Quantitative internal standards procedure

To use KBr discs for quantitative measurements it is best to employ an internal standard procedure in which a substance possessing a prominent isolated infrared absorption band is mixed with the potassium bromide. The substance most commonly used is potassium thiocyanate, KSCN, which is intimately mixed and ground to give a uniform concentration, usually 0.1-0.2 per cent, in the potassium bromide. A KBr/KSCN disc will give a characteristic absorption band at 2125 cm 1. Before quantitative measurements can be carried out it is necessary to prepare a calibration curve from a series of standards made using different amounts of the pure organic compound with the KBr/KSCN. A practical application of this is given in Section 19.9. 

Define internal standard. Tell why an internal standard is important in a quantitative analysis by GC. Also tell what is plotted on the x- and y-axes when plotting the standard curve in internal standard procedures. 

Variation in sample volume is the factor that most affects the precision of quantitative measurements and the use of an injection valve may overcome this and permit the use of external standards. However, it is still often desirable to use an internal standardization procedure as this will reduce the effects of any variation in the detector responsiveness over a period of time. 

The trend in industrial hygiene work is to identify the particular species responsible for an occupational health problem, although assessment of exposures to inorganic materials previously has most often been based on elemental analysis When a solid inorganic compound is to be identified and quantified, X-ray diffraction should be among the approaches considered This paper has outlined the use of X-ray powder diffraction as a tool for the identification and quantitation of crystalline particulates It has been shown that the substrate standard method is the preferred quantitative procedure for several reasons (1) easy adaptability to most analytes (2) fast analysis time (as compared to the internal standard procedure) and (3) accurate determination of matrix absorption effects While there are a number of reasons why a given compound may not be amenable to this technique, it is likely that the list of analytes will be added to in the future ... 

GC Analyses, The n-alkanes in the fuel samples were determined with a 100 meter OV-101 wall coated glass capillary column. The inlet split ratio was 50 1, the column oven was temperature programmed from 80 to 240°C, and the inlet temperature was 310°C. The internal standard procedure was used for quantitation. 

Quantitation may be done crudely on the spots separated by planar chromatographic techniques such as TLC or slab gel electrophoresis (see Chapter 13). One might compare the optical density, the fluoresence, or the degree of stationary phase fluoresence suppression by the unknown spot to a series of standards of known concentration. In contrast, the electrical signals from the variety of detectors used in various column chromatography instruments can be precisely, reproducibly, and linearly related to the amount of analyte passing through the detector cell. If all parameters of injection, separation, and detection are carefully controlled from run to run, and especially if appropriate quantitative internal standards are incorporated in the procedure, accuracy and precision better than +1 % may be attained. 

For coupling of HSGC with MS, the internal standard procedure has proved particularly successful for quantitative analyses. Besides the headspace-specific effects, possible variations in the MS detection are also compensated for. The best possible precision is thus achieved for the whole procedure. The MHE procedure can be used in the same way. 

The isotope dilution analysis method provides a definitive technique that does not depend on the comparison to one or more external calibration standards for quantitation. The method is similar to the standard addition technique in that a known quantity of analyte material is added to the sample to be analyzed, thereby performing the quantitation in situ. This approach ehminates matrix interference problems. The method is also similar to the internal standardization procedure, with the exception that a form of the analyte itself serves as the internal standard. AU of these factors combine to provide analysis characteristics comparable to the most basic gravimetric methods. 

A quantitative analysis for vitamin Bi was carried out using this procedure. When a solution of 100.0 ppm Bi and 100.0 ppm o-ethoxybenzamide was analyzed, the peak area for vitamin Bi was 71 % of that for the internal standard. The analysis of a 0.125-g vitamin B complex tablet gave a peak area for vitamin Bi that was 1.82 times as great as that for the internal standard. How many milligrams of vitamin Bi are in the tablet ... 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

It should be noted here that the difficulty of accurately injecting small quantities of liquids imposes a significant limitation on quantitative gas chromatography. For this reason, it is essential in quantitative GLC to use a procedure, such as the use of an internal standard, which allows for any variation in size of the sample and the effectiveness with which it is applied to the column (see Sections 9.4(5) and 9.7). 

The use of an internal standard probably gives the most accurate quantitative results. However, the procedure depends upon finding an appropriate substance that will elute in a position on the chromatogram where it will not interfere or merge with any of the natural components of the mixture. If the sample contains numerous components, this may be difficult. Having identified a reference standard, the response factors for each component of interest in the mixture to be analyzed must be determined. A synthetic mixture is made up containing known concentrations of each of the components of interest and the standard. If there are (n) components, and the (r) component is present at concentration (Cr) and the standard at a concentration (Cst). 

An internal standard is desirable in any quantitative trace environmental analysis. The ideal internal standard should behave in a manner identical to that of the analyte in all the procedures followed for isolation, purification, and determination without producing interference. This is a difficult requirement to meet for nitrosamines, especially for NDMA. 

