Towards a comprehensive 2‐D‐LC–MS separation

Abstract

A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column. This cycle is repeated throughout the chromatogram. The use of dual secondary columns provides the secondary columns with additional time to generate high-speed chromatograms. Each secondary column generates alternate chromatograms which when combined generate the entire chromatogram. The primary column separation is comparable to conventional HPLC, whereas the secondary column separation is fast. With both the columns operating in reverse phase mode, one would expect strong correlation in the two-dimensional retention and hence inefficiency in separation. However, differences in column operation modes, interaction mechanisms, and vendor silica result in a complementary separation. The system was evaluated by comparing it to one-dimensional counterparts and coupled column chromatography. Although some correlations were observed in 2-D-LC-MS, peaks do show two-dimensional distribution with superior UV and MS data as co-elution is minimized. Also, the ease of converting conventional systems to 2-D-LC-MS is discussed.

Development of different comprehensive two dimensional systems for the separation of phenolic antioxidants

“…2) employed a PEG column with a gradient of 0 to 5% acetonitrile in 70 min and isocratic post-gradient elution with 5% acetonitrile F = 0.2 mL/min in the 1-D and 2-D ZR-CARBON columns in parallel [67,68]; the valve was switched every 6 min and each fraction was trapped alternately on one of the two columns; after the end of the trapping period each column was back-flushed under isocratic conditions (40% of methanol in 0.010 M CH 3 COONH 4 + 0.05 M H 3 PO 4 , pH = 3, F = 1 mL/min,) at a high temperature (T = 1208C).…”

“…1) employed a PEG column in the first dimension under isocratic conditions (1% ACN in 0.010 M CH 3 COONH 4 , pH = 3) at a flow rate of 0.3 mL/min. Two trapping X-Terra columns were used instead of the sampling loops [66,67] (the 10-port valve was switched every 9 min and each fraction was trapped alternately on one of the two trapping columns. The retained compounds were back-flushed from the trapping columns onto a short monolithic Chromolith SpeedROD RP-18e analytical column in the second dimension, using a gradient of 5 to 20% acetonitrile in 8 min and isocratic post-gradient elution with 5% acetonitrile at F = 2 mL/min, T = 408C).…”

Column selectivity for two‐dimensional liquid chromatography

“…The approach was applied to the analysis of drugs and degradation products and illustrated by nine examples using various combinations of silica-based RP columns in the two dimensions [147]. Another recent work reports a RP6RP 2-D system for separation of barbiturates and other drugs, using a hybrid silica gel organic support X-Terra MS C18 column in the first dimension and two SB-Phenyl columns in parallel alternating setup in the second dimension, coupled to a mass-spectrometric detector [148]. Heart-cutting approach was employed for 2-D separation of active medicinal constituents from natural plant extracts using a semipreparative bonded nitrile column in the first dimension and a C18 column in the sec-…”

“…In the same time, the compounds retained from the previous fraction on the second trapping column are back-flushed onto the analytical column in the second dimension [132,148] (Cacciola, F., Jandera, P., Blahovµ, E., Mondello, L., J. Sep. Sci., submitted).…”

“…Another possibility is to omit the sampling loops and to connect the first-dimension column via a switching valve to two identical second-dimension columns operating in parallel setup (a fraction from the first-dimension column is trapped alternately on one of the two columns; meanwhile the second column is back-flushed and the solutes transferred in preceding fraction from the firstdimension column are separated) [33,148] (Cacciola, F., Jandera, P., Blahovµ, E., Mondello, L., J. Sep. Sci., submitted).…”