Retention time fundamentals

During their passage through the column, sample molecules spend part of the time in the mobile phase and part in the stationary phase. All molecules spend the same amount of time in the mobile phase. This time is called the column dead tine or holdup time (t.) and is equivalent to the tine required for an unretained solute to reach the detector frsolute retention time (t,) is the time between the instant of saiq>le introduction and when the detector senses the maximum of the retained peak. This value is greater than the column holdup time by the amount of time the solute spends in the stationary phase and is called the adjusted retention time (t, ). These values lead to the fundamental relationship, equation (1.1), describing retention in gas and liquid chromatography. 

By definition, the e]q>erlmentally determined average mobile phase velocity Is equal to the ratio of the column length to the retention time of an unretalned solute. The value obtained will depend on the ability of the unretalned solute to probe the pore volume. In liquid chromatography, a value for the Interstitial velocity can be obtained by using an unretalned solute that Is excluded from the pore volume for the measurement (section 4.4.4). The Interstitial velocity Is probably more fundamentally significant than the chromatographic velocity in liquid chromatography (39). 

We discussed the fundamentals of mass spectrometry in Chapter 10 and infrared spectrometry in Chapter 8. The quadrupole mass spectrometer and the Fourier transform infrared spectrometer have been adapted to and used with GC equipment as detectors with great success. Gas chromatography-mass spectrometry (GC-MS) and gas chromatography-infrared spectrometry (GC-IR) are very powerful tools for qualitative analysis in GC because not only do they give retention time information, but, due to their inherent speed, they are also able to measure and record the mass spectrum or infrared (IR) spectrum of the individual sample components as they elute from the GC column. It is like taking a photograph of each component as it elutes. See Figure 12.14. Coupled with the computer banks of mass and IR spectra, a component s identity is an easy chore for such a detector. It seems the only real... 

While retention time is used for peak identification, it is dependent on the flow rate, the column dimension, and other parameters. A more fundamental term that measures the degree of retention of the analyte is the capacity factor or retention factor (k ), calculated by normalizing the net retention time (% > retention time minus the void time) by the void time. The capacity factor measures how many times the analyte is retained relative to an unretained component. ... 

As readily observed in most chromatograms, peaks tend to be Gaussian in shape and broaden with time, where W, becomes larger with longer This is caused by band-broadening effects inside the column, and is fundamental to all chromatographic processes.The term, plate number (N), is a quantitative measure of the efficiency of the column, and is related to the ratio of the retention time and the standard deviation of... 

HPLC precision is critical in pharmaceutical analysis.For most pharmaceutical assays under a good manufacturing practice (GMP) environment, retention time and peak area precision of <2.0% RSD must be demonstrated before any samples can be analyzed. This section reviews the fundamental principles of HPLC precision and offers practical guidelines for its enhancement. The reader is referred to Reference 18 for a more detailed treatment of this topic. 

The most fundamental one is that the fractionation problem associated with polymer complexity is ignored. Therefore, for a complex polymer, since each detector is really seeing a wide variety of molecules, only average prc erty values at each retention time can be obtained and a polymer may appear to be uniform in composition when there is actually significant composition variety. 

The slope of a plot of the partition coefficient vs. the reciprocal of the temperature (in Kelvin) is PUR. This is the fundamental equation of gas and liquid chromatography. In our laboratory, we coat a capillary column with a polyurethane of interest and measure the retention time of chemicals passing through it. The retention time is colinear with the partition coefficient. 

Although PTGC was used as early as 1952 for the separation of alkyl chlorides, the technique did not achieve general acceptance until after the fundamental work of Dal Nogare and co-workers (27-29) and the activities of Martin and co-workers (30), who first made commercial PTGC equipment available and showed how one could predict retention times based on isothermal data by the use of graphical methods. 

Figure 13.6 shows a schematic for IGC operation. Inverse, in this instance, refers to the observation that the powder is the unknown material, and the vapor that is injected into the column is known, which is inverse to the conditions that exist in traditional gas chromatography. After the initial injection of the known gas probe, the retention time and volume of the probe are measured as it passes through the packed powder bed. The gas probes range from a series of alkanes, which are nonpolar in nature, to polar probes such as chloroform and water. Using these different probes, the acid-base nature of the compound, specific surface energies of adsorption, and other thermodynamic properties are calculated. The governing equations for these calculations are based upon fundamental thermodynamic principles, and reveal a great deal of information about the surface of powder with a relatively simple experimental setup (Fig. 13.6). This technique has been applied to a number of different applications. IGC has been used to detect the following scenarios ...

In the inverse GC at infinite dilution or near zero surface coverage, the surface characterization of the solid are realized by injecting very small quantities of probes of known properties into a column packed with the solid. The retention time can be the fundamental parameter characterizing the equilibrium distribution of the probe concentration between the probes where all probe molecules behave independently. Therefore, the retention time is a function only of the adsorbate-adsorbent interaction [111]. 

The overall quality of the separation of two solutes is measured by their resolution Rs), a combination of the thermodynamic factors causing separative transport and the kinetic factors causing dispersive transport and is an index of the effectiveness of the separa-tion. Defined by Rs = (r, - tr,a)l (w,b + where a and b refer to the two solutes, is the retention time of solute X, and is the peak width at the base of solute X in units of time, it is frequently estimated by use of the fundamental resolution equation. 

Physico-chemical properties constitute the most important class of experimental measurements, also playing a fundamental role as - molecular descriptors both for their availability as well as their interpretability. Examples of physico-chemical measurable quantities are refractive indices, molar refractivities, parachors, densities, solubilities, partition coefficients, dipole moments, chemical shifts, retention times, spectroscopic signals, rate constants, equilibrium constants, vapor pressures, boiling and melting points, acid dissociation constants, etc. [Lyman et al, 1982 Reid et al, 1988 Horvath, 1992 Baum, 1998]. 

The principle of this pulse method and its general equations are easily extended to the case of several components in a mixture. The method was used by Lindholm et al. [24] to determine the quaternary isotherms of the enantiomers of methyl- and ethyl-mandelate on the chiral phase Chiral AGP. One of the serious roadblocks encountered in the use of the pulse tracer method is that the amplitudes of most of the system peaks decrease rapidly when the plateau concentration increases. Since the signal noise increases in the same time, it becomes rapidly impossible to make any accurate measurements of the retention time of these peaks. On the basis of fundamental work by Tondeur et al. [114], the origin of this variation of the relative intensity of the system peaks was explained by Forss n et al. [47], who then derived an effective rule to determine the composition of a perturbation pulse that generates system peaks that are detected easily. The concentrations of the components in the injected perturbation pulse should... 

There have been numerous attempts to determine HLB numbers from other fundamental properties of surfactants, e.g., from cloud points of nonionics (Schott, 1969), from CMCs (Lin, 1973), from gas chromatography retention times (Becher, 1964 Petrowski, 1973), from NMR spectra of nonionics (Ben-et, 1972), from partial molal volumes (Marszall, 1973), and from solubility parameters (Hayashi, 1967 McDonald, 1970 Beerbower, 1971). Although relations have been developed between many of these quantities and HLB values calculated from structural groups in the molecule, particularly in the case of nonionic surfactants, there are few or no data showing that the HLB values calculated in these fashions are indicative of actual emulsion behavior. 

Another fundamental measure of retention is retention volume, Vr. It is sometimes preferable to record values of Vr rather than tn since tr varies with the flow rate F, while Vr is independent of F. If we wish to describe the volume of mobile phase required to elute a retained compound, then the retention volume is the product of the retention time and the mobile-phase flow rate. 

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