Quantitative analysis chromatographic

I had two objectives to give the chemist new to a particular area the perspective to begin an analysis, and to provide the more experienced analyst an up-to-date reference book, made accessible by a thorough index. In recognition of usage in modem analytical chemistry laboratories, instramental techniques are described in preference to wet chemistry for both qualitative and quantitative analysis. Chromatographic procedures are emphasized. For the literature prior to 1972, I recommend study of the comprehensive volume of Rosen and Goldsmith. 

Quantitative Calculations In a quantitative analysis, the height or area of an analyte s chromatographic peak is used to determine its concentration. Although peak height is easy to measure, its utility is limited by the inverse relationship between the height and width of a chromatographic peak. Unless chromatographic conditions are carefully controlled to maintain a constant column efficiency, variations in... 

In addition to modem spectroscopic methods ( H nmr spectroscopy, ftir spectroscopy) and chromatographic methods (gc, hplc), HBr titration (29) is suitable for the quantitative analysis of ethyleneimine samples which contain relatively large amounts of ethyleneimine. In this titration, the ethyleneimine ring is opened with excess HBr in glacial acetic acid, and unconsumed HBr is back-titrated against silver nitrate. 

The synthesis and the quantitative gas chromatographic analysis of stable, yet volatile, A/-trifluoroacetyl- -butyl esters of amino acids has been estabhshed (124). An extensive review of subsequent advances ia gas chromatographic iastmmentation has been provided (125). 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Ion chromatography has been successfully applied to the quantitative analysis of ions in many diverse types of industrial and environmental samples. The technique has also been valuable for microelemental analysis, e.g. for the determination of sulphur, chlorine, bromine, phosphorus and iodine as heteroatoms in solid samples. Combustion in a Schoniger oxygen flask (Section 3.31 )is a widely used method of degrading such samples, the products of combustion being absorbed in solution as anionic or cationic forms, and the solution then directly injected into the ion chromatograph. 

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 ... 

The mixture is identical in each example. The peaks are shown separated by 2, 3, 4, 5 and 6 (a) and it is clear that a separation of 6a would appear to be ideal for accurate quantitative results. Such a resolution, however, will often require very high efficiencies which will be accompanied by very long analysis times. Furthermore, a separation of 6o is not necessary for accurate quantitative analysis. Even with manual measurements made directly on the chromatogram from a strip chart recorder, accurate quantitative results can be obtained with a separation of only 4a. That is to say that duplicate measurements of peak area or peak height should not differ by more than 2%. (A separation of 4a means that the distance between the maxima of the two peaks is equal to twice the peak widths). If the chromatographic data is acquired and processed by a computer, then with modem software, a separation of 4a is quite adequate. 

Quantitative estimates of the mass of a particular solute present in a sample are obtained from either peak height or peak area measurements. The values obtained are then compared with the peak height or area of a reference solute present in the sample at a known concentration or mass. In this chapter quantitative analysis by LC will be discussed but the procedures described should not be considered as entirely appropriate for other types of chromatographic analysis. Those interested in general quantitative chromatographic analysis including GC and TLC are referred to the book by Katz (4). 

It is seen that there is not a great difference between the use of peak heights or peak areas for quantitative analysis, except possibly for very early peaks, where the results seem to indicate that peak height measurements might be more precise. However, it again must be emphasized that the measurements made by Scott and Reese were overall precision measurements that will include all variations in the chromatographic system. The difference between the two methods of measurement may well be significant, but the absolute values for precision will not, by any means, be solely dependent on the method of peak measurement. 

Quantitative Analysis using Chromatographic Techniques" (Ed. E. Katz), John Wiley and Sons, Chichester-NewYork-Brisbane-Toronto-Singapore (1987). 

Figure 5.59 Molecular structures of the diarrhetic shellfish poisons (a) pectenotoxin-6 (PTX6) (b) okadaic acid (OA) (c) dinophysistoxin-1 (DTXl) (d) yessotoxin (YTX). Reprinted from J. Chromatogr., A, 943, Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins , Ito, S. and Tsukada, K., 39-46, Copyright (2002), with permission from Elsevier Science.

