Guest Author: Seyed Sadjadi, Senior Scientist
We are going to explore the reasons why C18 stationary phase is the most used when it comes to reversed-phase and how it works.
It is probably more appropriate to refer to C18 stationary phase as one of the most prolific chemistries available. There is certainly no shortage of the ever-growing varieties of this phase among chromatography column providers. However, before we delve into the subject at hand, here is a short review of how C18 stationary phase came to existence.
In the beginning, Mikhail Tsvet, a Russian botanist, tried to isolate and separate the natural pigments (carotenoids, chlorophyll, etc.) in plant tissues. As one might have guessed, what Tsvet used for his separation experiment was NOT highly refined, fully porous, perfectly shaped spherical, and uniformly sized silica particles bonded with a C18 ligand and packed tightly into a high grade stainless-steel tube! He used materials that nature made available to him—minerals and rock salts (calcium salts)—as the stationary phase and a mixture of ethanol as his mobile phase. Tsvett named this separation technique “chromatography” which today is a significant branch of analytical chemistry.
In the early chromatography experiments (Tsvet’s design), the hydrophobic analytes were first to elute from the column followed by polar and very hydrophilic compounds. This elution order was referred to as normal phase chromatography (NP). Several decades later and with many more scientists involved in this field, additional separation modes have been introduced into chromatography to resolve and purify many classes of compounds in many different matrices. As exciting (!!!) as it may be to cover all these new additions, we will only concentrate on one, reversed phase chromatography(RP).
In a chromatographic system the ionic and very polar analytes elute from the column first, followed by more hydrophobic analytes. The compounds elute in the opposite order as compared to Tsvet’s experiments. Accordingly, this system is called “reversed-phase” chromatography.
The ultimate goal of the RP technique is to separate analytes with varying degree of hydrophobicity. In a nutshell, this task is accomplished by:
- Use a hydrophobic stationary phase to retain the analytes.
- Modifying and optimizing a multi-component solvent system with differential ability to act as a good solvent for the target analytes. This includes both organic solvents and buffer solutions, when applicable. Not all compounds are strongly acidic or basic, but instead may have polar groups that require a narrow pH range to control their behavior.
Hydrophobic Stationary Phase
Hydrocarbons are a perfect example of hydrophobicity and are in effect, oily substances. More specifically, straight-chain alkanes are well-suited to serve as a good stationary phase. We should note that as the carbon atoms in the chain increases, the physical properties of the hydrocarbon changes as well—C1 through C4 are gaseous; C5-C17 are liquid; C18 and larger are solids. Other than alkanes, there are other candidates, such as aromatic and cyclic alkanes, that could equally be viable here as a stationary phase. These other stationary phases will be discussed in other articles. The popularity of the C18 may be due to early availability of the starting material for the bonding process. This fortunate coincidence lead to further discovery of how well-suited C18 was to the chromatographic process. Either way, both factors helped promote C18 as the undisputed champion of the RP stationary phases.
The next step is to attach the alkane group, specifically C18 or octadecyl, onto a suitable surface. Silica has outperformed every other media that was considered, and silica particles are available in many shapes (regular spherical or irregular), sizes (0.9 o 10 µm and larger), porosities (as large as 1000 Ȧ), and is either fully porous or has a solid core by design.
After the bonding process is completed (many of the details of this process are kept confidential), a C18 ligand attaches to the silica at a molecular level and a more generic physical placement inside a silica particle pore as the images depict below.
Figure 1. C18 ligand bonded to silicon dioxide
Figure 2. A generic depiction of two C18 ligand molecules on the surface of silica pores
RPC Retention Mechanism
The beauty and simplicity of a C18 stationary phase is that it offers a very simple hydrophobic interaction. As the solutes in the mobile phase travel through the silica pores, they can be attracted and held by the hydrocarbon through a rather weak hydrophobic (and van der Waal force) interaction. Figure 3 is a representative of such an interaction.
Figure 3, A simple hydrophobic interaction between a C18 ligand and a compound.
Figure 4 shows that there is a hydrophobic interaction between the C18 ligand, the compound’s benzene ring, and its propyl group moiety (encircled). The attraction between the C18 ligand and the amine moiety on the other side of molecule is minimal due to the presence of a positive charge on nitrogen.
A parameter that indicates the degree of a compound’s hydrophobicity is called log P. This value is the equilibrium constant of a compound after it is placed in a mixture of water and n-octanol. A positive log P value indicates the target compound is more soluble in n-octanol and thus has more hydrophobic nature. A negative log P indicates a water-soluble molecule and thus has a more hydrophilic nature. Figure 4 shows a list of various classes of compounds with approximate log P ranges.
Figure 4. log P scale for various class of compounds
Empowered with this information, the task at hand becomes rather clear: to retain a compound on a C18 column, the compound must become as neutral or as hydrophobic as possible. Obviously, nothing can be done to analytes that are already neutral or have no possibility of becoming charged. However, for weakly acidic and basic compounds, we can use a buffer to control the extent of their charge state. Another chemical property to use here is the pKa (and pKb). Weak acids and bases in solution exist in two forms: their neutral form, and one in which they are deprotonated (acids) or protonated (bases). At a specific pH value, these two conjugate forms are in equal concentrations. This pH value is referred to as pKa and pKb. The graphical presentation below (Fig 5) illustrates the pK values and its relationship to increasing or decreasing hydrophobicity of compounds as a function of pH.
Figure 5. Graphical presentation of weakly acid and basic conjugates as function of pH.
In general practice, it is recommended that the mobile pH to be set 2 units above or below the pKb or pKa, respectively, to ensure that compound exists in one form with the highest possibility of retention on column. Of course, there are exceptions to just about everything and every rule.
To this point, we have only covered one component of the mobile phase, specifically, the aqueous portion. This led us to use several chemical properties to help increase the analyte retention on a C18 stationary phase. Now, it is natural to discuss how to elute the analytes from a C18 stationary phase.
To overcome the hydrophobic interaction under reversed-phase conditions, Methanol (MeOH), Acetonitrile (ACN) and Tetrahydrofuran (THF) are the primary solvent choices. In order of strength, MeOH is considered the weakest solvent and THF the strongest. This strength translates into how quickly the analytes will elute with each solvent. Given identical proportion in the mobile phase (MP), a single analyte will elute much faster, with THF as the solvent, than the others. However, the solvent strength does not yield proportional selectivity when more than one analyte is considered. In the next article, we will address the difference in solvent selectivity in reversed-phase chromatography.
In conclusion, C18 is one of simplest and most convenient stationary phases available for reversed-phase chromatography.