Thermo Scientific Hypercarb Columns - Applications Notebook

Significance of the Topic

Porous graphitic carbon (PGC, sold as Hypercarb) offers a distinctive liquid chromatography stationary phase with unparalleled retention of highly polar and structurally related compounds. Unlike conventional alkyl-silica or polymeric phases, PGC combines a crystalline, fully porous carbon surface with strong dispersive and charge-induced dipole interactions, enabling separation of analytes from small polar diastereomers to complex glycans. Its stability over pH 0–14 and compatibility with aggressive solvents broadens application areas across biochemical, environmental, food safety and clinical analysis.

Goals and Overview

This Applications Notebook issue compiles the physical and chemical properties of PGC, explains its unique retention mechanisms (including the Polar Retention Effect on Graphite) and reviews a series of application notes. These range from microscale SPE of hydrophilic peptides to nanoflow PGC-LC-MS analysis of glycopeptides, carbohydrate profiling, pesticide metabolite quantification, and inorganic anion determination. The objective is to showcase method development, analytical performance and practical workflows exploiting PGC’s selectivity and robustness.

Methodology and Instrumentation

The cornerstone PGC support features 3–5 µm spherical particles, ~250 Å pores and ~120 m2/g surface area. Retention arises from planar analyte alignment on the flat graphite surface and charge-induced dipole interactions for polar groups. Common instrumentation includes:

• Thermo Scientific Hypercarb columns (analytical, capillary, nanobore and preparative formats)
• HPLC/UPLC systems (Surveyor, Accela) with PDA or UV detection
• Online SPE and nanoscale SPE tips packed with Hypercarb for desalting and cleanup
• Mass spectrometers: ion trap (LCQ Deca XP), triple quadrupole (TSQ), Orbitrap hybrids with CID/ETD capabilities
• High-temperature column ovens and microflow splitters for elevated-temperature and nanoflow applications

Main Results and Discussion

PGC demonstrates superior retention of very polar analytes, including underivatized carbohydrates, nucleotides, small polar peptides and glycans, often eluting on C18 at dead-volume. Sample notes include:

• Microscale SPE tips with Hypercarb enabled >70% recovery of 2 short hydrophilic peptides from buffer.
• PGC columns retained and resolved mono- and multiphosphopeptides 3× more strongly than C18, improving ESI-MS sensitivity and spectral cleanliness.
• Hybrid LC-MS methods on PGC coupled with ETD on Orbitrap XL facilitated site-specific glycopeptide characterization, benefiting from formation of higher charge-state ions via metal adducts.
• NanoLC-MS of non-reducing wheat stem oligosaccharides achieved baseline separation of dp2–dp20 in <30 min; zeptomole sensitivity was realized via nanoflow PGC columns.
• Automated column-switching LC-APCI-MS/MS quantified acrylamide at 2.5 pg on-column LOQ without SPE cleanup.
• Elevated-temperature Hypercarb LC at 160 °C achieved 11 herbicide analytes in 2 min at <80 bar backpressure.
• Dynamic coating of Hypercarb with cetyltrimethylammonium enabled anion exchange separations of F–, Cl–, Br–, NO3–, PO43– and SO42– in drinking water.

Benefits and Practical Applications

• Exceptional selectivity for isomeric and very polar analytes without derivatization.
• Wide pH and solvent compatibility, including high-temperature operation.
• Strong retention of analytes that co-elute at dead-volume on C18 permits desalting and cleanup prior to MS detection.
• Flexible formats: from preparative SPE cartridges to nanobore columns for trace-level analysis.
• High throughput via short run times, elevated-temperature LC and on-line sample preparation.

Future Trends and Possibilities

Advances are expected in micro- and nano-scale PGC columns combined with ultrahigh-resolution MS to push limits of detection into the attomole/zeptomole range. Emerging areas include modulation of surface charge for mixed-mode separations, integration into multidimensional LC workflows for proteomics and glycomics, electrochromatography applications, and online coupled systems for real-time monitoring in environmental and clinical settings. Preparative-scale PGC may see growth in chiral and stereoselective separations.

Conclusion

Porous graphitic carbon (Hypercarb) stands out as a versatile stationary phase for challenging separations of polar, isomeric and labile analytes across diverse fields. Its robust surface, broad solvent and pH tolerance, and unique retention mechanisms make it an essential tool for modern analytical laboratories seeking high selectivity, sensitivity and throughput.

Reference

1. L. Pereira, J. Liq. Chrom. & Rel. Technol., 2008, 31, 1687–1731
2. J. Knox, P. Ross, Adv. Chromatogr., 1997, 37, 73–119