Method development with four solvents

The complexity of the method in terms of number of steps and solvents needed depends on the sorbent chemistry. The development in a simplified scenario involves running an analyte in several concentrations in multiple replicates and assaying for recovery and performance. This procedure is described in detail for several silica and polymeric sorbents by Wells.42 However, if a number of sorbents are to be evaluated, the process becomes time-consuming if multiple 96-well plates (each with one sorbent packed in all the wells) must be screened separately. This process may take a week or more and consume an analyst s precious time as well. The most plausible solution is to pack different sorbents in the same well plate and use a universal procedure that applies to all of them. An example of such a multisorbent method development plate is the four-sorbent plate recently introduced by Phenomenex demonstrated124 to require only 1 to 2 hr to determine optimal sorbent and SPE conditions. 

A similar RP-HPLC method has been developed for the determination of isoflavones in soybean powder. Ground soybean powder (100 g) was extracted four times with 300 ml of hexane, then the defatted powder was extracted with six solvents or solvent mixtures such as acetone, methanol-water (4 1, v/v), methanol-ethyl acetate-acetone (1 1 1, v/v), acetone-0.1 M HC1 (5 1, v/v), methanol-water (4 1, v/v) plus 0.1 M HC1 (5 1, v/v), and methanol-ethyl acetate-acetone (1 1 1, v/v) plus 0.1 M HC1 (5 1, v/v). An aliquot of 0.5 g defatted or non-defatted soybean powder was shaken for 2h or sonificated for lh with 5 ml... 

My preference today would be for a high-pressure mixing system if I had to run very complex mixtures on a routine basis because these systems give the best reproducible gradients as a rule. As a routine research instrument or a methods development system, I would prefer a low-pressure, four-solvent dynamically mixed system using a dual-headed pump. I would use the deoxygenation apparatus (Fig. 6.4) to degas my solvents with helium and run them under a helium demand valve to conserve helium. 

Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column re-equilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But, many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations. 

Many manufacturers have used the capabilities of on-board processing power to provide a user friendly interface for the operator. This results in a host of useful (and some not so useful) features for the analyst, and allows the pump to be remotely controlled by a computer, or another component of the HPLC system. This can facilitate method development for instance, as the pump can be programmed to change automatically the composition of the mobile phase after a number of runs. It does this by changing the proportion of time that it draws from each of a number (up to four) of solvent reservoirs, that can contain diflferent modifiers or buffers. Computerisation of pumps can also allow unattended runs to be performed with a greatly increased measure of confidence. In a networked... 

Another application of solvent extraction to redox speciation studies is the method developed for iodine (Malmbeck and Skamemark 1995). It was used for online speciation of iodine in the Forsmark BWR power plant in Sweden. Iodine can enter the reactor water in two ways, either by fission of tramp uranium (uranium that is adsorbed on the outer surface of the fuel pins) or via leaking fuel pins. In this method, a tiny stream of water was withdrawn fi-om the reactor system and used as feed for a 20 stage mixer-settler battery. The mixer-settlers were arranged in four batteries with 4, 4, 6, and 6 mixer-settler units. Part of such a mixer-settler battery is shown in O Fig. 52.4. 

Ohno et al. (1996) developed a reversed-phase TLC on octadecylsilica (Cig) gel method that complementarily employs four solvent systems to separate 45 water-soluble dyes, most of which are used for colouring cosmetics or food in Japan. They applied that method in combination with scanning densitometry to separate and identify dyes in a cosmetic lotion, a bath preparation, and imported candies. Another reversed-phase TLC-scanning densitometry method, which involves two developing solvent systems, has been used by Ohno et al. (2003) to separate and identify 11 oil-soluble cosmetic dyes. That method was applied to the separation and identification of colouring agents present in two kinds of nail polishes and other cosmetic products. 

---