A Critical Review of Methods for Hold-Up Time (Volume) Determination

Known methods for determining hold-up time, essential for calculating retention factors and related chromatographic patterns, are reviewed. The retention factor, initially defined in the theoretical plate model for chromatography on non-porous sorbents, can be adapted to porous sorbents containing mobile phase components in stagnant zones within the sorbent pores. However, in this case, the zone of an unretained solute moves slower than the actual velocity of the mobile phase. The most commonly used approaches from previously proposed methods are considered, taking into account new experimental data. The possible presence of gallery pores in sorbents raises concerns about the gravimetric method. Furthermore, this method, like many others, does not take into account differences in retention mechanisms between adsorption and absorption. The use of solvents wetting the stationary phase likely leads to the penetration of solvent molecule into the attached phase and should yield a hold-up volume applicable to the absorption mechanism. If the solvent poorly wets the stationary phase, the obtained hold-up volume loses clear physical meaning. Calculations of hold-up time based on the retention of deuterated water are more appropriate to the determination of hold-up time in adsorption-based retention but may result in overestimated values due to water adsorption on residual silanol groups. The use of deuterated organic solvents for the same purpose raises additional concerns. Using inorganic salts as hold-up volume markers may lead to inaccuracies due to exclusion effects, which should also be considered in other methods. The homologous series method is not entirely reliable for several reasons discussed in the text. Similarly, the system peak method cannot provide unambiguous confirmation, as it may correspond to retention with an uncertain degree of exclusion effects. Consequently, the problem remains unresolved, and the development of new specialized approaches is required.