2024-09-27
Determination of Polyethylene Glycol by Gel Permeation Chromatography
1. Introduction
Purpose: Determination of molecular weight and distribution of polyethylene glycol (PEG) by high performance gel permeation chromatography (GPC) method.
Method:
Xtimate SEC-120, 5 μm, 7.8x300 mm gel permeation chromatography column
Differential refractive index detector (RID)
Mobile phase: ultrapure water
Flow rate: 1.0ml/min
Column temperature: 35 ℃;
Injection volume: 10μl.
Calibration curves are established and the molecular weight and distribution results of each sample are calculated by GPC software.
Result: The linearity of PEG is good when the molecular weight was in the range of 400-20000. The reproducibility of the experiment is good, with 6 consecutive injections of PEG6000, the RSD value of retention time is 0.105%, and the RSD value of peak area is 0.335%.
Conclusion: High performance gel permeation chromatography (GPC) is a reliable method for the determination of molecular weight and distribution of PEG, which has the advantages of accurate and high reproducibility when used to evaluate the polydispersity property of polymer compounds.
Keywords: HPLC, GPC, RID, Polymer, Polyethylene Glycol
2. Experiment Method
2.1 Instrument Configuration
Table 1 Configuration List of High Performance Liquid Chromatograph
| No | Modular | Qty |
| 1 | High Performance Liquid Chromatography LC3200 Series | 1 |
| 2 | PB3200 Binary Pump | 1 |
| 3 | RID3300 | 1 |
| 4 | CT3200 Column Oven | 1 |
| 5 | AS3200 Autosampler | 1 |
2.2 Test Conditions
Chromatography Column: Xtimate SEC-120，5μm，7.8x300mm
Column Temperature: 35°C
Detector: RID
Flow Rate: 1.0mL/min
Mobile Phase: Water
Injection Volume: 10μL
2.3 Instrument/Reagents and Consumables
Reagents:
Ultrapure Water
Standard: PEG400; PEG2000; PEG6000; PEG10000; PEG20000
Auxiliary Equipment
Analytical Balances
Solvent Extraction Unit
Ultrasonic Cleaner
Experimental Materials
Filter Membrane: aqueous filter membrane 0.45μm
2.4 Preparation of PEG Standard
Pipette 0.20g each of PEG400, PEG2000, PEG6000, PEG10000 and PEG20000 standards, add 10mL of water to dissolve, mix well, and prepared the concentration of 20mg/mL samples to be tested.
3. Result and Discussion
3.1 Different Molecular Weight Standards
Fig 1 Chromatogram of PEG400
Table 1 Chromatographic Parameters of PEG400
| No | Compounds | Retention Time | Peak Area | Theriacal Plate Number | Trailing Factor |
| 1 | PEG400 | 10.315 | 501.732 | 2346 | 1.185 |
Table 2 Chromatographic Parameters of PEG2000
| No | Compounds | Retention Time | Peak Area | Theoretical Plate Number | Trailing Factor |
| 1 | PEG2000 | 8.659 | 499.892 | 1926 | 1.230 |
Fig 3 Chromatogram of PEG6000
Table 3 Chromatographic Parameters of PEG6000
| No | Compounds | Retention Time | Peak Area | Theoretical Plate Number | Trailing Factor |
| 1 | PEG6000 | 7.215 | 499.482 | 1.171 |
Fig 4 Chromatogram of PEG10000
Table 4 Chromatographic Parameters of PEG10000
| No | Compounds | Retention Time | Peak Area | Theoretical Plate Number | Trailing Factor |
| 1 | PEG10000 | 6.612 | 483.657 | 2550 | 1.265 |
Fig 5 Chromatogram of PEG20000
Table 5 Chromatographic Parameters of PEG20000
| No | Compounds | Retention Time | Peak Area | Theoretical Plate Number | Trailing Factor |
| 1 | PEG20000 | 6.081 | 497.803 | 1103 | 1.799 |
Fig 6 Overlap Chromatograms of Different Molecular Weight
Note: The above data shows that the retention time of PEG20000 is 6.081 minutes, and PEG400 is 10.315 minutes, larger molecules are eluted first and smaller molecules are eluted later.
3.2 Repeatability
Fig 7 Repeatability Overlap Chromatograms of PEG6000 (n=6)
Table 7 Repeatability Chromatographic Parameters of PEG6000 (n=6)
| No | Samples | Retention Time | Peak Area |
| 1 | 6000 | 7.233 | 498.821 |
| 2 | 6000 | 7.234 | 503.367 |
| 3 | 6000 | 7.225 | 499.891 |
| 4 | 6000 | 7.221 | 499.560 |
| 5 | 6000 | 7.219 | 501.374 |
| 6 | 6000 | 7.215 | 499.482 |
| Average | - | 7.225 | 500.416 |
| RSD(%) | - | 0.105 | 0.335 |
Note: The repeatability is good. The RSD of retention time is 0.105% and the RSD of peak area is 0.335% for 6 injections of PEG6000.
3.3 Standard Curves
Fig 8 Standard Curves Chromatograms of Different Molecular Weights
Table 8 Standard Curves Chromatographic Parameters of Different Molecular Weights
Note: The molecular weight and distribution results of each sample are calculated by GPC software. The linearity of PEGmolecular weight is good in the eange of 400 to 20,000, and the linear correlation coefficient is 0.999.
4. Conclusion
This polyethylene glycol (PEG) test is performed by gel chromatography by using an LC3200 series high performance liquid chromatograph with differential refractive index detector. the retention time of PEG20000 is 6.081 minutes, and PEG400 is 10.315 minutes, larger molecules are eluted first and smaller molecules are eluted later. The repeatability is good. The RSD of retention time is 0.105% and the RSD of peak area is 0.335% for 6 injections of PEG6000. The linearity of PEG molecular weight is good in the range of 400 to 20,000, and the linear correlation coefficient is 0.9999. High performance gel permeation chromatography (GPC) is a reliable method for the determination of molecular weight and distribution of PEG, which has the advantages of accurate and reproducible results when used to evaluate the polydispersity property of polymer compounds.
Note: The sample should be left at room temperature for more than 12h and should be mixed gently, do not ultrasonic or shake vigorously to accelerate dissolution.
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