Preparative chromatography experiment

The dimensions of the exit tube from the detector are not critical for analytical separations but they can be for preparative chromatography if fractions are to be collected for subsequent tests or examination. The dispersion that occurs in the detector exit tube is more difficult to measure. Another sample valve can be connected to the detector exit and the mobile phase passed backwards through the detecting system. The same experiment is performed, the same measurements made and the same calculations carried out. The dispersion that occurs in the exit tube is normally considerably greater than that between the column and the detector. However, providing the dispersion is known, the preparative separation can be adjusted to accommodate the exit tube dispersion and allow an accurate collection of each solute band. 

The chemical structure of PBS also may be altered by exposure to radiation and such changes may contribute to the solubility rate difference between an exposed and an unexposed PBS film. U-type films were prepared from unirradiated powders while IP-type films were prepared from irradiated powders. Inspection of Table II or Figure 2 shows that the three U-type films have slightly larger solubility rates than IP-type films of comparable M. The solubility rate differences between IP and U-type films are small relative to the differences between IP and IF type films. The solubility rate difference between a U and an IP film of comparable M must arise from chemical structural differences between irradiated and unirradiated powders. These radiation-induced changes may also be responsible for differences observed in the elution behavior between irradiated and unirradiated PBS samples in gel permeation chromatography experiments. Irradiated PBS samples yield abnormally broad elution curves while unirradiated samples elute normally I3.8I. 

Oligosaccharides ranging from two to six repeating units were prepared by testicular hyaluronidase digestion of hyaluronate, and isolated by Sephadex G-50 chromatography. Experiments were carried out to determine the minimum chain length of hyaluronate to which core molecules would bind. When core molecules were mixed... 

The work carried out on this compound highlights nearly all of the main problems that are likely to be encountered during the development of preparative chromatography procedures. For any preparative work (of significant size) the following four point checklist can be summarised from the above experiences ... 

Countercurrent chromatography (CCC) is based on the use of two-phase liquid solvent systems. One of the characteristics of a two-phase system is its settling time, which is the time required for the mixture of both phases to be completely separated into two layers, usually in the Earth s gravitational field. The measurement of the settling time itself is helpful in preparing the experiment, for instance, when preparing the mobile and stationary phases in the same vessel. However, it is also intrinsically linked to the hydrodynamic behavior of the two-phase system and, therefore, to its physical parameters such as densities, viscosities, and interfacial tension. It was, therefore, used as a parameter for predicting the hydrodynamic behavior of various solvent systems in J-type CCC devices. 

Using your HOAc solution, pour a 1.5 x 28 cm Sephadex G-15 column (see Experiment 5.4 for preparing chromatography columns). 

Although high solubility should be preferred for good productivities, time pressure and thermodynamics can require the acceptance of poor solubilities. Nearly half of R D laboratories involved in preparative chromatography examine 10 to 100 samples per day and the number of candidates for preparative separation is still increasing (Neue, Mazza et al. 2003). Obviously, time and large amounts of sample can not be spent for systematic evaluations and tedious solubility studies. Very often the amount of sample is also too small to carry out extensive experiments. 

Before starting extensive experiments, a procedure recommended by Kaiser and Oel-rich (1981) to rule out adsorbents by fast experiments should be employed. Each elution experiment takes about 20 s. For this purpose samples are applied on a 50 X 50 mm TLC plate at 9 points, which are exactly 10 mm apart. Five microlitres of methanol are drawn into a micro-capillary with a Pt-Ir point. By applying the point of the filled capillary on one of the sample points on the plate, methanol is introduced onto the plate. A miniature radial chromatogram of ca. 7 mm diameter is produced. If the sample components remain at the point of application, the use of this adsorbent type is ruled out for HPLC usage. To make sure, the procedure is repeated with 5 pi of acetonitrile and tetrahydrofuran, respectively. If the products still remain at the point of application, the situation will not be changed by using any other mobile phase that is suitable for preparative chromatography work. 

As a standard approach for preparative chromatography, binary solvent combinations are always investigated before considering ternary mixtures. Figure 4.21 shows the results of experiments with binary mixtures where selectivity was obtained. Other binary combinations were tested, but none resulted in a good spot shape and an acceptable separation between the isomers. Therefore, some additional tests were performed with ternary solvent combinations. 

