The Basics
The most widely used mode of chromatography is Reversed-Phase Liquid Chromatography (RPLC). Accounting for more than 90% of all analysis of low molecular weight samples, RPLC employs a non-polar (hydrophobic) stationary phase column and a polar mobile phase. As a mixture is pumped through a RPLC column, a “differential migration” of its components sets up (which is the basis for the separation), driven by dispersive forces (hydrophobic or London type Van der Waals interactions) (1 ).
Carbon makes the Column Non-Polar
The element carbon is foundational to stationary phase materials used in RPLC. The media packed in these columns obtain their non-polar character from carbon bonded to or composing its surface. Hydrocarbons, compounds made exclusively of carbon and hydrogen, are non-polar as a general rule. They derive this property due to their atoms’ similarity in electronegativity. We will explain the concept of electronegativity in greater detail in another blog.
Hydrophobic Interactions Drive Analyte Retention in RPLC
The most widely used stationary phase material for RPLC is octadecylsilane, covalently bound to silica particles (known as ODS or C18) (2,3). Here, the naturally hydrophilic surface of bare silica is transformed into a highly hydrophobic structure through the attachment of carbon atoms. Alkyl chains, 18 carbons in length, are bonded to the surface of silica and protrude out from the support like pendants (Figure 1). As a sample mixture is pumped through C18 supports, the individual analyte molecules interact with the carbon groups of these stationary phase alkyl chains. The unique charge distribution of each individual sample molecule defines the strength of its interaction with the stationary phase and residence time “on the column”. The rule of thumb for standard LC analysis is retention time is ordered with analyte hydrophobicity. Typically, the more hydrophobic an analyte is, the longer its retention time on a RPLC column.
Figure 1: Typical silica surface modification scheme. Octadecylsilane bound to silanol groups.
For example, a mix containing Phenol, Toluene and 2,4-Dimethyl-1-pentene will separate and elute off the column in that order since it is the order of their corresponding hydrophobicity (Figure 2). Each of these molecules have a unique molecular configuration and will show increasing amounts of affinity toward the alkyl chains. In RPLC, hydrophobic dispersive forces drive analytes out of the mobile phase and onto the stationary phase. So, under the action of these weak forces, the more closely aligned in charge an analyte is with the stationary phase, the longer it would like to reside with it.
Figure 2. Analytes Ordered from Least to Most Hydrophobic.
With Increased hydrophobicity (non-polar charge), analytes show stronger affinity to the Stationary Phase and Longer Retention Times.
Images generated using software tools at https://chemagic.org/molecules/amini.html
Carbon Used in LC Comes in Many Forms
Variations in carbon structures are possible. Bond configuration defines the shapes of the molecules observed as well as how they behave in a chromatographic system. For example, aromatic benzene-like structures can be used as an alternative to C18. Both variants contain only carbon and hydrogen, yet they take very different conformations and show different selectivity toward analytes. The formation of these carbon structures is described by a concept called orbital hybridization. We will explain this in greater detail in another blog, yet for now it is sufficient to know that the carbon atoms in the most popular stationary phases C18, C8, C4 are sp3 hybridized. The carbon atoms in benzene, phenyl, and graphite-based structures are sp2 hybridized.
sp3 Hybridized Carbon in Long Alkyl Chains Behaves Like an Organic Liquid
The sp3 hybridized carbon bound to the most popular stationary phase, C18, sets up a near-net zero charge over the length of its chains. This phase behaves like an organic liquid with respect to analyte retention and may be modeled most closely as partitioning events, rather than an adsorption phenomenon . Studies have shown that the mechanism for solutes distributing themselves between a mobile phase and alkyl chains bonded to silica particles (length n = 12 or more) is best described as solutes partitioning between water and oil mixtures like in shake flask experiments (4).
The Charge and Shape of an Analyte Dictates its Interactions with Aromatic sp2 Carbon
The surface of porous graphitic carbon (PGC) and other graphite-based stationary phases show differences in their charge distributions compared with C18. As an aromatic sp2 hybridized carbon, graphite has a network of delocalized electrons in the P orbital that sets up a negative charge over the central regions in its carbon ring structures. This phenomenon is confirmed chromatographically by PGC showing higher affinity to retain polar analytes than C18. The term polar retention effect on graphite (PREG) was coined to explain these observations. It has been proposed that electronic interactions between polarizable groups of analytes and the surface of graphite account for this retention. PREG is hypothesized to stem from some charge induction or electron-ion pair donating/accepting interactions with the pi cloud of graphite.
In addition to the charge contributions, the planar shape of aromatic sp2 carbon contributes to analyte selectivity. A shape-selective property has emerged as a significant factor in retention. It has been shown that analytes are selected based on their structural conformation. Analyte–graphite interactions become stronger when the analyte shape allows for multiple contact points with the stationary phase media. In the case of naphthalene, the double ring system results in strong pi–pi interactions with maximal molecular surface area contact with the graphitic surface. On the other hand, triptycene, a paddle wheel-shaped molecule, cannot have all its surface area interact with the stationary phase because of its structure. Figure 3 illustrates the differences each analyte has available to contact the planar surface of graphite. The rigid planar surface of graphite results in strong retention of planar molecules and reduced retention of branched molecules with limited availability for contacting the sorbent surface (5,6).
Figure 3. Planar Surface Area Determines Analyte Retention in Graphitic Stationary Phase Media.
Shape-selective property of graphite defines strong retention of planar molecules and reduced retention of branched molecules that have limited contact with the sorbent surface.
The Bottom Line
The interaction between a target analyte and the stationary phase column in LC is a complex series of events governed by a host of forces. Textbooks are devoted to describing the effects of these forces, including the nature of the analyte itself, its conformational structure, concentration, and dosing volume, the mobile phase composition, pH, temperature, salt content, flow rate, and both the chemical and physical attributes of the stationary phase media. However, carbon’s role in RPLC cannot be overstated. The properties of carbon primarily define the retention and selectivity of the stationary phase media. In its most popular form, sp3 alkyl chains of carbon create a highly hydrophobic phase that promotes retention and selectivity ordered on analyte hydrophobicity. In its alternative form, carbon organized as sp2 aromatic sheets of graphite selects analytes based on hydrophobicity, the presence of polarizable groups, and structural conformation. Advances in stationary phase media are continually increasing the power of RPLC in separating unique compounds and closely related products. This field is developing rapidly and pushing the limits of what we thought was possible in analytical science. Although new materials are always being developed, the element carbon will have a central role in LC for the foreseeable future.
Author: Michael Jack Parente
References
1. Kazakevich, Y. & Lobrutto, R. HPLC for Pharmaceutical Scientists 445 - 447 (2006).
2. Michel, M. & Buszewski, B. Porous graphitic carbon sorbents in biomedical and environmental applications. Adsorption 15, 193-202 (2009).
3. Sander, L. C., Rimmer, C. A. & Wilson, W. B. Characterization of triacontyl (C-30) liquid chromatographic columns. Journal of Chromatography A 1614, 460732 (2020).
4. Vailaya, A. & Horváth, C. Retention in reversed-phase chromatography: partition or adsorption? Journal of Chromatography A 829, 1-27 (1998).
5. Corman, C., Muraco, C., Ye, M. & Michel, F. Insights into the Shape-Selective Retention Properties of Porous Graphitic Carbon Stationary Phases and Applicability for Polar Compounds. (2023).
6. De Matteis, C. I. et al. Chromatographic retention behaviour of n-alkylbenzenes and pentylbenzene structural isomers on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and QSRR. Journal of Chromatography A 1217, 6987-6993 (2010).
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