Orthogonal separations

Multidimensional LC separation has been defined as a teehnique whieh is mainly eharaeterized by two distinet eriteria, as follows (1). The first eriterion for a multidimensional system is that sample eomponents must be displaeed by two or more separation teehniques involving orthogonal separation meehanisms (2), while the seeond eriterion is that eomponents that are separated by any single separation dimension must not be reeombined in any further separation dimension. 

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

Multidimensional LC-LC, using two high-resolution eolumns with orthogonal separation meehanisms, has only a few applieations in environmental analysis. The limitations that sueh a multidimensional system has with regard to seleetivity must... 

As compared to GC, pSFC possesses inferior figures of merit (efficiency, speed, sensitivity, detection, resolution), but allows a greater sample capacity and is more widely applicable. pSFC can be used as an orthogonal separation technique to verify the accuracy of GC methods. pSFC should be thought of as an extension of LC because of similarities in equipment and approach. The... 

Similar mathematical solution can be derived from a Poisson distribution of random events in 2D space. The probability that 2D separation space will be covered by peaks in ideally orthogonal separation is analogical to an example where balls are randomly thrown in 2D space divided into uniform bins. The general relationship between the number of events K (number of balls, peaks, etc.) and the number of bins occupied F (bins containing one or more balls, peaks, etc.) is described by Equation 12.3, where N is the number of available bins (peak capacity in 2DLC). 

The mechanisms of the first- and second-dimension separations are independent here that is, the alkyl chain length separation is independent of the PEO chain length. The retention in this case is often called orthogonal between the two dimensions. A discussion of orthogonal separations is given in Chapters 2, 3, 6, and 12. This independent nature of the two separation dimensions allows the full analysis of the alkyl and PEO components per molecule. Additional examples of the independence in separation axes will further demonstrate the importance of 2DLC for surfactant analysis. 

CE provides analysis based on orthogonal separation principles compared to other techniques as well as high resolving power. Like slab gel electrophoresis, CE is a family of techniques that resolve sample components by differences in intrinsic molecular characteristics such as size, mass, charge, differential interaction, and isoelectric point (pi). 

Marsh, A., Clark, B. J., and Altria, K. D. (2004). Orthogonal separations of nicotine and nicotine-related alkaloids by various capillary electrophoretic modes. Electrophoresis 25, 1270—1278. 

Degradation product(s) co-eluting with the parent compound in the parent compound assay. Check for peak homogeneity via PDA-UV, LC MS, and/or use an orthogonal separation technique, as discussed earlier. Modify conditions to separate if necessary. 

Capillary electrophoresis (CE) provided an orthogonal separation technique. The retention mechanism can be manipulated with buffers or addition of surfactants to form micelles for the analysis where size and charge differences... 

Purity is indicated by a clean, single, sharp peak on analytical RP-HPLC. The presence minor peaks of shoulders or a distorted peak shape, are indicators that the material needs further purification. To further confirm purity an orthogonal separation method should be used. For example, analytical ion-exchange HPLC, Fig 2E, for which the separation is based on ionic rather than nonpolar interactions. Analytical isoelectric focusing can also be used. 

---