Orthogonal HPLC methods

FIGURE 4 Comparison of two orthogonal HPLC methods for the same sample. 

K. Herman, Orthogonal HPLC methods for quantitating related substances and degradation products of pramlintide, AAPS PharmSciTech 1 (2000), article 6. 

Often you have a good column, but for some reason it is not available or its production has been stopped or new production batches of the columns show a completely different selectivity and the separation suddenly no longer works. You may also have to prove the correctness of the first method using a preferably orthogonal HPLC method, or co-elution occurs in a method development. We have all experienced this during the laboratory routine. The question here is How do 1 find the most similar or most dissimilar column to my current one ... 

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. 

Once a new drug candidate has been identified, a TLC method is needed as an orthogonal tool with respect to HPLC methods. Similar to HPLC method development, certain criteria must be met ... 

Packed column SFC and CE are both able to make inroads into the application area served by HPLC, but from opposite extremes of polarity and with little overlap. CE is likely to be more efficient and faster, but mostly applicable to very polar molecules and ions. SFC qualifies as a more reproducible, trace technique, with greater selectivity and multiple detection options. HPLC and CE have been compared [365], Owing to their orthogonality, CZE and SFC are worth developing, not in competition or as an alternative to HPLC, but as an additional method in order to augment the information obtained from the analysis. With the broad scope of possible eluents and stationary phases, HPLC has fewer constraints than SFC and CZE. The parameters influencing selectivity may be used as a guide to optimisation (Table 4.44). 

Because HPLC and HPCE are based on different physico-chemical principles, HPCE may be expected to address areas in which HPLC has shortcomings [884]. One such area is time of separation. In terms of speed of analysis, selectivity, quantitation, methods to control separation mechanism, orthogonality, CE performs better than conventional electrophoresis and varies from HPLC (Table 4.49). CE has very high efficiency compared to HPLC (up to two orders of magnitude) or GC. For typical capillary dimensions 105—106 theoretical plates are common in CE compared to 20 000 for a conventional HPLC column and... 

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