My LC Blog: Reversed Phase HPLC

As mentioned in my last article, reversed phase chromatography is used in the vast majority of HPLC applications, which is why I'll explain it in more detail. Also, method development and optimization will focus on this technique.

Reversed phase LC (RP LC)

The reversed phase LC mode is the opposite or "the reverse" of normal phase HPLC. A non-polar stationary phase is used with polar mobile phases for the separation of hydrophobic compounds, meaning analytes that possess low to moderate polarity. Separation occurs according to differences in hydrophobicity, so that polar compounds will be carried through the column by the polar mobile phase faster, than non-polar ones that interact with the non-polar stationary phase → polar compounds elute earlier than non-polar analytes, as shown in figure 1.

However, it's not quite as simple as that as there are other interactions at play that have to be considered. Typical stationary phases in RP LC use hydrophobic functional groups that are chemically bonded to silica gel as the base packing material (figure 2). 

Octadecylsilane (ODS) or C18 groups are the most popular bonded phase, but also others, more specialized columns are worth mentioning when we talk about method development, such as C8, Phenyl, Pentafluorophenyl (PFP) or polar embedded phases. However, each of these differing phases, in addition to hydrophobic retention, exhibit other retention mechanisms such as dipole : dipole, π-π, silanophilic, electrostatic attraction and repulsion and hydrogen bonding interactions. Therefore, they can produce differing chromatographic selectivity (i.e., peaks move about) – which is another term that will be explained at a later stage when we come to method development and column selection. 

Reasons why RP LC is preferred over NP LC are use of less toxic mobile phases and applicability to water-soluble analytes, it is also more reproducible. The polar mobile phase in RP LC is usually water, often including a buffer to control the pH, in combination with an alcohol or less polar solvent. Retention time of a non-polar compound decreases with decreasing polarity of the mobile phase, as the solvent strength increases, and the hydrophobic analytes hence have a greater affinity for the mobile phase. Mixtures of solvents are used to achieve the goal of eluting all the compounds in as short a time as possible, with baseline resolution of the individual peaks. And this is where it becomes interesting - seeing that this goal often can't be achieved with one fixed ratio of polar and less polar solvent - the term gradient chromatography comes to mind. But I think, first it's time for another glossary.

• C18 column: These columns are also referred to as octadecylsilane (ODS), where the stationary phase is composed of linear alkyl chains consisting of 18 carbon atoms bonded to the silica support (figure 2).
• Silica gel: Most common base packing material in LC columns, also used as polar stationary phase in NP LC. Silica gel, or just silica refers to porous silicon dioxide. Due to its slightly acidic character, it adds to the complexity of retention mechanisms in RP LC.
• Solvent strength (elution strength): The ability of the mobile phase to elute the analyte retained on the stationary phase - the stronger the solvent, the faster it carries the analyte through the column. Figure 3 lists a few solvents typically used in RP LC in order of solvent strength.

Now - back to gradient chromatography. More often than not, a sample consists of several compounds with large differences in hydrophobicity, so that some - more polar analytes - only show little retention on a C18 column, while others don't elute for a long time, or not at all. In that case, it would be great if one could increase the elution strength of the mobile phase during the run. And in fact - this "Gradient Chromatography" is common practice in RP LC and feasible with modern HPLC equipment. So, let's look at that in my next article. But if you have questions before that - don't hesitate to ask!