Arc HPLC Aqueous SEC/GPC Separation of PEO/PEG

This application brief assesses the performance of the Waters Arc HPLC System for Gel Permeation Chromatography (GPC), also known as Size Exclusion Chromatography (SEC) of polyethylene oxide/polyethylene glycol (PEO/PEG). 

Benefits

• The Arc HPLC System, running Empower 3 Software, delivers precise flow rate to obtain accurate, reproducible peaks in GPC analysis
• The Ultrahydrogel Linear Column provides polymer size exclusion separation nominal 7 million Daltons

Gel permeation chromatography requires a high-performance liquid chromatography (HPLC) system that delivers precise flow and reproducible results. The GPC technique plots retention time against the log of the molecular weight, thus, the pump must produce a consistent isocratic flow to provide reproducible chromatographic peaks.¹ The most often used method for determining the molecular weight of an unknown sample is by comparing the sample against a relative calibration curve of narrow molecular weight polymer standards.²

The flow rate is one of many listed Arc HPLC GPC method parameters in Table 1. This application uses polyethylene glycol and polyethylene oxide standards (PEG/PEO) for the calibration curve (Figure 1).

The excellent flow rate precision of the Arc HPLC Pump is demonstrated in an overlay of six injections of a PEO/PEG mixed molecular weight standard as shown in Figure 2. Relative standard deviations of the peak retention time and peak molecular weight (Mp) at <0.3% are obtained on a Waters Arc HPLC System using two linear GPC columns and a 30-minute run time (Table 3 and 4).

Empower 3 has many built-in features that enable sophisticated reporting of data usually done in spreadsheets. A popular reporting area is column plate count, which is included with the system suitability feature. By reporting the column plate counts, the column efficiency can be charted and observed for any changes in performance (Table 5). 

The Arc HPLC with Empower 3 Software is a very capable GPC data processing tool for fundamental and complex chromatograms and calculations. In this experiment, the fundamental GPC calculations are demonstrated. The narrow PEO/PEG standards are used as calibration standards, and as broad unknowns for this experiment. The choice of method and standards used in this application are common for aqueous GPC of polymers like carbohydrates.³ The linear Ultrahydrogel Column is chosen for a wide molecular range nominal to seven million Daltons, and two of the same column are used for higher resolution of polymer peaks.⁴  The Empower 3 Software’s partial list of reportable peak result fields includes Mn, Mw, Mp, Mz, Mz+1, and polydispersity (Table 5). Relative and modified universal calibration types employing point-to-point and first- through fifth-order fits are available, and an example of relative calibration is shown in Figure 1.

The Arc HPLC Pumps in isocratic mode provide the excellent flow precision necessary for high-quality gel permeation chromatography.

All basic GPC calculations can be calculated using the Empower 3 Software with the GPC option, and numerous complex GPC calculations are accessible.

Short analysis time of 30 minutes was demonstrated using two linear columns.

1. Arc HPLC System, https://www.waters.com/waters/en_US/Arc-HPLC-System/nav.htm?cid=135068659&locale=en_US.
2. Frequently Asked GPC/SEC Questions, https://www.waters.com/waters/en_US/Frequently-Asked-GPC-SEC-Questions/nav.htm?cid=10167847&locale=en_US.
3. Yakindra Prasad Timilsena, Raju Adhikari, Stefan Kasapis, Benu Adhikari, Molecular and Functional Characteristics of Purified Gum From Australian Chia Seeds, Carbohydrate Polymers, Volume 136, 2016, Pages 128–136, ISSN 0144-8617, https://doi.org/10.1016/j.carbpol.2015.09.035.
4. Ultrahydrogel Columns. Waters Care and Use Manual, WAT011556, Revision F March 2015.
5. Empower GPC Software. Waters Getting Started Guide, 71500031303, Revision A May 2004.
6. Waters Corporation. Empower Tips. https://blog.waters.com/category/empower-tips.