Use of Retention Data

As a first approximation, the most commonly used method of peak identification is that of matching the retention times, or occasionally capacity factors, of the sample components with those of standard reference 

In most data systems, a given peak in a sample mixture is identified if its retention time falls within a user-defined retention time window. The retention time window is typically centered about the retention time of that peak in a standard solution (i.e., 5%). However, the size of the window may be altered depending on the number of other components in the sample that may fall within a given window and the reproducibility of the method. The more reproducible the retention times, the smaller are the windows that may be defined. 

The relative retention of a compound on two different systems can also be used for tentative peak identification. This method is based on the premise that the possibility of different compounds showing identical behavior under different conditions is relatively small. Thus, use of different detectors, for example, fluorescence and UV detection, or different techniques, such as chromatography and electrophoresis, will help to confirm the identities of peaks in a sample. 

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Sz. Nyiredy, Zs. Fater and B. Szabady, Identification in planar chromatography by use of retention data measured using characterized mobile phases , ]. Planar Chromatogr. 7 406-409 (1994). 

Quarry, M. A., Grob, R. L., and Snyder, L. R., Measurement and use of retention data from high-performance gradient elution. Contribution from "nonideal" gradient equipment,. Chromatogr., 285, 1, 1984. 

The use of retention data in combination with odour detection provides a valuable aid to the computer assisted GC-MS analysis of osmogenes. Experience in use of the technique frequently allows quite accurate predictions to be made on sample composition based on retention data and odour quality. 

The usefulness of retention data from gas chromatography can be enhanced by reporting standardized times or retention indices (RI), which involves expressing retention in terms of a ratio of the retention time (RT) of an analyte to the RT of a standard. Retention scaling based on the Kovats (1965) method requires the chromatographic separation of a homologous series of normal paraffins, esters, and others, producing an index that is the ratio of the RT of an analyte minus the RT of a less retentive standard to the RT difference between... 

The use of retention data on two or more G( . columns can improve the chances of correctly identifying an unknown compound. The columns can be used in separate experiments or sometimes they can be employed in tandem. The use of two or more columns in series is termed multidimeimonal chromatography. Likewise, the responses of two or more OC detectors can greatly aid in qualitative identification. 

The use of retention data for identification is useful in those cases where the chemical type of the sample is known and when there are a number of pure compounds of this type available for the determination of reference date. However, more often than not, in complex mixtures there are several possible interpretations for each peak and so the agreement between the retention characteristics of a known standard and any component can never be regarded as conclusive evidence of identification. To remove this ambiguity, use of an ancillary technique for identification of components is required. The ancillary technique most used in modem Instruments is mass spectrometry. This type has become so popular that directly coupled gas chromatography - mass spectrometry (GC-MS) units have become the nomi of the day. 

As already mentioned, there are two so called "dead volumes" that are important in both theoretical studies and practical chromatographic measurements, namely, the kinetic dead volume and the thermodynamic dead volume. The kinetic dead volume is used to calculate linear mobUe phase velocities and capacity ratios in studies of peak variance. The thermodynamic dead volume is relevant in the collection of retention data and, in particular, data for constructing vant Hoff curves. 

In fact, this procedure can be used for any aliphatic series such as alcohols, amines, etc. Consequently, before dealing with a specific homologous series, the validity of using the methylene group as the reference group needs to be established. The source of retention data that will be used to demonstrate this procedure is that published by Martire and his group [5-10] at Georgetown University and are included in the thesis of many of his students. The stationary phases used were all n-alkanes and there was extensive data available from the stationary phase n-octadecane. The specific data included the specific retention volumes of the different solutes at 0°C (V r(To)) thus, (V r(T)) was calculated for any temperature (Ti) as follows. 

Often, the retention time is used but, as discussed above in Section 2.3, this absolute parameter changes with column length and flow rate and this precludes the use of reference data obtained in other laboratories. To make use of these reference data, the capacity factor (k ), which removes such variability, must be employed. 

Temperature control is important for the accurate measurement of retention data, and has to be used with refractometer detectors (Section 2.4.5). Increasing the temperature can increase the speed of the separation, especially in exclusion chromatography, and usually increases the efficiency of the column (though the gain in efficiency can be lost if the mobile phase is not properly equilibrated). Complicated separations can often be optimised by increasing the temperature, but this is done very much on a trial and error basis, and most work in hplc is still done without temperature control. 

The description of the degree of retention data correlation is more complicated than it appears. For example, the 2D retention maps cannot be characterized by a simple correlation coefficient (Slonecker et al., 1996) since it fails to describe the datasets with apparent clustering (Fig. 12.2f). Several mathematical approaches have been developed to define the data spread in 2D separation space (Gray et al., 2002 Liu et al., 1995 Slonecker et al., 1996), but they are nonintuitive, complex, and use multiple descriptors to define the degree of orthogonality. 

