner and it might be assumed that it would be relatively easy to calculate the retention volume of a solute from the distribution coefficient, which, in turn, could be calculated from a knowledge of the standard enthalpy and standard entropy of distribution. Unfortunately, these properties of a distribution system are bulk properties. They represent, in a single measurement, the net effect of a large number of different types of molecular interactions which, individually, are almost impossible to separately identify and assess quantitatively. 

In contrast to traditional ambient fixed station "parametric approaches to river quality evaluation for trends determination and water quality standards attainment, data from the Willamette River, Oregon, USA were used to demonstrate the value of quantitative, semi-quantitative and qualitative approaches to mechanistic assessment of river water quality. 

Under many experimental conditions, the mass spectrometer functions as a mass-sensitive detector, while in others, with LC-MS using electrospray ionization being a good example, it can behave as a concentration-sensitive detector. The reasons for this behaviour are beyond the scope of this present book (interested readers should consult the text by Cole [8]) but reinforce the need to ensure that adequate calibration and standardization procedures are incorporated into any quantitative methodology to ensure the validity of any results obtained. 

Since the carrier effect is not general for all analytes and all additives, quantitative studies using the particle-beam interface should only be carried out after a very careful choice of experimental conditions and standard(s) to be used, with isotopic-dilution methodology being advocated for the most accurate results. 

Recognizing these problems, UK food regulatory authorities have generally abandoned the use of quantitative microbial counts as enforceable standards of food quality. Despite this, the European Pharmacopoeia has introduced both quantitative and qualitative mierobial standards for non-sterile medicines, which might become enforceable in some member states. It prescribes varying maximum total microbial levels and exclusions of particular species according the routes of administration. The British Pharmacopoeia has now ineluded these tests, but suggest they should be used... 

In addition, the SRM contains a DNA molecular size standard for sizing the allele fragments a set of quantitative DNA standards in concentrations of 6-250 ng/6 pL and a visualization marker set which produces twelve bands ranging from 594 to 35 937 base pairs and which is used to assess the DNA separation on the electrophoretic gel. 

The MHLW described the validation protocol criteria in the 2006 official guidelines to standardize the Japanese official method for specific allergenic ingredient detection. The outlines of the validation protocol criteria for the food allergenic ingredient quantitative and qualitative detection methods are shown in Tables 4.7 and 4.8, respectively. 

Ensure that quantitation yields accurate and precise results by monitoring the background, recoveries and standard deviations. 

Instrument calibration is done during the analysis of samples by interspersing standards among the samples. Following completion of the samples and standards, a linear calibration curve is estimated from the response of the standards using standard linear regression techniques. The calibration constants obtained from each run are used only for the samples quantitated in that run. Drastic changes or lack of linearity may indicate a problem with the detector. 

At least four chromatographic standards prepared at concentrations equivalent to 50-70% of the limit of quantitation (LOQ) up to the maximum levels of analytes expected in the samples should be prepared and analyzed concurrently with the samples. In LC/MS/MS analysis, the first injection should be that of a standard or reagent blank and should be discarded. Then, the lowest standard should be injected, followed by two to four blanks, control samples, fortifications or investigation samples, followed by another chromatographic standard. This sequence is then repeated until all the samples have been injected. The last injection should be that of a standard. In order to permit unattended analysis of a normal analysis set, we recommend that samples and standards be made up in aqueous solutions of ammonium acetate (ca 5 mM) with up to 25% of an organic modifier such as acetonitrile or methanol if needed. In addition, use of a chilled autosampler maintained at 4 °C provides additional prevention of degradation during analysis. 

Stock solutions of approximately 1 mg mL were prepared by dissolving the appropriate amounts of the analytical standards in acetonitrile. Working standard solutions for fortification were prepared in volumetric flasks by appropriate dilutions of the stock solutions for each analyte or combination of analytes. During analysis, SCA is converted to DMS and HMS is derivatized therefore, the analytical standard solutions for quantitation and instrument calibration contained sulfentrazone, DMS and derivatized HMS. A measured volume of a standard solution containing sulfentrazone, DMS and HMS (prepared from stock solutions) was derivatized simultaneously with the samples. 

The determination of the relationship between detector response and the sample concentration is termed the calibration of the method. There are two types of methods in use for the quantitative analysis of a sample, i.e., the external standard and the internal standard method. An external standard method is a direct comparison of the detector response of a pure compound (standard) to a sample.2 The calibration of the method is performed by preparing standards of varying concentration and analyzing them by a developed method. Method 1 (below) was developed for toluene, and standards of varying concentration were prepared and analyzed. The results obtained are summarized in Table 2 see Figure 3. 

In direct insertion techniques, reproducibility is the main obstacle in developing a reliable analytical technique. One of the many variables to take into account is sample shape. A compact sample with minimal surface area is ideal [64]. Direct mass-spectrometric characterisation in the direct insertion probe is not very quantitative, and, even under optimised conditions, mass discrimination in the analysis of polydisperse polymers and specific oligomer discrimination may occur. For nonvolatile additives that do not evaporate up to 350 °C, direct quantitative analysis by thermal desorption is not possible (e.g. Hostanox 03, MW 794). Good quantitation is also prevented by contamination of the ion source by pyrolysis products of the polymeric matrix. For polymer-based calibration standards, the homogeneity of the samples is of great importance. Hyphenated techniques such as LC-ESI-ToFMS and LC-MALDI-ToFMS have been developed for polymer analyses in which the reliable quantitative features of LC are combined with the identification power and structure analysis of MS. 

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