Reproducibility and Reliability of GPC Analysis of Polystyrene on the Arc™ HPLC System

In this application note, we demonstrate the ability to adapt the Arc HPLC System for GPC analyses and demonstrate the compatibility with strong solvents, including THF. The reproducibility, reliability, and durability of the system were evaluated for polystyrene GPC analysis over five days. The resulting data demonstrated consistent retention times, peak areas, and calculated molecular weight values for the polystyrene sample, highlighting the reproducibility of the Arc HPLC System equipped with the strong solvent compatibility kit, and producing reliable GPC analyses results.

Benefits

• Reliable and reproducible Polystyrene GPC analysis over an extended period using the Arc HPLC System with strong solvent compatibility kit
• Integration of Arc HPLC System with Empower™ CDS GPC option for MW analysis of polystyrene sample

In Gel Permeation Chromatography (GPC) reliable retention times are critical to determine the expected molecular weight of the sample. Given that the sample peak elution is compared to a calibration curve, reproducible retention times are required for the analyses, including both standards and samples, as changes in retention can affect sample characterization including M_n and M_w values. In addition, GPC requires mobile phases that solubilize polymers, and are typically strong organic solvents, such as tetrahydrofuran (THF). Many traditional HPLC systems contain materials that are incompatible with these strong organic solvents, from swelling or otherwise degrading over time. For GPC analysis, the instrument must contain parts durable under strong solvents conditions. To address this concern, the strong solvent compatibility kit for the Arc HPLC System replaces all components that can be adversely impacted by strong solvents, enabling extended use of the system for GPC analysis with strong organic solvents. In this study, system reproducibility using this strong solvent compatibility kit was examined for the GPC analysis of polystyrene over an extended analysis time. The resulting data demonstrated consistent retention times and calculated molecular weight values for the polystyrene sample. This highlights the flow rate precision of the Arc HPLC System equipped with the strong solvent kit, producing reliable GPC analyses results.

Sample Description

Standard Solutions

Polystyrenes Standards, ReadyCal, (p/n: WAT058930) was obtained from Waters™ Corporation and contains three sets of narrow standards:

• M_p Range 1 (3,470 to 2,520,000)
• M_p Range 2 (1,306 to 1,210,000)
• M_p Range 3 (370 to 552,000)

ReadyCal standards were prepared by addition of 2 mL THF to each vial. The final concentrations were 1 mg/mL for M_p = 2,520,000 and 1,210,000 and 2 mg/mL for the remaining standards. The vials were capped and left for three hours in the fume hood for standards to dissolve without any vigorous shaking.

Sample Solution

4 mg of the polystyrene sample was weighed and dissolved in 2 mL of THF in a similar way to the standards.

To assess the reproducibility of the Arc HPLC System with the strong solvent compatibility kit, GPC analysis of polystyrene was performed with THF mobile phase over a period of five days. Both polystyrene narrow standards and broad sample were analyzed using a bank of four GPC columns in a controlled temperature environment. The bank of columns, including two mixed bed columns, was selected to characterize a MW range from 4 M to 50 Da. The method conditions were optimized to resolve all peaks of the polystyrene narrow standards, as well as characterize the unknown polystyrene sample (Figure 1).

To analyze the separation, Empower CDS Software GPC option was used for data analysis and reporting.¹ As described earlier, a set of narrow polystyrene standards with the molecular weights (M_p) from 370 to 2,520,000 were analyzed each day to create the calibration curve. Correlation between the log molecular weight (MolWt) and retention time was achieved by applying a third order fit, producing a correlation coefficient of 0.9997, as shown for Day 5 (Figure 2).

Reproducibility of Standards and Calibration Curve

As described earlier, the GPC analysis of polystyrene was continuously run for a period of five days, with five replicate injections of standards and samples analyzed daily. To show the consistency of the calibration, two standard peaks closest to the retention time of the broad sample (M_p = 277,000 and 130,000 respectively), were evaluated (Table 1). The intra-day retention time precision for each day ranged from 0.01–0.32% for the standard at 277,000 and was 0.01% for the standard at 133,000 Da for each day. Furthermore, the flow rate precision was assessed over an extended time and inter-day variability was evaluated. The mean retention times of these standards were within 0.01 minute, demonstrating reproducible performance over the course of the study.

As described earlier, the standards were analyzed daily over the period of five days, with calibration curves created each day. During this period, the calibration curves for the analysis showed minimal variation and produced identical R² values, demonstrating the consistency of calibration (Table 2).

Reproducible GPC Analysis of Polystyrene Sample

In addition to calibration curves, it is also critical that a system provide reproducible GPC characterization, including polydispersity. Analysis of a known polystyrene sample was performed daily, with calculations using the respective standards from each day. Using the GPC Option in Empower CDS, the sample was characterized with reporting of M_n, M_w, M_p, M_z, M_Z+1, and polydispersity.

The results show comparable results obtained over the time period (Table 3). High retention time repeatability was observed with RSDs of less than 0.02% for each day. In addition, acceptable correlation was observed for the sample’s polydispersity, M_n, M_w, and M_p values. All results were within 10% of the reported values for the broad sample (M_p=146,600, M_w=141,700, M_n=133,300). In addition, the polydispersity values were within 5% of the expected value. The day-to-day reproducibility is demonstrated by the overlay of the distribution plot obtained for the broad sample on each of the five days, as displayed in Empower (Figure 3).

GPC analysis of a polystyrene sample in THF was performed over five days on the Arc HPLC with strong solvent compatibility kit and Empower CDS with GPC option. Reproducible retention times were observed on the Arc HPLC System, demonstrating good flow rate precision. The system also produced consistent day-to-day results for both calibration curves and sample analysis, with molecular weight (M_n and M_w) values within 10% of expected value. These results demonstrate the Arc HPLC System equipped with the strong solvent compatibility kit, combined with Empower CDS, and GPC Option, is a reliable system for routine GPC analysis.

1. Empower GPC Software Getting Started Guide, Waters Corporation User Guide, 71500031303