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Polar InvestigationsPorous graphitic carbon offers many benefits for the analysis of polar compounds.by Luisa Pereira, Principal Scientist, Chromatography Consumables and Speciality Products, Thermo Fisher Scientific
Figure 1. Effect of the solute shape on the strength of the interaction with the graphite surface: (a) Good alignment of planar molecule to the flat graphite surface; (b) Poor alignment of non-planar molecule to the flat graphite surface. Click to Enlarge. | Porous graphitic carbon (PGC) has unique properties as a stationary phase in high performance liquid chromatography (HPLC). Its chemical surface properties distinguish PGC from more conventional LC packings such as bonded-silica gels and polymers. PGC behaves as a strongly retentive alkyl-bonded silica gel for non-polar analytes; however, its retention and selectivity behavior toward polar and structurally related compounds is very different.
PGC (Hypercarb) provides unique retention and separation of very polar compounds. Its surface is stereo-selective with the capability to separate geometric isomers and other closely related compounds. Hypercarb is stable throughout the entire pH range of 0 to 14 and is not affected by aggressive mobile phases. Its compatibility with all solvent systems enables separation of a wide range of polarities within a single chromatographic run. The selectivity of the Hypercarb packing is different from the selectivity of silica and polymeric phases. Its retention mechanism is different from conventional C18 columns.
This article briefly reviews the PGC properties, the polar retention mechanism, and application areas where this materials’ HPLC unique capabilities for the analysis of polar compounds of biological interest have been used.
Physical and chemical properties of PGC
PGC particles are spherical and fully porous with a porosity of ~75%. The surface of PGC is crystalline and highly reproducible and does not contain micropores. At the molecular level, PGC is made up of sheets of hexagonally arranged carbon atoms linked by the same conjugated 1.5-order bonds that are present in any large polynuclear aromatic hydrocarbon. In principle, there are no functional groups on the surface because the aromatic carbon atoms have fully satisfied valencies within the graphitic sheets.
Table 1 lists the more important physical properties of PGC. The requirements placed on its physical properties are similar to other HPLC supports where factors such as narrow particle size distribution are essential to the ultimate performance of the phase if good bed uniformity and low operating pressures are to be achieved.
Table 2. Comparison of Values of Log kw on Reversed Phase Stationary Phases. Click to Enlarge. | PGC also has a tight pore size distribution with a mean value around 250 Å, allowing good mass transfer of a wide range of analyte shapes and sizes. Surface homogeneity and absence of highly adsorptive sites are essential for good peak symmetry. PGC meets all the conventional operating criteria of a chromatographic support.
Retention mechanisms on PGC
On a molecular scale, the surface of the graphite is flat and highly crystalline unlike that of alkyl-bonded silicas, which possess a brush-type surface with bonded phase and residual silanols. Consequently, the PGC mechanism of interaction is very different. The retention by graphite from aqueous/organic eluents is determined by the balance of two factors: 1. hydrophobicity, which is primarily a solution effect that tends to drive analytes out of solution, and 2. the interaction of polarizable or polarized groups in the analyte with the graphite (these are additional to the normal dispersive interactions).
The strength of interaction depends on both the molecular area of an analyte (and, therefore, shape of the analyte) in contact with the graphite surface and upon the nature and type of functional groups at the point of interaction with the flat graphite surface. The more planar the analyte, the closer its alignment is to the graphite surface, and so the greater the number of points of interaction possible—hence, maximum retention. Retention is reduced for highly structured, 3-D and rigid molecules that can contact the surface with only a small part of their surface, compared with planar molecules with the same molecular mass (Figure 1).
Retention of polar compounds in RP-LC
Figure 2. Separation of 2´-deoxynucleoside 5´-monophosphates on PGC capillary column. Experimental conditions: Column – Hypercarb 5 µm, 100 x 0.32 mm; Mobile phase – A: water + 0.1% formic acid, B: ACN + 0.1% formic acid; Gradient – 10 to 30% B in 10 min; Flow rate – 6 µL/min; Detection – UV at 254 nm. Column temperature: 25 C. Analytes: 1. dCMP; 2. dUMP; 3. dAMP; 4. dGMP. Click to Enlarge. | In a traditional reversed phase (RP) system, analyte retention increases as its hydrophobicity increases. This is due to the increased dispersive interactions that take place between the stationary phase and the analyte.
