1Wyatt multi angle laser light scattering instrumentPre-test preparation
Solvent preparation
Water phase system: Use ultrapure water or prepare a salt solution (about 1L), add 0.02% NaN3 antibacterial agent, and filter through a 0.22 μ m membrane.
Organic phase system: HPLC grade solvent is used, and 0.22 μ m filter membrane is also used for filtration (imported reagents are processed according to actual conditions).
Sample preparation
Prepare concentration based on molecular weight range (quantitative ring 100 μ L):
Molecular weight~1000kDa: 0.5-1mg/mL
Molecular weight~100kDa: 1-2mg/mL
Molecular weight~10kDa: 3-5mg/mL
Molecular weight~5kDa: 5-10mg/mL
The sample needs to be filtered through a 0.22 μ m or 0.45 μ m filter membrane, or impurities can be removed by centrifugation, and mobile phase preparation should be used as much as possible.
Instrument inspection
Confirm that the power cord connection, power switch, and switch (applicable for network cable signal connection) are turned on.
2、 Instrument startup and balancing
Boot Sequence
Turn on the pump, column temperature chamber (set temperature), injection system (manual/automatic sampler), differential or ultraviolet detector, viscosity detector, multi angle laser light scattering detector power supply, and computer in sequence.
System flushing and balancing
Turn on the pump and rinse the system with ultrapure water for about 5 minutes (purge pump), then close the purge valve.
Use the latest formulated mobile phase flushing system (organic systems directly use mobile phase).
Adjust the flow rate to 0.1mL/min (if connected to a viscosity detector, wait for it to enter the working interface before turning on the pump, and keep IP&DP in purge on state).
If the GPC/SEC column is not connected to the system, the flow rate can be directly adjusted to 0.1-1.0mL/min (when there is no viscosity detector, the purge valve of the differential detector needs to be in the "Purge On" state).
If the GPC/SEC column has been connected to the system, the starting flow rate should be 0.1mL/min, and the flow rate should be increased by 0.1mL/min every 1-2 minutes to adjust the flow rate to 0.4-0.5mL/min and fully balance the system.
Balance time: It is recommended to rinse and balance the aqueous system one day in advance, and start the experiment the next day; The equilibrium time for organic phase systems is generally 3-12 hours.
3、 Testing operations and data collection
System balance and baseline stability
After the system is fully balanced, gradually adjust the flow rate to the test flow rate.
Adjust the Purge valve of the differential detector to the "Purge Off" state, and return to zero after the signal stabilizes.
The operation method of Visco Star is shown in the attachment, wait for the baseline to stabilize.
Call the experimental template
Call the corresponding test template in the Astra software (it is recommended to use the template/method created by the installation engineer).
Single needle injection: File → New → Experiment from Template/Method → My Templates/Methods, select the corresponding experimental template.
Automatic sampler injection: File → New → sample set Template/layout sequence → My Templates/method, select the corresponding experimental template.
Sample injection and data collection
Switch the injection valve from "load" to "inject" state and inject the sample through the injection system.
The Astra software automatically starts collecting data, and after the data collection is completed, the software stops according to the set time or manually stops (Experiment → Stop).
data saving
After data collection is completed, click File → Save as..., name the file and save it.
4Wyatt multi angle laser light scattering instrumentData processing and report viewing
Data processing flow
Baseline adjustment: Double click Baseline, check LS 11 (90 ° angle), adjust baseline endpoints to ensure baseline level, and click OK/Apply to save.
Peak setting: Move the mouse to the starting point of the peak, hold down the left button and drag to the falling point of the peak, release the left button to define the peak area, slightly adjust the peak area edge, and click OK/Apply to save.
DN/DC value input and normalization: Enter the DN/DC value and select normalise (if<50kDa standard samples such as BSA and dextran have been normalized in the template under this system, there is no need to repeat the operation).
View report: After completing the above steps, view the report to obtain the test results.
Instrument calibration (conducted regularly)
Delay volume correction: Calibrate the volume of the pipelines between each detector to ensure alignment of peak positions, usually completed by installation engineers, and the results are saved in the Astra software template.
90 ° angle signal correction (toluene correction): Obtain the correction coefficient for the scattered light intensity and voltage values at a 90 ° angle, recommended once a year or every six months.
Eighteen angle normalization correction: Using a 90 ° angle as the reference, calibrate the other 17 detectors using standard samples with Rg<10nm (such as 30kDa Dextran or bovine serum albumin BSA) for calibration.