High-performance liquid chromatography is one of the most common methods in pharmaceutical quantitative and qualitative analysis, yet the lack of some of these top 10 tips for HPLC analysis in pharmaceuticals it can prove to be overly complicated. These tips aid in optimizing the analysis process other than pushing the wrong elements of the analysis, for example, the temperature. These tips encompass understanding what HPLC analysis entails. In this case, it entails optimizing selectivity.
1. Tip number one is avoiding trying to re-invent the wheel, which would result in lots of experimentation and thus time wastage. Instead, one should consult the existing literature on what has been done before to identify the appropriate conditions. Such literature will also often consist of information on the most suitable HPLC analysis, normal phase HPLC or the Reverse phase HPLC.
2. Another tip is the determination of the sample preparation required. Sample preparations vary from dissolution, pre-concentration, or filtration among others depending on the preparation that optimizes selectivity for specific samples.
3. The correct choice of the chromatography process is important in getting reliable analysis results. Acidic and basic analytes should be analyzed using reverse phase ion suppression. The most suitable chromatography type for low to medium polarity is the normal phase HPLC. On the other and, ion exchange chromatography is best for inorganic anions and cations.
4. Complex samples should be handled using the gradient HPLC. It is the most appropriate method in this case because it offers greater resolution the higher number of peaks in complex samples. Also, gradient HPLC eliminates the shortcoming of out of range capacity factors under isocratic conditions.
5. The sizing of the columns should be correct. Unless in the case of complex samples. The columns should measure 10-15 cm for packing particle size of 3 or 5 micrometers.
6. During the selection of the detectors, sample properties should be put into consideration. These properties include the presence of chromophores, which enable UV detection. Detection limits should also be put into consideration. Another consideration would be the necessity of chemical derivation to increase sensitivity.
7. Fluorescence and UV wavelengths are important parameters in the optimization of the process. The UV wavelength should be set to the maximum, while the Fluorescence wavelength that results in the maximum emission should be referred to from existing literature, or deduced through the use of expert system software and empirical methods.
8. Cleanliness is vital in the chromatography process. As such the column should always be kept clean via the use of column protection, filter samples, filter buffered mobile phases, sample clean up, and appropriate flushing.
9. Peak issues are a common occurrence that could have multiple causes. The secret lie in determining the cause of the peak issue and resolving it, for example tailing with increased retention could be caused by a disrupted flow path or poorly packed bed.
10. Upon the choice of the most suitable High Performance Liquid Chromatography method, a follow-up validation process should follow to ensure the credibility of the process. This process should also involve double checking the necessary procedures and preparations upon which observation gives a highly reliable HPLC analysis.
