Mobile Phase Mixing

The flame ionization detector Is the most popular of the flame-based detectors. Apart from a reduction in sensitivity compared to expectations based on gas chromatographic response factors [138] and incompatibility with the high flow rates of conventional bore columns (4-5 mm I. 0.), the flame ionization detector is every bit as easy to use in SFC as it is in gas chromatography [148,149]. It shows virtually no response to carbon dioxide, nitrous oxide and sulfur hexafluoride mobile phases but is generally incompatible with other mobile phases and mixed mobile phases containing organic modifiers except for water and formic acid, other gas chromatographic detectors that have been used in SFC include the thermionic ionization detector (148,150], ... 

The second approach was taken by practicing liquid chromatographers. They routinely dealt with thermally labile, highly polar molecules and frequently sacrificed resolution, and speed in their separations because of the aqueous mobile phases that were required. With the enhanced diffusion and decreased viscosity of supercritical fluids over liquids, chromatographic run-time and resolution could be improved when supercritical fluids were used. But solubility in pure carbon dioxide mobile phases, which has the solvating powers from hexane to methylene chloride under normal density ranges, was a problem for these polar molecules. To compensate for this, experimentalists started working with mixed mobile phases. These mixed phases were based on... 

THF is highly flammable and its vapor pressure at ambient temperature is rather high. It may preferentially evaporate from various mixed mobile phases. The preferential evaporation brings about the base line drifts due the evaporation from the sample container and/or the pronounced system peaks due to the evaporation from the sample solution. 

Another type of nonideal SEC behavior, which will not be covered in this chapter, is related to the use of mixed mobile phases (multiple solvents). Because solute-solvent interactions play a critical role in controlling the hydrodynamic volume of a macromolecule, the use of mixed mobile phases may lead to deviations from ideal behavior. Depending on the solubility parameter differences of the solvents and the solubility parameter of the packing, the mobile phase composition within the pores of the packing may be different from that in the interstitial volume. As a result, the hydrodynamic volume of the polymer may change when it enters the packing leading to unexpected elution results. Preferential solvation of the polymer in mixed solvent systems may also lead to deviations from ideal behavior (11). 

Our initial attempts to separate the Cj and Dj isomers of 5 (G = 1) used octadecyl polysiloxane (ODS) as high performance liquid chromatography (HPLC) stationary phase and mixtures of acetonitrile/HjO or methanol/HjO as mobile phases. Under these classical reverse-phase conditions, the resulting efficiencies were extremely poor because of the low solubility of 5 (G = 1) in both mobile phases. By contrast, mixed mobile phases which contained acetonitrile (ACN) with some percentages of a cosolvent such as tetrahydrofuran (THF) substantially improved... 

If the first peak from the 40% run takes more than 20min or the peaks are too far apart, wash everything off with 100% acetonitrile. Mix mobile phase 80% and 40% in equal volumes to get 60%, reequilibrate, and shoot again. I usually found that I could get acceptable chromatography by the third run or I needed to make a solvent a change by going to methanol/watcr. 

Injection samples need to be as concentrated as possible and this leads to problems. A column acts as a sample concentrator. If the solution starts out saturated, it will supersaturate on the column, precipitate, and plug the column. I have seen a column with a 3-cm-deep plug that had to be bored out with a drill bit and a spatula. A couple of injector loops full of the stronger solvent in a mixed mobile phase will clear this if there is still some flow, but the separation will have to be repeated. It is better to dissolve the compound, then add a half volume of additional solvent, ensuring that there will be no precipitation on injection. 

For a 25-cm column, deduct 10% from the hrst peak s %B and equilibrate the column with this dial-a-mix mobile phase (i.e., if the first peak came off at 80% B, dial-a-mix 70% B). For a 15-cm column, deduct 7% from the first peak %B to find your dial-a-mix isocratic. Equilibrate the column with this mobile phase. 

Run 15pL of each collect tube in the isocratic dial-a-mix mobile phase chosen in step 4. 

The following table provides guidance in the selection of mobile phases that are to be used in conjunction with ultraviolet spectrophotometric detection.1-2 The data in this table differ from the data in the other solvent tables in this volume in that the wavelength dependence of absorbance is provided here. Moreover, common mixed mobile phases are considered here. The percentages that are given are on the basis of v/v. 

