Effect of first dimension phase selectivity in online comprehensive two dimensional liquid chromatography (LC×LC)

Abstract

In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC×LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC×LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP×RP.

Fig. 1

Selective triangle (S*–B–C) of the six reversed stationary phases in this study. F5: Discovery HS-F5; C3: Zorbax 300SB-C3; C8: Zorbax 300SB-C8; CN: Zorbax SB-CN; Bonus: Zorbax Bonus-RP; HC–COOH: home-made HC–COOH.

Fig. 2

1DLC chromatograms of maize extract samples using six different phases under Elution Condition 1. (a) F5: Discovery HS-F5; (b) C3: Zorbax 300SB-C3; (c) C8: Zorbax 300SB-C8; (d) CN: Zorbax SB-CN; (e) Bonus: Zorbax Bonus-RP; and (f) HC–COOH: home-made HC–COOH.

Fig. 3

LC × LC chromatograms of maize extract samples using six different first dimension phases paired with second dimension carbon phase under Elution Condition 1. (a) F5: Discovery HS-F5; (b) C3: Zorbax 300SB-C3; (c) C8: Zorbax 300SB-C8; (d) CN: Zorbax SB-CN; (e) Bonus: Zorbax Bonus-RP; and (f) HC–COOH: home-made HC–COOH.

Fig. 4

LC × LC contour plots of maize extract samples using six different first dimension phases paired with second dimension carbon phase under Elution Condition 1. (a) F5: Discovery HS-F5; (b) C3: Zorbax 300SB-C3; (c) C8: Zorbax 300SB-C8; (d) CN: Zorbax SB-CN; (e) Bonus: Zorbax Bonus-RP; and (f) HC–COOH: home-made HC–COOH.

Fig. 5

LC × LC f_coverage plots of maize extract samples using six different first dimension phases paired with second dimension carbon phase under Elution Condition 1. (a) F5: Discovery HS-F5; (b) C3: Zorbax 300SB-C3; (c) C8: Zorbax 300SB-C8; (d) CN: Zorbax SB-CN; (e) Bonus: Zorbax Bonus-RP; and (f) HC–COOH: home-made HC–COOH.

Fig. 6

Comparison of C3 and Bonus phases in the first dimension when paired with carbon phase in the second dimension using whole grain bread extract samples under Elution Condition 1. (a) LC × LC chromatogram when C3 in the first dimension; (b) LC × LC chromatogram when Bonus in the first dimension; (c) LC × LC contour plot when C3 in the first dimension; (d) LC × LC contour plot when Bonus in the first dimension; (e) LC × LC f_coverage plot when C3 in the first dimension; and (f) LC × LC f_coverage plot when Bonus in the first dimension.

Fig. 7

Comparison of C3 and Bonus phases in the first dimension when paired with carbon phase in the second dimension using juice extract samples under Elution Condition 1. (a) LC ×LC chromatogram when C3 in the first dimension; (b) LC × LC chromatogram when Bonus in the first dimension; (c) LC × LC contour plot when C3 in the first dimension; (d) LC × LC contour plot when Bonus in the first dimension; (e) LC × LC f_coverage plot when C3 in the first dimension; and (f) LC × LC f_coverage plot when Bonus in the first dimension.

Fig. 8

Comparison of C3 and Bonus phases in the first dimension when paired with carbon phase in the second dimension using yeast supernatant samples under Elution Condition 1. (a) LC × LC chromatogram when C3 in the first dimension; (b) LC × LC chromatogram when Bonus in the first dimension; (c) LC × LC contour plot when C3 in the first dimension; (d) LC × LC contour plot when Bonus in the first dimension; (e) LC × LC f_coverage plot when C3 in the first dimension; and (f) LC × LC f_coverage plot when Bonus in the first dimension.

Fig. 9

Comparison of C3 and Bonus phases in the first dimension when paired with carbon phase in the second dimension using human urine extract samples under Elution Condition 2. (a) LC × LC chromatogram when C3 in the first dimension; (b) LC × LC chromatogram when Bonus in the first dimension; (c) LC × LC contour plot when C3 in the first dimension; (d) LC × LC contour plot when Bonus in the first dimension; (e) LC × LC f_coverage plot when C3 in the first dimension; and (f) LC × LC f_coverage plot when Bonus in the first dimension.