There is an abimdant literature on the comparison between experimental and calculated band profiles for binary mixtures. The most popular methods used have been the forward-backward finite difference scheme and the OCFE method. The former lends itself readily to numerical calculations in many cases representative of the present preoccupations in preparative chromatography. We present first a comparison between the band profiles obtained with the ideal and the equilibrium-dispersive model to illustrate the dispersive influence of the column efficiency. Related to the comparison between these two models is the issue of the use of the hodograph transform of experimental results discussed in Section 11.2.2. Computer experiments are easy to carry out and most instructive because it is possible to show e effects of the change of a single parameter at a time. Some... 

Nxuneroxis publications have discussed the issue of optimization of the experimental conditions in preparative chromatography. Most papers in this area are based on empirical observations. They report on the conclusions derived by people who have acquired long-term experience and familiarity with the method. Each author has dealt with a variety of problems, but the scope and range of these problems vary considerably from author to author. Without a solid theoretical background to sift through this experience and place it into perspective, the validity of the conclusions and, more importantly, the range in which they are valid are still much in doubt. 

Your instructor will select one experiment for teams to perform validation studies. An example is a gas chromatography experiment such as Experiment 32, but for one analyte. A flow injection analysis (FIA) experiment, such as Experiment 37, would be a good choice as well, since multiple measurements can be made rapidly. The team will determine linearity, accuracy, precision, sensitivity, range, limit of detection, limit of quantitation, and robustness (repeatability) of the method. In addition, a control chart will be prepared over at least one laboratory period. The instructor will have available a reference standard to use for accuracy studies. Plan for two laboratory periods for the completed study. A report of the method will be prepared and documented. Before beginning the experiment, you should review method validation in Chapter 4. 

The importance of sample preparation for HPLC cannot be overemphasized. This is particularly true for ionizable metabolites whose retention under reverse phase conditions will vary with pH and for polar metabolites whose retention is relatively poor. The nearer the solvent composition of the sample is to that of the mobile phase, the greater the chance of success in a preparative HPLC experiment. If possible, the proportion of organic solvent in the sample should be kept to less than, or equal to, its proportion in the mobile phase for reverse phase chromatography. 

The obtained analytical results can be directly transferred to a preparative chromatography column. For the standard method depicted in Figure 3.51, a combination of acetonitrile and methanol is used as the organic modifier. Therefore, some additional experiments, investigating the effect of each type of organic modifier individually, are advisable because, very often, large differences in selectivity can be observed. 

Solvents for analytical and preparative chromatography should be pure reagents. Commercial-grade solvents often contain small amounts of residue, which remains when the solvent is evaporated. For normal work, and for relatively easy separations that take only small amounts of solvent, the residue usually presents few problems. For large-scale work, commercial-grade solvents may have to be redistilled before use. This is especially true for hydrocarbon solvents, which tend to have more residue than other solvent types. Most of the experiments in this laboratory manual have been designed to avoid this particular problem. 

The parameters of preparative chromatography that can be adjusted by the chromatographer for optimization are listed in Table 16. Other parameters cannot be modified by the user but are rather related to the nature of the chromatography medium. When an optimization routine does not yield the expected results, it is best to switch to another medium, based on either a different mass transfer principle or another matrix material. The adsorption process that occurs at a chromatography surface is very complex and is poorly understood. This is especially true for non-specific adsorption. This is why it is necessary to carry out the optimization with the real solutions. Experiments with artificial samples often do not result in conditions that can be transferred to the real situation. The most important targets for optimization are purity and productivity. After the required purity has been achieved, the productivity can be optimized. Productivity includes costs, column size, and operation time. It also includes the lifetime of the column material. 

Due to the complex interactions, simple calculations as can be performed for single solutes are of no - or very little - value in multi-component preparative chromatography. All results which have led to our better understanding of mass overload have arisen from a combination of experiment and computer simulation. The key to... 

Preparative chromatography can be practised on two different scales laboratory (recovery of grams of sample) and production (recovery of kilograms of sample). Preparative Scale Supercritical Fluid Chromatography (PS-SFC) has been practised on the laboratory scale from the very beginning of SFC in 1962 [1], More information on these historical experiments has been gathered by Berger et al. [2] and Bevan [3]. 

A mixture of the ketone (4.62 g), iridium tetrachloride (1.23 g), trimethyl phosphite (15 ml), propan-2-oI (200 ml) and water (50 ml) is heated under reflux for 21 hr. Much of the solvent is then distilled off ca. 215 ml) and the organic products remaining are isolated by extraction with ether. If reduction is essentially complete, the product at this stage may be sufficiently pure for most preparative purposes. Pure components can be obtained by chromatography over alumina, a representative experiment (on the above scale) gives unchanged ketone (0.13 g), cw-alcohol (4.36 g) and tmns-2 co o (0.16 g) (eluted in this order by pentane, and then by pentane containing ether). 

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