Conversely, as the polarity of the analyte increases, analyte-solvent interactions begin to dominate and retention is reduced. This simple observation holds true for all reversed-phase systems with the exception of the PGC.
For Hypercarb columns, it has been observed that in some cases retention increases as the polarity of the analyte increases. This effect has been called “the polar retention effect on graphite” or PREG. The effect of PREG makes Hypercarb columns particularly useful for the separation of highly polar compounds, such as carbohydrates, and compounds with several hydroxyl, carboxyl, and amino groups, which are difficult to retain on conventional alkyl-silica phases.
Retention of polar compounds on PGC
PREG defines the ability of molecules having lone-pair or aromatic-ring electrons to apparently interact through an electron transfer mechanism to the electronic cloud of the graphite (Figure 1). PREG is particularly pronounced when the polar groups are attached to a benzene ring and other larger aromatic systems. Knox et al. have attributed this to some type of orbital overlap between the conductivity electrons in graphite and lone-pair and/or π electrons in analytes.
Coquart and Hennion correlated log k and log P for PGC, C18-silica and polystyrene divinylbenzene (PS-DVB), as part of their study of the use of Hypercarb as an adsorbent for removing polar contaminants from water samples. The correlation with C18-silica was very good, for PS-DVB the data points were more scattered and for PGC the data points for polar compounds lay well above the line for the alkyl benzenes or alkanes. They measured the log k values for a range of compounds in their mono-, di- and tri-substituted forms at different concentrations of methanol-water eluents. The data was then extrapolated to generate log k in pure water, log kw (intercept at the y axis) in equation 1:
Equation 1: Log k = log kw + ACorg
Figure 3. Separation of underivatized amino acids on PGC capillary column. Experimental conditions: Column – Hypercarb 5 µm, 100 x 0.32 mm; Mobile phase – A: water + 0.1% formic acid, B: ACN + 0.1% formic acid; Gradient – 5 to 100% B in 5 min; Flow rate – 7 µL/min; Detection: +ESI. Analytes: 1. Ornithine; 2. Methionine; 3. Phenylalanine; 4. Tryptophan. Click to Enlarge. | where A is the gradient of the line and Corg is the percentage of organic solvent in the water-organic solvent mixture. These results (Table 2) showed that the retention of monosubstituted benzenes is similar for C18-silica and PGC and lower than on PS-DVB. The log kw values obtained when solutes have two polar substituents using PS-DVB are always lower than those measured for each corresponding monosubstituted benzene, whereas the contrary is observed with PGC. The di-substitued benzenes are not retained on C18-silica. These results also highlight potential applications areas where PGC offers selectivity and retention where other chromatographic supports cannot.
The unique retention properties of PGC for the retention of polar compounds are put to good use in the example applications described in the following section.
Applications
The chromatographic analysis of very polar compounds is of interest in many biological, pharmaceutical and environmental studies but challenging due to the lack of retention in typical reversed-phase systems. Conventional reversed-phase columns do not provide enough retention, and therefore elution occurs near or at the solvent front with very low-capacity factors. In order to achieve adequate retention, addition of an ion pair reagent or, alternatively, the use of ion exchange chromatography or HILIC (hydrophilic interaction liquid chromatography) is necessary. The mobile phases used in ion exchange and ion-pair RP are generally not compatible with detection techniques such as MS and ELSD. In spite of the recent popularity of HILIC, its interaction mechanisms are still not fully understood, and injection-to-injection retention time reproducibility can be an issue. The PREG allows for retention of very polar compounds on PGC using standard RP mobile phases that facilitate good sensitivity when using MS detection. In this section examples of the retention and separation polar compounds of biological interest are discussed.
