48 CHROMATOGRAPHY
Stability even under tough conditions
Fredrik Lime & Cecilia Mazza discuss the efficient isolation of main compounds and impurities using pH.
A
s businesses strive to analyse and purify more samples during the same
timeframe, the turnaround time becomes an area of focus in the laboratory and in manufacturing of natural products and synthetic drugs.
With this mindset, there is also an increased expectation on the flexibility of the stationary phase’s operating conditions, column lifetime and the reproducibility of the results. In addition, when a company works on a new drug candidate, being able to use the same stationary phase and scale the methods accordingly throughout discovery, development and production is a unique opportunity to minimise uncertainties and to save project time.
Fig. 2. Chromatographic result of the purification of crude Tryptamine.
Consequently, laboratories participating in drug discovery and commercialisation projects favour the use of a stationary phase that is available in a wide range of particle sizes to improve productivity across the entire company.
For regular silica-based stationary phases, exposure to extreme pH, especially basic, has a negative impact on the chemical stability, and therefore column lifetime.
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However, the silica/organosilane surface of the Kromasil Eternity platform offers a chemical stability with long-lasting stationary phase, even under tough pH conditions and higher temperatures.
Substances with ionisable groups can exhibit retention time differences under reversed-phase chromatography depending on the degree of ionisation of the compounds and their interactions with the stationary phase under given mobile phase composition. Hence, by changing pH, selectivity between ionisable substances can be altered so that resolution is optimised for a given separation and purification.
As in many cases pharmaceuticals are basic, they are ionised under low or neutral pH conditions, resulting in low retention, poor loadability and broad peaks. Being able to run at high pH means basic compounds become
Fig. 1. Chromatographic results for testing stationary phase loadability with diphenhydramine.
more retained and peaks are narrower, revealing higher chances for better resolution and loadability.
In the isolation of compounds for characterisation of substances, Phase I, II or III studies as well as production, loadability is a key parameter. Loadability of the base diphenhydramine was tested using EternityXT C18. Fig. 1. shows the chromatographic results for diphenhydramine at low and high pH. At low pH of 3.7, the molecule is ionised, leading to a large band broadening even at very low loadings.
Te same loading at high pH of 10.5 (in the upper right chromatogram) produces a sharp peak without any tendency to broaden as a function of concentration overload. To
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