Retention times samples

Colunms can tolerate a limited amount of analyte before becoming overloaded, causing peak distortion and broadening, and shifts in retention time. Sample capacity ranges are from approximately 100 ng for a 0.25-mm-i.d. column with 0.25-/rm-thick film, up to 5 ixg for a 0.53-mm-i.d. column with a 5-/xm-thick stationary phase. 

A correlation between retention times and boiling points is established by calibration with a known mixture of hydrocarbons, usually normal paraffins, whose boiling points are known (see Figure 2.2). From this information, the distribution of boiling points of the sample mixture is obtained. 

Retention Behavior. On a chromatogram the distance on the time axis from the point of sample injection to the peak of an eluted component is called the uncorrected retention time The corresponding retention volume is the product of retention time and flow rate, expressed as volume of mobile phase per unit time ... 

With conventional nonspectroscopic detectors, other methods must be used to identify the solutes. One approach is to spike the sample by adding an aliquot of a suspected analyte and looking for an increase in peak height. Retention times also can be compared with values measured for standards, provided that the operating conditions are identical. Because of the difficulty of exactly matching such conditions, tables of retention times are of limited utility. 

Internal standards at a known concentration are added to the sample after its preparation but prior to analysis to check for GC retention-time accuracy and response stability. If the internal standard responses are in error by more than a factor of two, the analysis must be stopped and the initial calibration repeated. Only if all the criteria have been met can sample analysis begin. 

Continuing calibration for a Series Method is performed using calibration check compounds. Surrogate compounds are added to the matrix before sample preparation to evaluate recovery levels. To check GC retention times, internal standards are added to a sample after its preparation for analysis. 

Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

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. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

A plot of In n versus L is a straight line whose intercept is In and whose slope is —l/Gt. (For plots on base-10 log paper, the appropriate slope correc tion must be made.) Thus, from a given product sample of known shiny density and retention time it is possible to obtain the nucleation rate and growth rate for the conditions tested if the sample satisfies the assumptions of the derivation and yields a straight hne. A number of derived relations which describe the nucleation rate, size distribution, and average properties are summarized in Table 18-5. 

For some products, a decision may need to be made whether samples of product lots produced by a toller will be maintained at their site or returned to the client company. Certain samples may become hazardous waste, with associated disposal costs, when the sample retention time expires. When samples are held on behalf of the other party, ultimate disposal agreements should be in place. 

Equation (5) was examined by Scott and Reese [1] employing mixtures of nitrobenzene and fully deuterated nitrobenzene as the test sample. Their retention times were 8.927 min. and 9.061 min., respectively, giving a difference of 8.04 seconds. The separation ratio of the two solutes was 1.023 and the efficiencies of the front and rear portions of the peaks were 5908 and 3670 theoretical plates, respectively. The detector was, not surprisingly, found to have the same response to both solutes, i.e., a = (3. Thus, inserting these values in equation (5),... 

The solvent used was 5 %v/v ethyl acetate in n-hexane at a flow rate of 0.5 ml/min. Each solute was dissolved in the mobile phase at a concentration appropriate to its extinction coefficient. Each determination was carried out in triplicate and, if any individual measurement differed by more than 3% from either or both replicates, then further replicate samples were injected. All peaks were symmetrical (i.e., the asymmetry ratio was less than 1.1). The efficiency of each solute peak was taken as four times the square of the ratio of the retention time in seconds to the peak width in seconds measured at 0.6065 of the peak height. The diffusivities obtained for 69 different solutes are included with other physical and chromatographic properties in table 1. The diffusivity values are included here as they can be useful in many theoretical studies and there is a dearth of such data available in the literature (particularly for the type of solutes and solvents commonly used in LC separations). 

To demonstrate the effect in more detail a series of experiments was carried out similar to that of volume overload, but in this case, the sample mass was increased in small increments. The retention distance of the front and the back of each peak was measured at the nominal points of inflection (0.6065 of the peak height) and the curves relating the retention data produced to the mass of sample added are shown in Figure 7. In Figure 7 the change in retention time with sample load is more obvious the maximum effect was to reduce the retention time of anthracene and the minimum effect was to the overloaded solute itself, benzene. Despite the reduction in retention time, the band width of anthracene is still little effected by the overloaded benzene. There is, however, a significant increase in the width of the naphthalene peak which... 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

Occasionally, samples are run that adsorb onto the packing material. Generally, if one of the performance characteristics of the column changes by 10% or more, it is prudent to clean the column. These performance characteristics are (1) asymmetry factor, retention time, resolution, and theoretical plates. 

A standard test probe is not absolutely necessary to monitor the column. Any well-resolved peak in the sample may be used. To use a sample component, baseline data must be established when the column is new and performing well. After establishing that the column is performing properly using the manufacturer s standard test procedure, calculate the assymetry factor, theoretical plates, and resolution of one or more of the sample components. Also note the retention time. This will become the baseline test mix, which will be used for later comparison. 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

FIGURE 7.14 A Fractogel EMD BioSEC Superformance column (600-16) was loaded with 500 /il of BSA, ovalbumin, and cytochrome c (5/5/3 mg/ml) at I ml/min. The test covered 100 individual runs with the standard proteins as samples. The buffer system used was 20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2. After each individual run the column was rinsed with I /VI NaOH (60 min with I /VI NaOH at 2 ml/min). No significant change in retention times and resolution was observed after 100 cycles. 

In most cases these flow markers are species that are mixed with the sample and coinjected with the analyte onto the GPC column. The retention time of this marker is used to adjust the time axis to compensate for any moderate pump variability during the running of the standards and the samples. 

A more difficult criterion to meet with flow markers is that the polymer samples not contain interferents that coelute with or very near the flow marker and either affect its retention time or the ability of the analyst to reproducibly identify the retention time of the peak. Water is a ubiquitous problem in nonaqueous GPC and, when using a refractive index detector, it can cause a variable magnitude, negative area peak that may coelute with certain choices of totally permeated flow markers. This variable area negative peak may alter the apparent position of the flow marker when the flow rate has actually been invariant, thereby causing the user to falsely adjust data to compensate for the flow error. Similar problems can occur with the elution of positive peaks that are not exactly identical in elution to the totally permeated flow marker. Species that often contribute to these problems are residual monomer, reactants, surfactants, by-products, or buffers from the synthesis of the polymer. 

Observed sample retention time % flow rate shift Time delay... 

Corrected sample retention times Delayed injection calculation Coinjection calculation % difference... 

Absolute configurations of the isoxazolidines obtained in the nitrone cydoaddition reactions described in Schemes 7.21 and 7.22 were determined to be 3S,41 ,5S structure by comparison of the optical rotations as well as retention times in a chiral HPLC analysis with those of the authentic samples. Selection of the si face at C/ position of 3-crotonoyl-2-oxazolidinone in nitrone cydoadditions was the same as that observed in the Diels-Alder reactions of cyclopentadiene with 3-croto-noyl-2-oxazolidinone in the presence of the J ,J -DBF0X/Ph-Ni(C104)2-3H20 complex (Scheme 7.7), and this indicates that the s-cis conformation of the dipolaro-phile has participated in the reaction. 

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