.
Category
- Chromatography » Liquid Chromatography
Booking Details
Facility Management Team and Location
Facility Features, Working Principle and Specifications
Facility Description
Gel permeation chromatography (GPC) is a commonly used technique to measure the molecular weight distribution of synthetic polymers soluble in both organic as well as aqueous solution. The facility can be used on semi-preparative scale (few hundred mg) to separate (macro)molecules of interest. The facility can be used for a broad class of polymers (polyethylene, conjugated polymers, polypeptides, and others), provided the molecules have good solubility.
Some of the important specification and features of the facility are listed below.
- Pump for Analytical and Semi-Preparative Flow Rates: LC-20AR
- High pressure binary gradient system
- Flow rate: 0.01 mL/min to 20 mL/min
- Flow rate accuracy: ±1%
- Maximum discharge pressure: 49 MPa (7200 psi)
- Photo Diode Array (PDA) Detector (SPD-M20A)
- Light source: Deuterium (D2) lamp, tungsten (W) lamps
- Number of diode elements: 512
- Wavelength: 190 – 800 nm
- Wavelength accuracy: 1 nm max
- Linearity: 2.0 AU (ASTM standard)
- Evaporative Light Scattering (ELSD) Detector
- SEDERE Sedex 85LT ELSD with preparative cc nebulizer
- Detection: photodiode
- Light Source: Blue LED
- Sensitivity: 1 ng
- Temperature range: ambient to 100 °C
- Eluent flow rate: 5 μL/min to 5 mL/min
- Available columns/accessories
- Calibration kt, (PS),S-M-10,1/pk (THF standard)
- PLgel 5um MIXED-C 300 x 7.5 mm (THF analytical column)
- PLgel 5um MIXED-D 300 x 7.5 mm (THF analytical column)
- PLgel 5um Guard 50 x 7.5 mm (THF analytical guard column)
- PLgel 3um MIXED-E 300 x 7.5 mm (THF analytical column)
- PLgel 3um Guard 50 x 7.5 mm (THF analytical guard column)
- PLgel 10um 500A MIXED-D 300 x 25 mm (THF preparative column)
- EasiVial PEG/PEO 2 ml (Aqueous standard)
- PL aquagel-OH MIXED-H 8 um 300 x 7.5mm (aqueous analytical column)
- PL aquagel-OH MIXED-M 8 um 300 x 7.5mm (aqueous analytical column)
- PL aquagel-OH Guard 8 um 50 x 7.5 mm (aqueous guard analytical column)
- PL aquagel-OH 8 um 300 x 25 mm (aqueous preparative column)
Instructions for Registration, Sample Preparation, User Instructions and Precautionary Measures
Prepare the sample by dissolving 0.5-1 mg of the compound in 1 mL of THF or Water (dependent on the mobile phase), sonicate and filter through a 0.45/0.25 micron syringe filter. Place the sample in the sample chamber.
Please note the sample should be fully soluble at ambient temperature (25 C) for at least a few hours.
1. Parts of the instrument.
- Solvent bottles: Store the solvents used in the analysis
- Dual Pumps (A and B): Deliver the mobile phase
- Sample Chamber: Holds the sample to be analyzed
- Controller and Balance Module (CBM): Manages the instrument's operating parameters, monitors the system's balance, and controls the flow rates, pressures, and temperatures to ensure precise and accurate analysis.
- Column: Separates the sample components based on size
- PDA (Photodiode Array Detector): Detects and measures the absorbance of the separated components.
- Fraction Collector: Divide the eluent into discrete fractions based on time or volume
- ELSD (Evaporative Light Scattering Detector): ELSD detects and measures components that don't absorb UV light, using scattered light to identify and quantify them.
2. Protocols to run a sample in a GPC (Gel Permeation Chromatography) instrument:
- Verify that the solvent level is above the filter level; if not, add the required solvent (ensure mobile phase sonication for 10 minutes prior to use).
- Initialize the instrument by switching on the module switches and subsequently CBM.
- Open the pump valve and purge the lines for 5-10 minutes to eliminate air bubbles. Similarly, purge the injection line to ensure bubble-free operation.
- After purging is finished, close the pump valve, power on the PC, and launch LabSolution software; navigate to Instrument and HPLC, and set the flow rate to 0.2 mL/min and increase it gradually to 1 mL/min over 10 minutes.
- Allow the solvent to run for a few minutes while monitoring the baseline on both detectors.
- If the ELSD detector is not in use, maintain it in the off mode.
- For ELSD detector operation, switch on the nitrogen generator, set the pressure to 3.5 bar and temperature to 50 °C, and enable the LED and nitrogen valve.
- Once a stable baseline is achieved and the system is functioning nominally, connect the column and closely monitor the baseline and backpressure (70 – 80 kgf for 600 mm MIXED-D column) to ensure optimal system performance and maintain equilibrium.
- Verify the parameters in the existing method file 'GPC-Org-THF_sample_RUN Method' located at {New volume (D)>2024>GPC_STD_Methods_DATA_600mm_column>Methods} to ensure these parameters match with those used for GPC standards to guarantee reliable and reproducible results for sample analysis. Parameters/settings:
Sample run time: 22 minutes
Pump A flow rate: 1.00 mL/min
Pump B flow rate: 0 mL/min
- Prepare the sample by dissolving 0.5-1 mg of the compound in 1 mL of THF or Water (dependent on the mobile phase), sonicate and filter through a 0.45/0.25 micron syringe filter. Place the sample in the sample chamber.
- Create a batch file, enter sample details like sample name, injection amount, and method file, and then start the sample run.
- After the sample run is complete, carefully remove the column, place stoppers on both ends and store it in the designated drawer. Then, connect the joint line.
- Switch the mobile phase to IPA, purge the line for 5-10 minutes, and run the mobile phase for at least 30 minutes through the system. Store the previously used mobile phase in a capped bottle with the date labelled.
- To turn off the instrument, Turn off all instrument switches and the nitrogen generator switch.
- To determine the sample's molecular weight via GPC post-run analysis:
i) Open the sample data file in GPC postrun.
ii) Load the calibration method file 'GPC-Org-THF_Calibration_600mm column' from the location: {New volume (D)>2024>GPC_STD_Methods_DATA_600mm_column>Methods}.
iii) Proceed with molecular weight analysis using the loaded calibration method
- Finally, shut down the computer and dispose of waste solvent in the waste drum and cap the waste bottle to prevent solvent vapor accumulation in the room.
Charges for Analytical Services in Different Categories
| Type of analysis | IIT Bombay users | Academic institutes | National R&D labs | Industries/Non-government agencies |
| Charges per sample for analytical molecular weight analysis | 100 | 300 | 750 | 1500 |
| Charges for semi-preparative purification/recycle run per hour | 250 | 750 | 1500 | 3000 |
All charges in INR
For external samples, in addition to above basic charges prevailing GST will be added
Applications
The facility can be used to – i) analyze the molecular weight distribution of synthetic polymers soluble in THF and water, and ii) separate molecules on a semi-preparative scale (1000 amu and beyond).