Elution times

The simulated distillation method uses gas phase chromatography in conjunction with an apolar column, that is, a column where the elution of components is a function of their boiling points. The column temperature is increased at reproducible rate (programed temperature) and the area of the chromatogram is recorded as a function of elution time. 

The resolution of capillary columns enables the separation of all principal components of a straight-run gasoline. The most frequently used stationary phases are silicone-based, giving an order of hydrocarbon elution times close to the order of increasing boiling point. 

Partition Ratio. The partition ratio is the additional time a solute band takes to elute, as compared with an unretained solute (for which k = 0), divided by the elution time of an unretained band ... 

A plot of the detector s signal as function of elution time or volume. 

Physical Methods. Vitamins D2 and D exhibit uv absorption curves that have a maximum at 264 nm and an (absorbance) of 450—490 at 1% concentration (Table 8). The various isomers of vitamin D exhibit characteristically different uv absorption curves. Mixtures of the isomers are difficult to distinguish. However, when chromatographicaHy separated by hplc, the peaks can be identified by stop-flow techniques based on uv absorption scanning or by photodiodearray spectroscopy. The combination of elution time and characteristic uv absorption curves can be used to identify the isomers present in a sample of vitamin D. 

Thus, if (tr) is the elution time (in seconds) of a solute having a retention volume (Vr), (ocq) is expressed as the change in flow rate per second, (At) is taken as 1 second and (nt) the number of seconds. 

Figure 1. Effect of Flow Program Rate on Elution Time...

Figure 3. Curves Relating Elution Time to Flow Program Rate for Solutes having Different Retention Volumes...

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

The precision of SEC must be established before a comparison of columns and calibration standards can be made. Consistency in flow rate or elution time is the first requirement to obtain precision in SEC. Consistency in flow rate or elution time can be monitored by the elution time of the PEO standards, which are run before and after the samples. Elution time or flow rate can be considered consistent if the elution times of the PEO standards before and after the samples agree within 0.1 min. 

FIGURE 20.14 Molecular weight versus elution time as a function of ionic strength for FVAm-HCI using CATSEC columns. 

The ability of a GC column to theoretically separate a multitude of components is normally defined by the capacity of the column. Component boiling point will be an initial property that determines relative component retention. Superimposed on this primary consideration is then the phase selectivity, which allows solutes of similar boiling point or volatility to be differentiated. In GC X GC, capacity is now defined in terms of the separation space available (11). As shown below, this space is an area determined by (a) the time of the modulation period (defined further below), which corresponds to an elution property on the second column, and (b) the elution time on the first column. In the normal experiment, the fast elution on the second column is conducted almost instantaneously, so will be essentially carried out under isothermal conditions, although the oven is temperature programmed. Thus, compounds will have an approximately constant peak width in the first dimension, but their widths in the second dimension will depend on how long they take to elute on the second column (isothermal conditions mean that later-eluting peaks on 2D are broader). In addition, peaks will have a variance (distribution) in each dimension depending on... 

Figure 4.8 The GC X GC experiment can be considered to be a series of fast second clno-matograms conducted about five times faster than the widths of the peaks on the first dimension. The ID elution time is the total chromatograpliic run time, wliile the 2D time is the modulation period (e.g. 4-5 s). This figure shows two overlapping peaks A and B, with the zones of each peak collected together. When these slices are pulsed to the second column, they are resolved. Here, we show peak B eluting later on column 1, but earlier on column 2, with the 2D peak maxima nacing out a shape essentially the same as the original peak on 1D.

The HPLC elution time was typieally under 260 min, and the CZE analysis took plaee in 60 s, whieh led to an overall run time of about 4 h. The 1 min CZE sampling interval was problematie, as the LC eolumn was probably slightly undersampled. A shorter CZE analysis time, whieh would provide a more frequent sampling rate, would improve this system a great deal. The seeond-dimension analysis time must be short relative to the first dimension, lest resolution in the first dimension be saeri-fieed. 

An example of the results obtained in the form of a chromatoelectropherogram can be seen in Figure 9.6. The contour type data display showed the three variables that were studied, namely chromatographic elution time, electrophoretic migration time, and relative absorbance intensity. Peptides were cleanly resolved by using this two-dimensional method. Neither method alone could have separated the analytes under the same conditions. The most notable feature of this early system was that (presumably) all of the sample components from the first dimension were analyzed by the second dimension, which made this a truly comprehensive multidimensional technique. 

For preparative or semipreparative-scale enantiomer separations, the enantiose-lectivity and column saturation capacity are the critical factors determining the throughput of pure enantiomer that can be achieved. The above-described MICSPs are stable, they can be reproducibly synthesized, and they exhibit high selectivities - all of which are attractive features for such applications. However, most MICSPs have only moderate saturation capacities, and isocratic elution leads to excessive peak tailing which precludes many preparative applications. Nevertheless, with the L-PA MICSP described above, mobile phases can be chosen leading to acceptable resolution, saturation capacities and relatively short elution times also in the isocratic mode (Fig. 6-6). 

In the ion-exchange technique, separated amino acids exiting (eluting) from the end of the chromatography column mix with a solution of ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is detected by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. 

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