In cases where a mixture has a large number of components, or pure standards are not available, published retention data must be consulted. The uncorrected retention time, tR (p. 86), is not suitable for this purpose because it cannot be compared with data from different columns and instruments. Valid comparisons can be made using relative retention data which are dependent only on column temperature and type of stationary phase. An adjusted retention time, / R, is first obtained by subtracting from tR the time required to elute a non-retained substance such as air (Figure 4.26)... 

Methods similar to those used in GC are applicable to HPLC. Thus, comparison of retention data is the most useful means of qualitative identification, the retention factor, , generally being used in preference to... 

Using the retention data and the chromatogram shown in Fig. 14.8, tabulate the following for each peak retention time ( r), adjusted retention time (t K), retention factor (k), partition coefficient (Kc) and number of theoretical plates (N). The column phase ratio was 250 and the gas hold up time ( m) was 0.995 min. 

In ion-pair chromatography, a great number of parameters influence the retention of a charged solute e.g., the type of solute, the type and the concentration of the pairing ion, the type and the concentration of the buffer, the mobile phase composition, etc. This makes ion-pair chromatography a versatile technique at the same time as it appears to be complicated and difficult to control. From the discussion above, it is clear that a few simple basic principles often can be used to understand the retention behavior when the experimental conditions are varied. In practical work, it may be desirable to make predictions of retentions from a limited set of retention data and without going into the more complicated theoretical models. For this purpose, an approximate equation was derived that considers most of the parameters in a simple and practically useful way. For the derivation of this simple version of the model and for a guide to its use and applicability, we refer to Ref. [8]. Here, we will only state the final equation and show one simple example of its use. 

A chromatographic peak provides valuable information, namely, the elapsed time from the injection point or the difference in elution times of two peaks (qualitative information), the peak shape (qualitative or quantitative information), and the peak size(quantitative information). The simplest qualitative tool is simply the comparison of retention data from known and unknown samples. A chromatogram illustrating the commonly used retention nomenclature is given in Figure 4.1. The retention time (tp>) is the time elapsed from injection of the sample component to the recording of the peak maximum. The retention volume (VR) is the product of the retention time and the flow rate of the carrier gas. Generally, the adjusted retention time (t ) or adjusted retention volume (V >) and the relative retention (rA/B) are used for qualitative analysis. Adjusted retention time (volume) is the difference between the retention time (volume) of the sample and an inert component (usually air). The relative retention is the ratio of the adjusted retention time (or volume) of a standard to the adjusted retention time (or volume) of the unknown, (see Chapter 2). 

Mass chromatography is a new form of gas chromatography that uses two gas density detectors operated in parallel and provides (a) mass of components within 1-2% relative without determination of response factors, (b) molecular weight of components within 0.25-1% in the mass range 2—400, and (c) a powerful identification tool by the combined use of retention time and molecular weight data. The theoretical basis of the technique and its scope as a molecular weight analyzer, a qualitative identification tool, and a quantitative analyzer in the polymer field are discussed. 

In a series of papers, Szabo et al. (1990 a,b) used a variety of stationary phases, including octadecylsilica (ODS), cyanopropyl, ethylsilica, and immobilized humic acid, to investigate the relationship between Koc and RP-HPLC capacity factors for 11 aromatic hydrocarbons. While capacity factors generated with all the stationary phases showed significant correlations with log Koc, the authors concluded that the immobilized humic acid column capacity factors, obtained by extrapolation of retention data from binary elements to 100% water, gave the best correlation. 

Table 13.7 Comparison of retention data for an environmental sample using different ultrafiltration methods...

Pollard, F. H., Nickless, G., Uden, P. C. Chromatographic studies of silicon compounds. 1. Use of a flame ionisation gauge in determination of retention data. 

If silylation alone (0.2 ml of BSTFA + 0.05 ml of TMCS) without the preceding methoximation is carried out, TMS enol ether—TMS esters are produced from keto acids. Using the procedure described, methoxime-TMS esters of keto acids and TMS ether—TMS esters of hydroxy acids are produced. Unsubstituted acids give TMS esters. The procedure eliminates possible losses of the derivatives, which can be caused by, e.g., evaporation of the solvent between the esterification and the silylation steps, and is quantitative. SE-30, OV-17 and OV-22 can be used and retention data on these stationary phases have been reported for 15 acids [159]. An example of the separation of the derivatives of some acids prepared by this procedure is illustrated in Fig. 5.12. 

The meals were extrinsically labelled by added 65 Zn. The rationale for this method is that a complete isotope exchange takes place between the added radioactive zinc isotope and the zinc present in the meal. Measurements of the uptake of radioactive iron isotopes in blood or in the whole body have been used for many years in studies of iron absorption. (12, 13, 14). The absorption in the present study is determined from measurement of the whole body retention of the radioisotope. However, this can not be done until the non-absorbed fraction of the isotope has left the body. During this periode of time some of the initially absorbed has been extrected. A correction of retention data... 

OV-17 is phenylmethyl silicone, a useful, moderately polar, silicone phase with a high maximum operating temperature (350°). It is more oxygen-sensitive than most silicone phases and is available in capillary columns. A considerable amount of retention data for drugs on this phase has been published. 

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