For quite a long time (80th-2000th) the classic C8/C18 stationary phases were the choice for almost every routine pharmaceutical HPLC application. However, two decades of domination have revealed a number of weaknesses of the reversed-phase mode, and the lack of separation specificity is one of them.
Specific HPLC separations are always group selective; this means that the elution windows for compounds having similar chemical structures do never overlap. In the extreme, only related compounds can be seen on chromatograms, other compounds being either excluded, unretained, or irreversibly retained by pre-column.
This perfectly suits the requirements for routine HPLC methods.
Therefore, a good routine HPLC method should be based on a specific, group selective HPLC separation.
On the opposite, the reversed-phase separations on the C8/C18 phases are the classic example of the most non-specific chromatography. Using such approaches for developing routine HPLC methods presents a source of ongoing problems that cannot be resolved without causing significant losses to a method’s performance and cost-effectiveness.
The first often encountered problem is associated with two very typical features of the routine analysis, as follows: i. sample matrices can be quite complex; ii. the most often used detection techniques are non-specific UV and RID.
The combination of the non-specific separation and the non-specific detection techniques cannot provide a sustainable solution to complex sample analysis.
Co-elutions of target compounds with matrix contaminants are quite often in this case, thus limiting the method’s performance.
Sometimes the sample handling can help in solving poor method specificity issues, but the introduction of a sample preparation procedure, especially a multistage one, always has a dramatic negative impact on the method’s cost-effectiveness.
The second problem has a purely chromatographic origin. In HPLC the poor inter-group selectivity is often encountered in combination with the excessive intra-group selectivity. Simply speaking, in the case of the analysis of the related substances, the classic reversed-phase conditions may provide the excessive separation power: the good resolution of compounds having moderate retention factors is always accompanied by improper, extremely high retention factors for the rest of the analytes.
In isocratic elution mode, it results in longer run times and thus reduced the method’s cost-effectiveness. Gradient elution mode can help in solving the issue, but this technique is not the choice for routine applications.
Nevertheless, the excessive intra-group reversed-phase selectivity is often counterbalanced by using gradient elution, which yields inconsistent, ineffective routine methods.
Although the limitations of gradient elution should be addressed separately, it is worthy to note here that the gradient separations are less robust than the isocratic ones, and they are much harder to reproduce and to transfer. More than that, whereas using gradient elution can help in reducing retention times, the thing to remember is that it also may crush the separation of critical pairs that are eluted on the gradient ramp.
Therefore, classic reversed-phase HPLC is not the choice for developing routine HPLC methods. Most of its drawbacks originate from its poor inter-group selectivity (lack of separation specificity, the importance of sample handling), and the excessive intra-group selectivity (unwanted gradient elution as a forced step).
More optimal, the routine method friendly balance between the inter- and intra-group selectivities can be provided by the different mixed modes.
RP/IC, RP/IC/HILIC, RP/CT, HILIC/IC, HILIC/RP – here are examples of mixed HPLC modes suitable for developing routine HPLC methods.
Regrettably, many compendial methods are based on the outdated paradigm for HPLC method development, and the classic reversed-phase chromatography is yet very much popular for developing routine applications.
However, it does not mean that such obsolete approaches should be further copied and used. Today’s HPLC column market can offer plenty of mixed-mode packings filled with 3-1.8 um fully porous and 2.7-1.8 core-shell particles, either. And the future of HPLC belongs to them.
See also:
Key Words: Outsourcing HPLC Method Development & Validation, Analytical Method Development Services, Pharmaceutical Outsourcing, CDMO, Pharmaceutical Analysis, ICH Method Development & Validation