Myth 1: You Can't Reverse an HPLC Column False. In practice, a high performance liquid chromatography (HPLC) column is packed at pressures much higher (often as much as twofold) than its maximum operating pressure. Thus, if the proper slurry solvent was used and the time allotted for the packed bed to be stabilized, a well-packed column should be able to work in both directions. Some reasons why one would want to flow a column in the opposite direction include backflushing operations in column switching, rinsing a column with stubborn strongly sorbed sample at its entrance (a shorter path length than to flush the insoluble materials through the entire column length), and flushing trapped particulates to decrease pressure buildup. There is one exception in reversing the flow of an HPLC column. If the manufacturer has used a higher porosity frit at the entrance to the column, by reversing the column it might be possible to flush particles out of the packed bed. When the column is packed at the factory, the porosity of frit at the outlet of the column must be lower than the smallest particle size of the column. For example, if the column packing has an average diameter of 5 μm and a particle size distribution of 3–7 μm, the frit used at the column exit must be less than 3 μm so that there is no possibility that particles will escape the packed bed when the column is being used. For that case, most manufacturers choose a 2-μm frit. Why would a manufacturer have different porosity frits at the column inlets and outlets? Simple. A higher porosity frit has fewer tendencies to plug than a lower porosity frit. A 0.5-μm frit will plug faster than a 2-μm frit. So, to prevent rapid pressure buildup and customer complaints, a manufacturer might use a more forgiving, larger porosity frit at the inlet. Usually, the column will be marked with an arrow that indicates that it should be used in one direction only. Before reversing an HPLC column, it is best to consult the column instruction sheet or check with the column manufacturer to see if the column can be reversed.
Myth 2: All C18 (L1) Columns Are the Same False. In the early days of HPLC, octadecylsilane (most often referred to as an ODS or C18 phase) was one of the first bonded stationary phases that became available for the new technique called "reversed-phase chromatography." It became the standard phase for reversed-phase chromatography and quickly was adopted by most practitioners. Because the pharmaceutical industry was an early adopter of HPLC and regulatory bodies did not want to bless a particular manufacturer''s brand of column, the FDA and USP developed a classification system that gave a generic designation for each new method that was submitted under a new drug application. For HPLC columns, an "L" designation was given and because C18 was used in a majority of the submittals, it became "L1." As additional phases were added, they were given their own "L" number (for example, L7 C8, L10 CN, L11 Phenyl, and so forth). Unfortunately, this designation system proved to be unreliable because each commercial C18 column was synthesized differently using silica gel as the base material and the resulting reversed-phase columns had different properties. For example, some manufacturers used octa-decylmonochlorosilane and a low surface area silica gel (Figure 1) while others used the same silane but bonded it to a higher surface area silica gel. These two C18 columns would behave differently with the latter having more C18 phase than the former. A lower coverage bonded phase sometimes had unreacted silanols that caused mixed retention mechanisms. Some manufacturers use di- and trichlorosilanes and polymerized the bonded phase to form a thicker layer with different diffusional properties. To cut down on unreacted silanols that remained after the bonding process, some manufacturers endcap these silanols with a small silane (for example, trimethylmonochlorosilane). Silanols are sometimes responsible for tailing of basic compounds at intermediate pH values. Some manufacturers even went further and double-endcapped with a second small silane to provide a more inert surface. Some manufacturers used polymeric base materials and then bonded C18 moieties to their surface resulting in a totally different C18 packing material, yet it was still considered an "L1." Others use organoalkoxysilanes that display a different degree of reactivity and can produce a slightly different C18 bonded phase than would an organochlorosilane reagent.
我的社区
签到
