Retention time calculation experiment

Experiments 10-27 are designed to check the autosampler injection precision, pump repeatability and detector/system linearity. One programs the system to automatically inject multiple replicate volumes of a certified test standard. One typically injects 6-10 replicates per volume. The standard component s peak areas are used for calculated injection precision (reproducibility) and system linearity whereas, the retention times are used to calculate pump repeatability. 

Calculate tM from the following experiment, employing the method above A mixture of linear alkanes, possessing six, seven and eight atoms of carbon, is injected into the chromatograph. The total retention times for these compounds were respectively, 271 s, 311 s, and 399 s, under a constant temperature of 80 °C. (Length of column 25 m, ID = 0.2 mm, <7f — 0.2 pm and the stationary phase is made up of polysiloxanes). 

The experimental hydrophobicity index, tp shows acceptable correlation with the calculated log P values, and has the advantages of all chromatographic methods (i.e. it can be automated, only a small amount of sample is required and impurities do not disturb the measurements). However, the method cannot be regarded as strictly high throughput, as the retention times have to be measured at several isocratic mobile phase compositions, which must be decided before the experiment. 

After the adsorption isotherm experiments have been completed, an isotherm equation must be chosen. This equation should fit the experimental data. Often are the experimental data (the experimental adsorption data acquired by the FA method or the perturbation retention times acquired by the PP method) only compared with the ones calculated using the adsorption isotherm parameters acquired from some adsorption isotherm models [131], This is sometimes the only validation done in this field [131], However, the adsorption isotherm parameters should preferably be validated in two step (1) the different isotherm models should be compared using statistical calculations, e.g., an F-test, and (2) by using the parameters to computer simulate elution profiles and then compare them with experimental ones. 

Both the capacity factor and the net retention time depend on the nature of the tracer substance, which is used to determine to- Therefore only k values that are based on experiments with the same tracer substance should be directly compared and used to calculate selectivities. 

Figure 18.14 Window diagram. The separation was performed with a linear gradient from 0 to 45% B and the optimum runtime needs to be found out. (a) Gradient in 15 min (b) gradient in 45 min with some elution orders reversed (c) window diagram calculated from the initial two experiments with a linear relationship between retention time and %B assumed the plot shows the resolution / of the peak pair which is critical under the respective conditions it is necessary to use long gradient runtimes to obtain a good resolution (d) optimized chromatogram with 0-45% B in 80 min, but separation is already finished after 45 min and 25% B.

Calculate the mobile-phase composition that will result in an acceptable retention time under isocratic conditions This can be done by assuming a value (e.g., 8) for the slope S, of the dependence of the logarithm of the retention factor on the solvent composition. Alternatively, a third gradient experiment can be performed with a different gradient steepness. In the latter case, one obtains both S, and the intercept k for each solute. 

The retention time could be calculated by the geometric characteristics of the dryer, but it is desirable to obtain it by experiments rather than through theoretical calculations. In this case study it is estimated on the basis of pilot plant data, and the volume is calculated by the above expression. The length L of the dryer (m) is given by the correlation... 

Here c and are the concentrations of the substance under test in the stationary and mobile phase respectively, F" and F are the volumes of the stationary and mobile phase in the column, and is the retention time of an inert substance. From measurements of and f the capacity ratio k, and from this the partition coefficient K, can be calculated. In Figure 37 experimentally determined log k values are plotted against the density p and the pressure p of supercritical carbon dioxide at 40 C as a mobile phase for SFC experiments with alkanes having between 10 and 30 carbon atoms a chemically bonded stationary phase (Carbowax 400 on Porasil C) was used and further details are given in the caption of Figure 37. > The curves show that with increasing density the retention time f and the capacity ratio k decrease and consequently the... 

In Fig. 24c, log k[ values calculated from retention times with the use of Equation (6) at a constant density of 0.44 g cm (same density as in Fig. 24a) are plotted as a function of the reciprocal absolute temperature, and in Fig. 24b at a constant temperature of 395 K against the density of the mobile phase. Within the ranges of the experiments, both plots give nice straight lines. These findings are, for example, of interest for the estimation and correlation of capacity ratios (and by this of retention times) and consequently for the development of index systems similar to those used in gas chromatography. 

Figure 1. Purification of rat CpnIO (101 residues) using Fmoc probe 2. (A) Analytical RP-HPLC (C4 medium) of crude underivatized rat CpnIO. (B) Addition of lipophilic probe 2 increases the retention time of the protein (labelled 2) thus facilitating purification from underivatized truncated sequences (labelled 1). (C) Purified protein derivatized with 2. (D Purified protein after treatment with 5% aqueous TEA to remove 2. (E) ESI-MS of purified rat CpnIO. (R Deconvoluted mass spectrum for purified rat CpnIO. The calculated mass of target product is 10770.57 Da (average). The found mass is 10771.0. (Q) RP-HPLC (C4 medium, gradient TFA-water into 100% TFA-AcCN, 60 min) of purified rat CpnIO. The insert shows the expanded peak. (H) CZE of purified rat CpnIO. The concentration of the major peak (protein in its native, heptameric state) is 84%. Separate size-exclusion chromatography experiments showed that the majority of the flanking peaks correspond to protein with correct sequence but having an aggregation state different from the major peak.

The experiments can then be carried out by injecting the predetermined volumes of the probes and measuring their retention times t. If the enthalpies of the interactions are required, then the injections need to be repeated at different column temperatures to enable the entropy change to be calculated. 

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