Mobile Phases for RPLC

TABLE 2 Equal Elution Strength of Mobile Phases for RPLC... 

Figure 5.8 Illustration of a two-dimensional optimization using a modified Simplex algorithm. A ternary mobile phase for RPLC is being optimized. The third component is acetonitrile. Figure taken from ref. [505]. Reprinted with permission.

J.S. Landy and J.G. Dorsey, Characterization of Micellar Mobile Phases for RPLC, Anal. Chim. Acta, 178 179 (1985). 

Unfortunately, common mobile phases for RPLC utilize organic solvents, which may be detrimental to the ICPs analytical performance. A decrease in sensitivity can result due to excessive solvent loading of the plasma. Higher plasma instability, increased background at certain masses due to the formation of molecular ions and carbon deposition on the sampling cone of ICP have been reported. Use of mobile phases that do not utilize file conventional organic solvents is therefore worthwile. Micellar mobile phases have been proposed for RPLC/ICP-MS speciation... 

Miller and Hawthorne [416] have developed a chromatographic method that allows subcritical (hot/liquid) water to be used as a mobile phase for packed-column RPLC with solute detection by FID, UV or F also PHWE-LC-GC-FTD couplings are used. Before LC elution the extract is dried in a solid-phase trap to remove the water. In analogy to SFE-SFC, on-line coupled superheated water extraction-superheated water chromatography (SWE-SWC) has been proposed [417]. On-line sample extraction, clean-up and fractionation increases sensitivity, avoids contamination and minimises sources of error. 

Where the use of multiple spectroscopic analysis on a single HPLC separation is an advantage, the benefit of using the simplest possible mobile phase for separations is manifest. While selecting compatible solvent systems for NMR and MS is sometimes complex, addition of IR (even off-line) places even more constraints on solvent composition. For SEC-NMR-IR CDCR is a suitable eluent [666] for RPLC-FTIR-UV-NMR-MS D2O-CD3CN is recommended. Superheated D20 has been proposed as the mobile phase [670],... 

Also, subcritical (hot/liquid) water can be used as a mobile phase for packed-column RPLC with solute detection by means of FID [942]. In the multidimensional on-line PHWE-LC-GC-FTD/MS scheme, the solid sample is extracted with hot pressurised water (without the need for sample pretreatment), and the analytes are trapped in a solid-phase trap [943]. The trap is eluted with a nitrogen flow, and the analytes are carried on to a LC column for cleanup, and separated on a GC column using the on-column interface. The closed PHWE-LC-GC system is suitable for many kinds of sample matrices and analytes. The main benefit of the system is that the concentration step is highly efficient, so that the sensitivity is about 800 times better than that obtained with traditional methods [944]. Because small sample amounts are required (10 mg), special attention has to be paid to the homogeneity of the sample. The system is... 

There are two other phases indicated in figure 3.8. The first is a so-called pyrocarbon material. Such a stationary phase is formed by pyrolizing an organic layer on a silica substrate. The idea is to combine the mechanical strength of silica with the chemical inertness of carbon. The value of 14 used here can be thought of as typical for carbonaceous materials. These materials do not seem to behave like non-polar phases in the tradition of chemically bonded phases for RPLC, but rather like phases of intermediate polarity. Hence, as for silica, they may be most useful in the reversed phase mode for the separation of very polar molecules using aqueous mobile phases. 

Figure 5.5 Example of a response surface for the optimization of the mobile phase in RPLC. Horizontal axis ternary mobile phase composition. Drawn line response surface using the resolution product as the criterion. Dashed lines retention surfaces for individual solutes (In k). For further details see section 5.5.2. Figure taken from ref. [504], Reprinted with permission. 

The latter was also the case for the optimization of the composition of a ternary mobile phase in RPLC by Issaq et al. [554]. The ternary mixture was formed by mixing two limiting (non iso-eluotropic) binary mixtures and a fourth order polynomial equation was fitted through five equally spaced data points. 

Toon and Rowland [557] used a similar method for the optimization of the composition of a binary mobile phase in RPLC. However, they did not use eqns.(5.16) and (5.17), but plotted lines for the observed front and back of the peak. Since these quantities are affected not only by the capacity factor and the peak width of the solute, but also by the sensitivity of the detection, the method of Colin et al. is to be preferred. 

A remaining problem for all spectrometric peak recognition methods is the reproducibility of the spectra recorded under different chromatographic conditions. For example, if the differences between the UV spectra for a given solute induced by variations in the mobile phase in RPLC are larger than the differences between the UV spectra of different solutes recorded under identical conditions, then clearly the application of multichannel UV detection, with or without the use of PCA techniques, will be of limited use. 

I. Rapado Martinez, M.C. Garcia Alvarez-Coque and R.M. Villanueva Camanas, Performance of Micellar Mobile Phases in RPLC for the Analysis of Pharmaceuticals containing P-Block rs and other Antihypertensive Drugs, Analyst, 121 1677 (1996). 

On the other hand, the environment of the surfactant-modified stationary phase is independent of micelle concentration in the mobile phase (for most surfactants and stationary phases), and similar to that of pure aqueous eluent systems. As a result, the alkyl-bonded stationary phase will have both invariable amphiphilic and anisotropic properties. In contrast, in aqueous-organic RPLC, the composition and structure of the alkyl-bonded phase change with the concentration of organic modifier in mobile phase. 

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

It has been shown for many RPLC methods that correlations between log Pod and retention parameters were improved by separating compounds in two classes, i.e. H-bond acceptor and donor compounds. Minick et al. [23] propose to add 0.25% (v/v) of 1-octanol in the organic porhon of the mobile phase (methanol was preferred in this study) and to prepare the aqueous portion with 1-octanol-saturated water to minimize this discriminahon regarding H-bond properties. For a set of heterogenous neutral compounds, the addition of 0.25% (v/v) of 1-octanol in methanol and the use of water-saturated 1-octanol to prepare mobile phase improve the correlahon between log few obtained on the LC-ABZ column and log Poc, [13]. 

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

Most small organic molecules are soluble in mixed organic-aqueous solvents and can be easily analyzed using RPLC. However, there are some polar compounds which are not soluble in typical RPLC solvent systems or are unstable in an aqueous mobile phase system. These compounds can be analyzed on an RPLC column with a nonaqueous solvent system. This technique is called "nonaqueous reversed phase chromatography" (NARP).20-21 The NARP technique is primarily used for the separation of lipophilic compounds having low to medium polarity and a molecular weight larger than... 

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