Peptide synthesizerCalibration is the core link to ensure the purity, yield, and experimental repeatability of synthetic products. Systematic calibration is required for key modules such as liquid inlet accuracy, temperature control, pressure stability, and detection systems. The following is a step-by-step calibration process that complies with industry standards and is applicable to mainstream solid-phase peptide synthesizers.
1、 Preparation before calibration: environmental and equipment pretreatment
Environmental condition confirmation: Stabilize the laboratory temperature at 20-25 ℃ (fluctuation ≤± 1 ℃), control the relative humidity at 40% -60%, and avoid direct airflow on the equipment. Start the equipment preheating one hour before calibration to ensure that each module is in a stable working state.
Preparation of tools and reference materials: Prepare certified standard equipment for metrology (such as 0.01mg electronic balance, standard thermometer, precision pressure gauge, 100 μ L standard pipette), as well as calibration reagents such as anhydrous methanol (chromatographic purity) and indanone standard solution (0.1mol/L).
Equipment cleaning and inspection: Disassemble the inlet pipeline and reaction column, rinse with anhydrous methanol three times, and then air dry; Check the pressure of the nitrogen cylinder (≥ 0.5MPa), replace the aging gasket, and ensure that there is no leakage in the pipeline.
2、 Core module calibration: precise control of key parameters
（1） Calibration of the liquid inlet system: ensuring the accuracy of reagent measurement
Flow calibration of inlet pump: Select commonly used inlet volumes (10 μ L, 50 μ L, 100 μ L), set the inlet rate to 10 μ L/s, insert the end of the inlet pipeline into a pre weighed clean centrifuge tube, and start the inlet program. Repeat each volume gradient three times, weigh the collected anhydrous methanol mass using an electronic balance, and calculate the actual inlet volume based on the methanol density (0.7918g/mL at 20 ℃), with an error margin of ≤± 2%. Otherwise, adjust the pump body pressure or replace the sealing ring.
Calibration of reagent distribution accuracy: Connect different reagent pipelines to the standard pipette interface, set a single dispensing volume of 50 μ L, repeat dispensing 10 times to the volumetric flask, and verify the total volume error using the volumetric method to ensure ≤± 1%; At the same time, check the time difference during synchronous allocation of multiple pipelines, which should be ≤ 0.5 seconds.
（2） Temperature and pressure system calibration: stable reaction environment
Reaction temperature calibration: Insert a standard thermometer into the interior of the reaction column (close to the resin area), set three commonly used temperature points of 25 ℃, 30 ℃, and 40 ℃ respectively, hold for 30 minutes, and then read the temperature value. Repeat 3 times for each point, with an error of ≤± 0.5 ℃. If it exceeds the standard, adjust the temperature control module parameters of the equipment or replace the heating element.
Nitrogen pressure calibration: Close the outlet of the reaction column, connect the precision pressure gauge to the nitrogen inlet, set the pressure to 0.1MPa, 0.2MPa, 0.3MPa, stabilize for 5 minutes, and read the value with an error of ≤± 0.01MPa. After calibration, test the nitrogen blowing rate to ensure that the resin flips evenly and without splashing.
（3） Calibration of detection and control modules: ensuring accurate process monitoring
Calibration of Indene Triptolide Detection System: Prepare standard solutions of Indene Triptolide with concentrations of 0.01mmol/L, 0.05mmol/L, and 0.1mmol/L, and automatically inject them into the equipment for detection. Record the absorbance values and plot the standard curve, with a correlation coefficient of ≥ 0.995. If the linearity is poor, clean the detection light path or replace the light source.
Program control calibration: Write a standard synthesis program (including 3 steps of deprotection, washing, and coupling), record the deviation between the actual execution time of each step and the set time, repeat each step 5 times, and the deviation should be ≤ ± 1 second. Check the pipeline switching response during step switching to ensure no residual cross contamination of reagents.
3、 Verification and recording after calibration: ensuring the effectiveness of calibration
No load operation verification: Execute the calibrated no-load synthesis program and run it continuously for 3 cycles to check the operating status of each module, ensuring no leakage, abnormal noise, parameter drift, or other issues.
Validation of standard peptide synthesis: Select a standard pentapeptide (such as Leu Enkephalin) for synthesis, and detect the purity of the product through HPLC. The purity should be ≥ 95%, and the deviation from historical calibration data should be ≤ ± 2% to confirm the calibration effect.
Calibration record archiving: Detailed record of calibration date, environmental conditions, calibration personnel, standard equipment number, calibration data and deviation values of each module, paste calibration qualification identification, and clarify the next calibration cycle (it is recommended to calibrate every 6 months or synthesize 500 times in advance).