Experiment 18 Chromatographic Separation

Two or more substances are separated by the differences in their affinity to paper or some other material. This affinity is related to the intermolecular forces between the substances. (See the chapter on Solids, Liquids, and Intermolecular Forces.) 

In some cases, the distance a spot travels on the chromatographic media is measured. This requires the use of a ruler. 

Instead of an ion exchange resin or silica gel, it is possible to use filter paper as a chromatographic medium. 

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The process of chromatographic separation is illustrated in the following experiment, in which a wider tube than usual is employed to give a reasonably rapid separation within the time normally available to students. The alumina employed is the usual active alumina as supplied by dealers. 

A variable-size simplex optimization of a gas chromatographic separation using oven temperature and carrier gas flow rate as factors is described in this experiment. 

The distribution coefficient can be determined by batch experiments in which a small known quantity of resin is shaken with a solution containing a known concentration of the solute, followed by analysis of the two phases after equilibrium has been attained. The separation factor, a, is used as a measure of the chromatographic separation possible and is given by the equation,... 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

The molecular mass of the protein was redetermined by infusing a 5-10 pmolp.l solution of the protein in 50% aqueous acetonitrile containing 0.2% formic acid at a flow rate of 6 p,lmin into an electrospray source. The scan rate employed on the mass spectrometer was from m/z 60 to m/z 1800 in 12 s. This is a relatively slow scan speed which will lead to a more precise molecular weight determination. Scan speeds of this order may be, and indeed should be, utilized for infusion experiments if sufficient sample is available but it is unlikely to be feasible when chromatographic separations, particularly those involving capillary columns, are employed because of the restriction imposed by the chromatographic peak width (see Section 3.5.2.1 above). 

The mass spectrometry employed electrospray ionization and each metabolite gave an [M + H]+ ion which was then used as a precursor ion for a product-ion MS-MS scan. For subsequent MS" experiments, the base peak of the previous MS-MS experiment was chosen under computer control and this allowed all analytes to be studied in a single chromatographic separation. 

The MRM experiments do not require chromatographic separation of the metabolites. Therefore, other LC conditions, columns, gradient, and injection volumes may be used provided that there is adequate sensitivity and specificity, and the chromatographic quality is not compromised. 

Coupled on-line techniques (GC-MS, LC-MS, MS/ MS, etc.) provide for indirect mixture analysis, while many of the newer desorption/ionisation methods are well suited for direct analysis of mixtures. DI techniques, applied either directly or with prior liquid chromatographic separations, provide molecular weight information up to 5000 Da, but little or no additional structural information. Higher molecular weight (or more labile) additives can be detected more readily in the isolated extract, since desorption/ionisation techniques (e.g. FD and FAB) can be used with the extract but not with the compounded polymer. Major increases in sensitivity will be needed to support imaging experiments with DI in which the spatial distribution of ions in the x — y plane are followed with resolutions of a few tens of microns, and the total ion current obtained is a few hundreds of ions. 

Two-dimensional liquid-chromatographic separations are also of great potential interest in polymer analysis. After separating macromolecules, according to only one type of heterogeneity, by one experiment, there is no chance to get a correlation between different... 

The NMR spectrum is recorded during the chromatographic separation. Data are collected as in a 2D experiment, the two dimensions being the chemical shift and the retention time of the chromatogram. 

Spectra which are better resolved (useful for example for the exact determination of coupling constants) can be obtained by carrying out stopped-flow experiments. Here we stop the chromatographic separation after 3 and 7.5 min, optimize the homogeneity (by shimming the magnet) and carry out the desired NMR experiments. 

To obtain an even better separation, SEC was performed in a second dimension following the LCCC analysis. As has been described previously, the resolution of a 2D experiment may be significantly higher as compared to the single chromatographic separations. A typical experimental result is shown in Fig. 17.14 (Gutzler et al., 2005). 

Feeding can be avoided in the previous experiments if the molecular replication process at a surface is coupled with chromatographic separation. The two building blocks A and B are added to the mobile phase, while the matrices C are formed at the surface of the stationary phase. The main difference from the process described previously is that the matrices are not bonded covalently to the surface, but reversibly. During elution (which supplies new building block molecules to the system) a certain amount of matrix molecules is washed off and must be replaced by replication (von Kiedrowski, 1999). 

Bog-Hansen, T.C., Prahl, P., and Lowenstein, H. (1978) A set of analytical electrophoresis experiments to predict the results of affinity chromatographic separations. Fractionation of allergens from cow s hair and dander./. Immunol. Meth. 22, 293. 

The advent of the atmospheric pressure ionization (API) source in the early 1990s allowed direct coupling of LC to MS. By the mid-1990s, this technology was a common in drug metabolism laboratories. The enhanced selectivity of tandem mass spectrometry (MS/MS) experiments reduced the need for exhaustive chromatographic separations prior to detection and this feature was exploited to significantly reduce analysis times. 

Fig. 4.6 Chromatographic separation factors for various uranium isotopes vs. 238U as a function of mass at 433 K. s = ln[(238U/1U)iv,aq/(238U/1U)vi,resin]- The field shift (FS) and vibrational (BM) contributions are of opposite sign. Triangles = calculated vibrational (Bigeleisen-Mayer) contribution, diamonds = calculated FS contribution, circles = measured effects, open squares = calculated effects. Note that agreement between calculation and experiment is quantitative. The correlation lines are drawn through even/even data points only (Data from Bigeleisen, J., J. Am. Chem. Soc., 118, 3676 (1996))...

In tandem MS, two or more stages of mass analysis are combined in one experiment. [79,80] Each stage provides an added dimension in terms of isolation, selectivity, or structural information to the analysis. Therefore, a tandem MS stage is equivalent to a chromatographic separation, provided the separation of isomers is not required. While chromatography distinguishes substances by their retention time, tandem MS isolates them by mass. [2,3,25] The principles of tandem MS have been discussed and some applications for stmcture elucidation and quantitation have already been shown (Table 12.1). However, the aspect of increased selectivity has not been addressed so far. [81]... 

PHIC was chromatographed with the same columns and solvents as PS and PDMS. The results of this experiment are shown in Table IV, and the comparison with earlier PS and PDMS results is summarized in the same table. The MM of PHIC did not change during the chromatographic separation with an experimental error of 5%. The composition of PHIC is D CsHia and the backbone is rigid. 

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