The isolation of organic compounds, either after chemical synthesis or extraction from natural sources, constitutes a critical procedure for obtaining materials of adequate purity for subsequent studies or analysis (1). As is well known, the chemical structure of substances plays a key role in selecting the method for accomplishing the purification. In this sense, chromatography-based techniques employed in academic and industrial laboratories are preferentially accomplished on silica gel (of various particle sizes), which—due to the intrinsic acidity of the constitutive Si-OH groups—might affect the chemical integrity of the components of the mixture to be purified (2). In some instances, not only can molecular modifications be triggered by silica gel, but also racemization phenomena on stereochemically defined entities may occur (3). Preliminary—somehow effective solutions to the problem—can be ascribed to the so-called “silica deactivation” methods consisting of pre-treating the stationary phase with an organic base (e.g., triethylamine) or a silylating agent (e.g., trimethylsilyl chloride), approximately in 1-5% v/v (4). This is, upon adding these species to the selected eluent, the prepacked silica column is flushed once or twice with this solvent system prior to commencing the column. This protocol has been successfully validated in several instances and features the objective advantage of differing from a canonical silica gel chromatographic purification by simply requiring an additional component to the selected eluent.
However, as documented in studies dealing with the obtainment of silica-sensitive substances, pre-treating this stationary phase could not suffice for the purpose. That is, the chemical environment in which the chromatography is performed may maintain some reactivity, ultimately leading to the degradation of the mixture’s components. Moreover, it should be noted that including a deactivating agent in the eluent system could alter the effectiveness of the purification, thus increasing the risk of recovering contemporaneously two (or more) components: in other words, no separation is achieved!
As mentioned above, the change of the nature of the chemical interaction between the stationary phase and the mixture object of separation may constitute an effective alternative. In this sense, aluminum oxide (usually referred to as Alox) represents an excellent material for accomplishing chromatographic purifications under gravimetric conditions. Because of the intrinsic characteristic of amphoteric oxide, four different types of Alox are known: basic (pH ca. 9.5), neutral (pH ca. 7.5), weakly acidic (pH ca. 6.0), and acidic (pH ca. 4.5). It appears evident that chemical groups (e.g., amines) susceptible to protonation/deprotonation events could be more or less retained depending on the specific Alox considered. A critical aspect to be highlighted for the successful use of Alox is related to the modulation of the retentive activity tuned by the content of water. The seminal work by Brockmann and Schodder in the early 1940s recognized five different scales of activity for Alox (I to V), with grade I assigned to the anhydrous one, which is also the most active form (5). The latter is almost always the only available form of Alox (basic, neutral, weakly acidic, and acidic) from commercial suppliers. As a consequence, it becomes crucial for the chemist to be familiar with manipulation techniques for having access to (all) the possible deactivated forms of Alox starting from the Brockmann I. Based on the current practice, water content is measured according to the table below.
That is, the trivial addition of water enables the transformation of BG-I Alox into one of the less active species to be used. For a productive utilization of Alox, our laboratory found it highly versatile to add—with a pipette—the calculated amount of water to the wall of a round-bottom flask containing the previously weighted stationary phase (6). Once this operation is concluded, the flask can be connected to a rotary evaporation apparatus (at 20°C) without the application of any reduced pressure. As such, the rotational movement serves exclusively for homogenizing the water content on the Alox surface and, thus, for deactivating the Brockmann grade I form. After approximately 2 hours of rotation (at medium speed), Alox is ready to be used for preparing the usual slurry—with the selected eluent—for chromatography. Several suppliers offer pre-coated Brockmann grade I Alox TLCs, which can be advantageously used for determining the most suitable mobile phase. Because of the inherent high activity of these TLCs, it is recommended for users considering the increasing of Rf values upon the progressive deactivation of Alox (I →→ V) in order to perform an efficient purification. Based on our experience, neutral Alox provides excellent results for separating complex mixtures containing densely functionalized amines (7) or activated halomethyl functionalities (8), including highly sensitive reaction intermediates. Finally, a note of attention for avoiding degradative effects after the (successful) chromatographic purification with Alox: Unstable compounds could undergo decomposition even in deuterated NMR solvents exhibiting some (very minor) acidity, such as CDCl₃. Replacing it with an inert one, such as benzene-d6, could be extremely valuable to hamper this undesired event and thus, to secure satisfactory NMR analyses.
Conflict of Interest statement
The authors report no conflict of interest.
Declaration of Funding
The authors gratefully acknowledge project PRIN 2022 (ref. 20228W9TBL) and generous support from All4Labels (Hamburg, Germany).
Data Availability Statement
Data sharing is not applicable, as no new data were generated or analyzed during this study.
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