inject 200 μL of effluent from the HPLC into the SEC column from the moment
of injection in the HPLC column (50 μL injection loop). The 2D-LC system was
handled with software provided by Polymer Char (Valencia, Spain). The data
acquisition and evaluation was performed by WinGPC-Software v. 7.0 (Polymer
Standards Service, Mainz, Germany).
• Columns. Chromatograph 1: Perfectsil 300, 250 mm  4.6 mm i.d., average
particle size 5 μm (MZ Analysentechnik, Mainz, Germany). Chromatograph 2:
PL Rapide H, 150 mm  7.5 mm i.d. (Polymer Laboratories, Church Stretton,
England).
• Mobile phase. Chromatograph 1: linear gradient TCB-cyclohexanone. The flow
rate was 0.1 mL/min. Chromatograph 2: TCB with a flow rate of 2.5 mL/min.
• Detectors. ELSD PL-ELS 1000 (Polymer Laboratories, Church Stretton,
England). The following parameters were set on the ELSD: air flow rate 1.5 L/
min, nebulizer temperature 160
C, evaporator temperature 260
C.
• Column temperature. 150
C.
• Sample concentration. 2 mg/mL. All samples were dissolved in TCB.
• Injection volume. 50 μL (first dimension).
3.4.2.4 Preparatory Investigations
The major prerequisite for the successful 2D-LC separation is a suitable chromatographic system. The successful separation of EVA copolymers with respect
to the VA content has been shown on a bare silica column in a mobile phase
composed of TCB and cyclohexanone [80]. EVA adsorbs on the stationary phase in
the starting solvent (TCB) and is subsequently desorbed by a TCB-cyclohexanone
solvent gradient.
3.4.2.5 Measurement and Evaluation
The contour plot in Fig. 3.43 shows the 2D-LC separation of a blend of the
homopolymers PVAc and PE and three EVA copolymers. The y-axis represents
the gradient HPLC separation and the x-axis represents the SEC separation. The
samples are separated with respect to the polarity. PE is the least polar component
and elutes first, while the last eluting component is PVAc, the most polar component. Between these two extremes, the three EVA copolymers elute with respect to
their VA content. The separation of two of the EVA copolymers (6.5 mol% and
20 mol% VA) was not optimal; nonetheless, the presence of two components with
different chemical compositions as well as with different molar masses can be
concluded. The spot between 5.6 mL and 6.0 mL in the contour plot is an artifact
produced by the WinGPC software.
In 2D-LC, typically only the second dimension providing the molar mass
information is calibrated. In the present application, however, both dimensions
shall be calibrated. The knowledge of the delay volume of the system, namely the
time required by the gradient to reach the detector, is needed for calibration of the
HPLC instrument.
The delay volume can be obtained by summing up the void volume and dwell
volume of the corresponding system. The void volume corresponds to the volume
3.4 Two-Dimensional Liquid Chromatography
127
of injection in the HPLC column (50 μL injection loop). The 2D-LC system was
handled with software provided by Polymer Char (Valencia, Spain). The data
acquisition and evaluation was performed by WinGPC-Software v. 7.0 (Polymer
Standards Service, Mainz, Germany).
• Columns. Chromatograph 1: Perfectsil 300, 250 mm  4.6 mm i.d., average
particle size 5 μm (MZ Analysentechnik, Mainz, Germany). Chromatograph 2:
PL Rapide H, 150 mm  7.5 mm i.d. (Polymer Laboratories, Church Stretton,
England).
• Mobile phase. Chromatograph 1: linear gradient TCB-cyclohexanone. The flow
rate was 0.1 mL/min. Chromatograph 2: TCB with a flow rate of 2.5 mL/min.
• Detectors. ELSD PL-ELS 1000 (Polymer Laboratories, Church Stretton,
England). The following parameters were set on the ELSD: air flow rate 1.5 L/
min, nebulizer temperature 160
C, evaporator temperature 260
C.
• Column temperature. 150
C.
• Sample concentration. 2 mg/mL. All samples were dissolved in TCB.
• Injection volume. 50 μL (first dimension).
3.4.2.4 Preparatory Investigations
The major prerequisite for the successful 2D-LC separation is a suitable chromatographic system. The successful separation of EVA copolymers with respect
to the VA content has been shown on a bare silica column in a mobile phase
composed of TCB and cyclohexanone [80]. EVA adsorbs on the stationary phase in
the starting solvent (TCB) and is subsequently desorbed by a TCB-cyclohexanone
solvent gradient.
3.4.2.5 Measurement and Evaluation
The contour plot in Fig. 3.43 shows the 2D-LC separation of a blend of the
homopolymers PVAc and PE and three EVA copolymers. The y-axis represents
the gradient HPLC separation and the x-axis represents the SEC separation. The
samples are separated with respect to the polarity. PE is the least polar component
and elutes first, while the last eluting component is PVAc, the most polar component. Between these two extremes, the three EVA copolymers elute with respect to
their VA content. The separation of two of the EVA copolymers (6.5 mol% and
20 mol% VA) was not optimal; nonetheless, the presence of two components with
different chemical compositions as well as with different molar masses can be
concluded. The spot between 5.6 mL and 6.0 mL in the contour plot is an artifact
produced by the WinGPC software.
In 2D-LC, typically only the second dimension providing the molar mass
information is calibrated. In the present application, however, both dimensions
shall be calibrated. The knowledge of the delay volume of the system, namely the
time required by the gradient to reach the detector, is needed for calibration of the
HPLC instrument.
The delay volume can be obtained by summing up the void volume and dwell
volume of the corresponding system. The void volume corresponds to the volume
3.4 Two-Dimensional Liquid Chromatography
127