Nucleotides, nucleosides and nucleobases
PGC columns have been found to be efficient for achieving the separation of various mixtures of normal and modified nucleobases, nucleosides and nucleotides. Figure 2 illustrates the separation of 2´-deoxynucleoside 5´-monophosphates using a simple gradient mobile phase of water and acetonitrile modified with 0.1% formic acid. The resolution of mixtures of nucleosides and their mono-, di- and triphosphates using a PGC stationary phase, under conditions suitable for liquid chromatography/mass spectrometry (LC-MS), has been previously reported. Different organic mobile phases and modifiers were evaluated for the gradient elution separation of 16 nucleosides and nucleotides. The separation was attempted on silica-based columns designed for the retention of polar compounds but these could not provide suitable separation for accurate quantitation of mixed nucleosides and their phosphates.
Amino acids and peptides
Underivatized amino acids have weak hydrophobic character, which makes the retention of the more polar species difficult in RP-LC, unless an ion pair reagent is used. Figure 3 illustrates the LC-MS analysis of four underivatized amino acids on PGC, without an ion pair in the mobile phase. Chaimbault and co-workers achieved the separation of 20 underivatized amino acids on a PGC column, using nonafluoropentanoic acid as an ion pair reagent and evaporative light scattering detection. They concluded that this chromatographic system had a faster equilibration time than the previous system based on a C18-silica stationary phase. The elution order observed on the PGC column was different from that on a C18-silica stationary phase, making the two chromatographic systems complementary for the identification of trace amino acids. Chaimbault et al. described a method for the simultaneous determination of the sulfur amino acids taurine, hypotaurine and thiotaurine in tissues of some marine invertebrates by LC-MS-MS. The PGC column allowed the use of isocratic conditions and negative electrospray detection for quantification of these metabolites in biological matrices.
Figure 4. Retention of saccharides on PGC. Experimental conditions: Column – Hypercarb 3 µm, 100 x 2.1 mm; Mobile phase: H2O + 0.1% ammonia / ACN (96:4); Flow rate: 0.2 mL/min; Temperature: 60 C; Detection: -ESI. Analytes: 1. Sucrose; 2. Maltose; 3. Lactose. Click to Enlarge. | The analysis of small hydrophilic peptides such as mono-, di-, tri-, tetrapeptides or phosphopeptides in RP-LC can be problematic due to lack of retention, but retention can be achieved on PGC. Chin and Papac used PGC for desalting flow-through fractions from a C18-silica column, containing di-, tri-, tetra- and penta-peptides, and the separation of phosphorylated from nonphosphorylated peptides.
Carbohydrates
Carbohydrate separations are challenging because of the great diversity of structure and water solubility that makes retention on bonded-silica and polymeric phases difficult. PGC has the advantage that the PREG can be exploited to retain and separate small saccharides and also oligosaccharides.
Figure 4 illustrates the separation of saccharides at high pH, with 0.1% ammonia, and high temperature to prevent double chromatographic peaks due to the formation of anomers. In this type of application, PGC also is stable under high temperature and high pH.
Recently Antonio et al. reported a LC-ESI-MS-MS method for the sensitive targeted analysis of key glycolytic intermediates, sugars and sugar phosphates from plants, using a PGC stationary phase and an MS-compatible mobile phase. The method performance was demonstrated for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 and its starchless phosphoglucomutase mutant over a 12-h light/12-h dark growth cycle.
PGC has been extensively used in the analysis of oligosaccharides. Typical applications include the desalting/purification and the profiling of these compounds released from glycoproteins using LC-MS analysis. For instance, PGC has been used to purify oligosaccharides from solutions containing salts, detergents, proteins, and reagents used for the release of the oligosaccharides from glycoconjugates, with complete recoveries reported. LC-MS with a PGC column was also successful for simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols.The authors of these works demonstrated that the method could be used to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator from human melanoma cells.
For more information, contact Luisa Pereira, Thermo Fisher Scientific, at luisa.pereira@thermofisher.com or +44 1928 534 324.
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