Funk et al. have used a low-pressure mercury lamp without filter to liberate inorganic tin ions from thin-layer chromatographically separated organotin compounds these were then reacted with 3-hydroxyflavone to yield blue fluorescent chromatogram zones on a yellow fluorescent background [22]. Quantitative analysis was also possible here (XoK = 405 nm, Xji = 436 nm, monochromatic filter). After treatment of the chromatogram with Triton X-100 (fluorescence amplification by a factor of 5) the detection limits for various organotin compoimds were between 200 and 500 pg (calculated as tin). 

In contrast to the well-established methods for identifying and quantifying naturally occurring chlorophylls, very few reports concern quantitative analysis of chlorophyllin copper complexes in color additives and in foodstuffs. Analytical methods proposed are based on spectral properties, elemental analysis, chromatographic separation, and molecular structure elucidation or a combination of these procedures. 

Tpnnesen, H.H. and Karlsen, J., Studies on curcumin and curcuminoids Vll. Chromatographic separation and quantitative analysis of curcumin and related compounds, Z. filr Lebensm. und Forsch. A, 182, 215, 1986. 

Instead, the idea of Coupled-Column Chromatography was employed (1. Here, this means the manual coll don of fractions from one chromatograph and their reinjection into another. Coll tion of GPC fractions and their analysis by other instruments or re-injection has often been utilized qualitatively in GPC. However, precise quantitative analysis is much less often reported (6). 

Modem planar chromatography is suitable not only for qualitative and quantitative analysis but also for preparative purposes. The separation efficiency of a thin-layer chromatographic system is independent of this intended purpose and is mainly determined by the quahty of the stationary phase, that is to say, by the applied coated layer. Therefore, progress in modem planar chromatography can be attributed not only to the development of the efficiency of the instmments but also to a large extent to the availability of high-quahty precoated layers. And today, as in the past, bulk sorbents for self production, especially of preparative layer chromatography (PLC) layers, are widely used. 

The main uses of TLC include (1) qualitative analysis (the identification of the presence or absence of a particular substance in the mixture), (2) quantitative analysis (precise and accurate determination of a particular substance in a sample mixture), and (3) preparative analysis (purification and isolation of a particular substance for subsequent use). All these analytical and preparative applications of TLC require the common procedures of sample apphcation, chromatographic separation, and... 

In situ densitometry has been the most preferred method for quantitative analysis of substances. The important applications of densitometry in inorganic PLC include the determination of boron in water and soil samples [38], N03 and FefCNfg in molasses [56], Se in food and biological samples [28,30], rare earths in lanthanum, glass, and monazite sand [22], Mg in aluminum alloys [57], metallic complexes in ground water and electroplating waste water [58], and the bromate ion in bread [59]. TLC in combination with in situ fluorometry has been used for the isolation and determination of zirconium in bauxite and almnimun alloys [34]. The chromatographic system was silica gel as the stationary phase and butanol + methanol + HCl -H water -n HF (30 15 30 10 7) as the mobile phase. 

The principal limitation in the use of electrophoretic techniques is the lack of availability of suitable detection systems for quantitative analysis and unequivocal identification of pesticide analytes. Traditionally, either ultraviolet/visible (UVA IS) or fluorescence detection techniques have been used. However, as with chromatographic techniques, MS should be the detection system of choice. A brief comparison of the numbers of recent papers on the application of GC/MS and LC/MS with capillary elec-trophoresis/mass spectrometery (CE/MS) demonstrates that interfaces between CE... 

E. Katz, (Ed.), "Quantitative Analysis Osing Chromatographic Techniques", Wiley, New York, NY, 1987. 

A number of recommendations have been made in the development of quantitative chromatographic methods. The American Society for Testing Materials — using as a benchmark the reversed phase separation of benzyl alcohol, acetophenone, benzaldehyde, benzene, and dimethylterephthalate — discovered substantial laboratory-to-laboratory differences in quantitative analysis.53 These compounds are routinely used to test column performance or for system suitability testing. A followup study, using benzyl alcohol, acetophenone, p-tolualdehyde, and anisole, showed that measurement of... 

Farine, S., Villard, C., Moulin, A., Mouren, G. M., and Puigserver, A., Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods, Int. ]. Biol. Macromol., 21, 109, 1997. 

A chromatographic separation step provides various advantages to the analytical procedure (i) each component is isolated from the others (which facilitates identification) (ii) minor components in mixtures may be detected more readily than by direct analysis techniques (iii) the chromatographic retention parameter provides additional confirmation that a particular component is present or absent and (iv) quantitative analysis. However, chromatography alone does not provide information on the identity of a totally unknown sample. 

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