Figure 2: Calibration curves for individual TSKgel SuperMultiporeHZ-N (3 µm) ●, SuperMultiporeHZ-M (4 µm) ●, SuperMultiporeHZ-H
(6 µm) ●. Column dimensions: 150 mm ×4.6 mm; mobile phase: tetrahydrofuran; flow rate: 0.35 mL/min; detection: UV absorbance
at 254 nm; temperature: 25 °C; samples: standard polystyrene kit (PStQuick kit-H) with benzene as solvent; injection volume:
5 µL. Courtesy of Tosoh Bioscience; adapted from reference 11.
Modern SEC columns now have unprecedented column efficiencies, approaching >130,000 plates/m for 3-µm packings using a small-molecule
test solute. With high-efficiency and large-pore-volume packings, we are now able to detect previously unresolved components
not seen with older column technology. High-efficiency columns are more sensitive to MWD distortions that arise from nonlinear
regions of the calibration plot. An example of the latter is seen in Figure 3 which shows a series of SEC chromatograms of
four different lots of a phenolic resin. These phenolic resins have wide MWDs, especially lot D. Starting at 8 min (2.8-mL
elution volume), individual oligomers appear up to the negative solvent peak at 10 min.
Figure 3: Composite SEC chromatograms of four phenolic resin samples analyzed with a two-column set of "single-pore size"
packings: TSKgel SuperHZ 3000 + 2000 (150 mm ×4.6 mm) ×2; mobile phase: tetrahydrofuran; flow rate: 0.35 mL/min; detector:
UV absorbance at 254 nm; temperature: 25 °C; samples: phenolic resins (3 µg/µL); injection volume: 10 µL. Courtesy of Tosoh
Bioscience; adapted from reference 11.
An unusual feature of four of these chromatograms is the distorted profile in lots B, C, and D occurring at the same 6-min
elution volume. Because these phenolic resins are produced using condensation polymerization, we would not expect perturbation
in the higher-molecular-weight region, unless, of course, the polymerization was altered during production, or a second phenol
resin was added to the original lot — unlikely events. It has been suggested by Tosoh Bioscience researchers (11) that this
distortion, not normally seen with lower-resolution columns, is caused by column–column inconsistencies when two differently
packed columns are coupled together; in Figure 3, a TSKgel SuperHZ-3000 column is added to a SuperHZ-2000 column. Although
both columns have the same dimensions, properties of the columns, like different pore volumes and severely overlapping molecular
weight ranges, can give rise to distorted, nonlinear slopes. (Please note that it may be possible to compensate for calibration
nonlinearity by defining the calibration curve with a sufficient number of polymer standards, especially in the region where
the inflection point occurs.)
Figure 4: Composite SEC chromatograms of four different phenolic resins analyzed with a two-column set of "multipore" (wide-pore)
packings: TSKgel SuperMultiporeHZ-N (150 mm ×4.6 mm) ×2; see Figure 3 for all other chromatographic conditions. Courtesy of
Tosoh Bioscience; adapted from reference 11.
If the same phenolic resins are analyzed this time with two identical TSKgel SuperMultipore HZ-N columns, which have the same
dimensions and the same particle size (3 µm) as those used to produce Figure 3 chromatograms, we arrive at the expected profiles
in Figure 4 (11): The distortions at the beginning of the chromatograms are absent. We must stress, however, that if the proper
molecular weight calibration plot is constructed with the SuperHZ3000 + 2000 set using a sufficient number of standards and
a nonlinear fit, both column sets shown in Figures 3 and 4 should give identical MWDs for all of the phenolic resins, without
Figure 5: Composite SEC chromatograms of a phenolic resin sample analyzed with a four-column set of (a) "multipore" (wide-pore)
packings: TSKgel SuperMultiporeHZ-M (150 mm ×4.6 mm) ×4; (b) "single-pore size" packings: TSKgel SuperHZ4000 + 3000 + 2500
+ 2000 (150 mm ×4.6 mm) ×4; see Figure 3 for all other chromatographic conditions. Courtesy of Tosoh Bioscience; adapted from
The final example of chromatogram distortion caused by nonlinear calibration behavior is given in Figure 5 for the SEC analysis
of a different batch of phenolic resin. When a set of four single-pore TSKgel SuperHZ columns was used, considerable distortion
was obtained, ostensibly caused by considerable molecular weight overlapping among columns that also have different calibration
curve slopes. With the use of a truly linear SuperMultiporeHZ-M four-column set, distortion disappears. If one were not aware
of this anomalous, but not infrequently occurring effect, it would have appeared that this phenolic resin had experienced
a dramatic upset during polymerization, and the entire lot of phenolic resin could have been lost at great expense.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC