Orthogonal separation techniques

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... 

In 1990, Bushey and Jorgenson developed the first automated system that coupled HPLC with CZE (19). This orthogonal separation technique used differences in hydrophobicity in the first dimension and molecular charge in the second dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 (xL/min volumetric flow rate, with a column of 1.0 mm ID. The effluent from the chromatographic column filled a 10 pU loop on a computer-controlled, six-port micro valve. At fixed intervals, the loop material was flushed over the anode end of the CZE capillary, allowing electrokinetic injections to be made into the second dimension from the first. 

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... 

A 2D or 3D approach employing two or more orthogonal separation techniques or separation methods with different mechanisms of separation will significantly improve the chances of resolving a complex mixture of cell proteins into their individual components. M D peptide separation will play an increasingly important role on the road to identifying and quantifying the proteome. 

No current analytical strategy is capable of fully resolving complex biological samples. For this reason, orthogonal separation techniques are often combined to maximize peptide separation before the mass spectrometric analysis. The aim is to reduce as much as possible the number of coeluting peptides introduced into the MS at any given time, so as to maximize peptide identification and proteome coverage. 

How can one be assured that all the samples components are resolved This is particularly important in critical assays such as pharmaceutical impurity testing to ensure that no impurity peaks are co-eluting with other components or hidden under the active ingredient peak. The standard practice is the use of an orthogonal separation technique or method to demonstrate that all impurities are accounted for. An orthogonal method is one based on different separation mechanism from the primary method. 

Table 2.4. Common Examples of Orthogonal Separation Techniques...

More complex experimental designs have also been described. Hyphenation of microchip electrophoresis with an orthogonal separation technique was reported by Zhang and coworkers. ... 

MSI offers a complementary technique as mass spectrometry can differentiate between various ions on the basis of their molecular weight. Additional selectivity is achieved by utilizing MS", high-resolution mass spectrometry, or orthogonal separation techniques, either separately or combined. Each additional stage of analysis serves to reduce the interfering chemical noise that originates from the tissue without tedious sample preparation. 

Principles of the Optimization of HPLC Illustrated by RP-Chromatogyaphy 129 Step 3 Coupling orthogonal separation techniques... 

Eq. (1) is a simplification for the sake of clarity based on isocratic elution and orthogonal separation techniques a more general qttantification is given in Ref [1]. 

---