It is clear that neither NMEA nor NDPA is appropriate for an internal standard in NDMA determination if criteria are interpreted strictly, but both compounds have been used for this purpose. Addition of a nitrosamine, not normally present in the sample, is helpful in detecting any gross errors in the procedure, but the addition should not be considered to be internal standardization. Utilization of NMEA or NDPA to indicate recovery of NDMA can lead to significant errors. In most reports of the application of these "internal standards", recovery of all nitrosamines was close to 100%. Under these conditions, any added compound would appear to be a good internal standard, but none is necessary. NDMA is a particularly difficult compound for use of internal standardization because of its anomalous distribution behavior. I mass j ectrometry is employed for quantitative determination, H- or N-labeled NDMA could be added as internal standard. Because the labeled material would coelute from GC columns with the unlabeled NDMA, this approach is unworkable when GC-TEA is employed or when high resolution MS selected ion monitoring is used with the equipment described above. 

The use of Equation (22) is very general, but it is also possible, with accurate measurements and data treatment, to perform the quantitative phase analysis in semi-crystalline materials without using any internal standard. This procedure is possible only if the chemical compositions of all the phases, including the amorphous one, are known. If the composition of the amorphous phase is unknown, the quantitative analysis without using any internal standard can still be used provided that the chemical composition of the whole sample is available [51]. This approach, until now, has been developed only for the XRD with Bragg-Brentano geometry that is one of the most diffused techniques in powder diffraction laboratories. 

This procedure allows quantitative phase analysis without using any internal standard, but it requires the knowledge of the composition of the sample and a careful treatment of the experimental data, which have to be corrected for the air scattering. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

Pd removal was determined as follows. An aliquot of a representative liquid or solid sample was accurately weighed and subsequently digested by refluxing in nitric and/or hydrochloric acid using a closed vessel microwave procedure (CEM MARS5 Xpress or Milestone Ethos EZ). Cooled, digested samples were diluted, matrix matched to standards, and referenced to a linear calibration curve for quantitation an internal standard was employed to improve quantitation. All samples were analyzed by an Inductively Coupled Plasma Mass Spectrometer or ICP/MS (Perkin Elmer SCIEX Elan DRCII) operated in the standard mode. 

The accuracy of the injection volume measurement can be very important for quantitation, since the amount of analyte measured by the detector depends on the concentration of the analyte in the sample as well as the amount injected. In Section 12.8, a technique known as the internal standard technique will be discussed. Use of this technique negates the need for superior accuracy with the injection volume, as we will see. However, the internal standard is not always used. Very careful measurement of the volume with the syringe in that case is paramount for accurate quantitation. Of course, if a procedure calls only for identification (Section 12.7), then accuracy of injection volume is less important. See Workplace Scene 12.1 for an example of a purge-and-trap procedure for injecting a GC sample. 

Select one of the quantitation procedures we have discussed (response factor method, internal standard method, or standard addition method) and describe ... 

In the strategy for GC, it is noted that there may be no need for weight or volume data for the sample because the sample itself may be injected directly and quantitation performed solely from the chromatographic information. It is also noted that the internal standard method is common, and the solution preparation and calibration procedure are altered accordingly. 

A rapid technique for the identification of surfactants in consumer products by ESI-MS was proposed by Ogura and co-workers [6], After a simple preparation procedure, infusion of the sample, which was prepared in a water/methanol mixture (50 50) containing 10 mM ammonium acetate, allowed assignment of the [M + NH4]+ ions of Cio- and Ci2-mono- and -diglucoside in the mass spectrum (ion masses as in Table 2.7.1). The approach even permitted quantitative analysis when deuterated internal standards were used. 

Procedure 3.1 Quantitation of barbiturates by HPLC using an internal standard... 

This method assumes that the response factor is constant over a range of concentrations and it is often more acceptable to determine the response factor for a range of test concentrations. In this method, a calibration curve is produced by incorporating a fixed amount of the internal standard in samples that contain known amounts of the test compound. For each concentration the ratio of peak heights is determined and plotted against concentration (Procedure 3.2). For quantitation of a test sample, the same amount of the internal standard is introduced in its usual way and the ratio of peak heights for the standard and unknown is used to determine the concentration of the unknown from the calibration curve. 

Procedure 3.2 Quantitation of ethanol by GLC using an Internal standard... 

It is critical when performing quantitative GC/MS procedures that appropriate internal standards are employed to account for variations in extraction efficiency, derivatization, injection volume, and matrix effects. For isotope dilution (ID) GC/MS analyses, it is crucial to select an appropriate internal standard. Ideally, the internal standard should have the same physical and chemical properties as the analyte of interest, but will be separated by mass. The best internal standards are nonradioactive stable isotopic analogs of the compounds of interest, differing by at least 3, and preferably by 4 or 5, atomic mass units. The only property that distinguishes the analyte from the internal standard in ID is a very small difference in mass, which is readily discerned by the mass spectrometer. Isotopic dilution procedures are among the most accurate and precise quantitative methods available to analytical chemists. It cannot be emphasized too strongly that internal standards of the same basic structure compensate for matrix effects in MS. Therefore, in the ID method, there is an absolute reference (i.e., the response factors of the analyte and the internal standard are considered to be identical Pickup and McPherson, 1976). 

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