Gradient elution separation scheme

Although all-trans-octadecatrienoate tended to elute with the cis-dienes, this was not a problem in practice with samples of hydrogenated fats containing dienoic and trienoic fatty acids. A gradient elution scheme was adapted from the isocratic mobile phase used for monoenes and polyenes for the separation of such mixtures. 

The gradient elution scheme is a scaled-up procedure originally described by Middleton (10) that has been extended to handle highly refractive materials such as coal liquids. This separation technique uses Alcoa F-20 alumina activated to a 5.5 wt % moisture level as the stationary phase. Details of this separation procedure are given elsewhere (2). This method separates SRC into 13 fractions and these fractions are listed in Table II along with some key chemical and physical descriptions of the cuts. The structural types indicated in Table II for Fractions 1-6 have been assigned based upon model compound studies and low resolution mass spectrometry (MS) (2), whereas the chemical types indicated for Fractions 7-13 are based upon IR observations and additional model compound studies. Recoveries in these separations are normally greater than 90%. 

An example for the separation for flavonoids with HP-RPC is the screening method employed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials by Lin et al20 These authors used an analytical 4.6 mm x 250 mm 5 pm C18 silica column at 25 °C with linear gradient elution (eluent A (0.1% FA in water and eluent B 0.1% FA in ACN) at 1.0 ml min-1. DAD was performed at 270, 310, 350, and 520 nm to monitor the UV/VIS absorption. The LC system was directly coupled to an ESI mass spectrometer without flow splitting and the mass spectra acquired in the positive and negative ionization mode. The same analytical scheme (aqueous MeOH extraction, reversed-phase liquid chromatographic separation, and diode array and mass spectrometric detection) can be applied to a wide variety of samples and standards and therefore allows the cross-comparison of newly detected compounds in samples with standards and plant materials previously identified in the published literature. 

Step gradient elution is also well suited for group, or ionic class separations. For example, a chelating resin might be specific for the platinum-group metals. With this resin, a step-gradient scheme could be devised to take up, and later elute, only the selected group of metal ions. 

Consider the case of a pair of weak organic acids with Kd values of 3.0 and 3.5, whose pK values are 4.2 and 4.8, respectively. What are the capacity factors of these acids using an aqueous eluent at pH 1 At pH 4.4 At pH 7.0 How many plates would be needed to separate the acid which elutes first before 0.5% of the second elutes at each of these pH values Is a gradient elution scheme feasible here ... 

The use of gradient elution methods is often necessary to allow the analysis of analytes of differing polarities, as is the case with mixtures of EDCs, many of which exhibit poor solubility in aqueous systems due to their hydrophobic nature. While gradient schemes can be automated with suitable instrumentation, this adds to the complexity of the separation. 

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