Internal standardization, quantitation

Addition of 3-O-methyl-glucose as a quantitative internal standard. 

A microcolumn packed with 3 g of activated silica gel is used for sample cleanup and fractionation. An aliquot of the extract (containing about 20 mg oil or TSEM) is then transferred to the silica gel cleanup column to remove polar components and other interferences. The column is eluted first with hexane, which recovers samrated hydrocarbons as Fraction 1 (FI). The mixture of hexane/DCM or hexane/benzene (1 1, v v) is used to elute the aromatic compounds as Fraction 2 (F2). Half of FI and half of F2 are combined into the Fraction 3 (F3). These three fractions are concentrated to appropriate volume (0.5 to 1.0 ml) by nitrogen purge. The quantitation internal standards are 5-a-androstane, di4-terphyl, and C30 17(3(H), 21(3(H)-hopane. The Fraction 3 is analyzed for quantitation of the TPH and the UCM by GC-FID. The Fraction 1 is analyzed for determination of n-alkanes by GC-FID and biomarker terpanes and steranes by GC-MS. The Fraction 2 is analyzed for the determination of alkylated PAH homologues and other EPA priority unsubstantiated PAHs by GC-MS. 

Packed column, isothermal, temperature programming, FID, qualitative, quantitative, internal standard. 

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. 

Table 11.2 Use of Retention and Quantitation Internal Standard to Correctly Identify Peaks and Correct for Sample Loss or Concentration Changes...

Keywords headspace VOCs water soil quantitation internal standard. Introduction... 

A sixth spectrophotometric method for the quantitative determination of Pb + levels in blood uses CQ+ as an internal standard. A standard containing 1.75 ppb Pb + and 2.25 ppb CQ+ yields a ratio of Sa/Sis of 2.37. A sample of blood is spiked with the same concentration of Cu +, giving a signal ratio of 1.80. Determine the concentration of Pb + in the sample of blood. 

A seventh spectrophotometric method for the quantitative determination of Pb + levels in blood gives a linear internal standards calibration curve for which... 

Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample s matrix is to explore the effect of pH on the quantitative analysis of an acid-base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 buffer and used to analyze samples buffered to different pHs in the range of 6-10. Results can be compared with those obtained using a standard addition. 

Troost and Olavesen investigated the application of an internal standardization to the quantitative analysis of polynuclear aromatic hydrocarbons. The following results were obtained for the analysis of the analyte phenanthrene using isotopically labeled phenanthrene as an internal standard... 

Caffeine is extracted from beverages by a solid-phase microextraction using an uncoated fused silica fiber. The fiber is suspended in the sample for 5 min and the sample stirred to assist the mass transfer of analyte to the fiber. Immediately after removing the fiber from the sample it is transferred to the gas chromatograph s injection port where the analyte is thermally desorbed. Quantitation is accomplished by using a C3 caffeine solution as an internal standard. 

When possible, quantitative analyses are best conducted using external standards. Emission intensity, however, is affected significantly by many parameters, including the temperature of the excitation source and the efficiency of atomization. An increase in temperature of 10 K, for example, results in a 4% change in the fraction of Na atoms present in the 3p excited state. The method of internal standards can be used when variations in source parameters are difficult to control. In this case an internal standard is selected that has an emission line close to that of the analyte to compensate for changes in the temperature of the excitation source. In addition, the internal standard should be subject to the same chemical interferences to compensate for changes in atomization efficiency. To accurately compensate for these errors, the analyte and internal standard emission lines must be monitored simultaneously. The method of standard additions also can be used. 

Samples of analyte are dissolved in a suitable solvent and placed on the IR card. After the solvent evaporates, the sample s spectrum is obtained. Because the thickness of the PE or PTEE film is not uniform, the primary use for IR cards has been for qualitative analysis. Zhao and Malinowski showed how a quantitative analysis for polystyrene could be performed by adding an internal standard of KSCN to the sample. Polystyrene was monitored at 1494 cm- and KSCN at 2064 cm-. Standard solutions were prepared by placing weighed portions of polystyrene in a 10-mL volumetric flask and diluting to volume with a solution of 10 g/L KSCN in... 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

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 ... 

This experiment describes a quantitative analysis for caffeine, theobromine, and theophylline in tea, pain killers, and cocoa. Separations are accomplished by MEKC using a pH 8.25 borate-phosphate buffer with added SDS. A UV detector set to 214 nm is used to record the electropherogram. An internal standard of phenobarbital is included for quantitative work. 

Radiochemical methods of analysis take advantage of the decay of radioactive isotopes. A direct measurement of the rate at which a radioactive isotope decays may be used to determine its concentration in a sample. For analytes that are not naturally radioactive, neutron activation often can be used to induce radioactivity. Isotope dilution, in which a radioactively labeled form of an analyte is spiked into the sample, can be used as an internal standard for quantitative work. 

To produce a quantitative result, chromatographic peak areas of identified target compounds are compared with peak areas of the internal standards, which are of known concentration. 

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. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

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. 

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). 

Quantitative analysis using the internal standard method. The height and area of chromatographic peaks are affected not only by the amount of sample but also by fluctuations of the carrier gas flow rate, the column and detector temperatures, etc., i.e. by variations of those factors which influence the sensitivity and response of the detector. The effect of such variations can be eliminated by use of the internal standard method in which a known amount of a reference substance is added to the sample to be analysed before injection into the column. The requirements for an effective internal standard (Section 4.5) may be summarised as follows ... 

Provided a suitable internal standard is available, this is probably the most reliable method for quantitative GLC. For example, the concentration of ethanol in blood samples has been determined using propan-2-ol as the internal standard. 

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. 

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