The following table provides a comprehensive guide to the selection of thin-layer chromatography media and solvents for a given chemical family. Mixed mobile phases are denoted with a slash, /, between components and where available the proportions are given. Among the references are several excellent texts,13 60 review articles,4 24 and original research papers and reports.25 59 6198 A table of abbreviations follows this section. 

Incomplete mobile phase mixing. 3. Mix mobile phase by hand or use less viscous solvent. 

To a first approximation, we can write for the solubility parameter of a mixed mobile phase [320]... 

So far, an empirical approach that neglects the specific problems of LSC has appeared more feasible. Antle [572] demonstrated the applicability of the Sentinel method to LSC, using mixed mobile phases corresponding to table 5.4a, i.e. mixing the individual binary mixtures according to their volume fractions. This yielded some success, although admittedly not all solvents were iso-eluotropic. 

Since the mobile phase is moving on a dry bed, several other undesirable effects occur. The adsorption of the first liquid (at the front) on the stationary phase is exothermic, causing the front to have a higher temperature than the rest of the system. Since the temperature of the system is not usually controlled but is allowed to assume the ambient value, some evaporation may occur at the solvent front. If the solvent is composed of a mixture of liquids, preferential evaporation of the most volatile one will cause a slight change in the solvent composition. In fact, the adsorption of a mixed mobile phase will probably also cause some changes in composition because the most polar component will be preferentially sorbed. The situation can become so severe that solvent demixing can occur. At best, a mixed solvent mobile phase is probably not uniform across the planar bed, and some temperature differentials probably exist as well. 

Similar arguments can be applied for the mixed mobile phase A/B. However, it must be emphasized that a good understanding of why solute-solvent and solvent-solvent interactions effectively cancel in many LSC systems has not yet been fully achieved. 

Some very hydrophobic samples, e.g., lipids, are strongly retained and not eluted in an acceptable time even with pure methanol or acetonitrile as the mobile phase. Such samples are usually adequately resolved by normal-phase chromatography, but they can be often equally well or even better separated by non-aqueous reversed-phase (NARP) chromatography in mixed mobile phases containing a more polar (e.g.. acetonitrile or methanol) and a less polar (e.g., tetrahydrofuran. dichloromethane. methyl-r-butyI ether) organic solvent. Ternary non-aqueous mobile phases may contain even hexane or heptane. The retention decreases with increasing concentration of the less-polar... 

Assumption 5 In the definition of the isotherm, the convention is adopted that the solvent (if pure) or the weak solvent (in a mixed mobile phase) is not adsorbed [8]. Riedo and Kov ts [9] have given a detailed discussion of this problem. They have shown that the retention in liquid-solid i.e., adsorption) chromatography can best be described in terms of the Gibbs excess free energy of adsorption. But it is impossible to define the surface concentration of an adsorbate without defining the interface between the adsorbed layer and the bulk solvent. This in turn requires a convention regarding the adsorption equilibrium [8,9]. The most convenient convention for liquid chromatography is to decide that the mobile phase (if pure) or the weak solvent (if the mobile phase is a mixture) is not adsorbed [8]. Then, the mass balance of the weak solvent disappears. If the additive is not adsorbed itself or is weakly adsorbed, its mass balance may be omitted [30]. 

For mixed mobile phases or buffers the mode of preparation should be described in detail. The eluent properties may differ depending on the order of the necessary steps such as the dissolution of various buffer salts, the pH adjustment or the addition of nonionic additives. When mixing water and methanol (to some extent also other water-miscible solvents), a volume contraction effect occurs the volume of the mixture is smaller than the individual volumes. Therefore it is necessary to measure the two solvents separately before mixing them. 

Most of the work to date has been done using relatively few mobile phases (n-pentane, carbon dioxide, and nitrous oxide). Future efforts are needed to explore the use of other more polar and/or mixed mobile phases. Furthermore, column surface deactivation has become more important as emphasis has focussed more on the analysis of polar and trace compounds. The greatly increasing... 

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