Step 1:Peptide synthesizerPreparation before operation - laying the foundation for success
Environmental and equipment inspection
Choose a dry, clean, and temperature stable location (20 ℃± 5 ℃) to avoid moisture, high temperatures, or dust interference.
Ensure that the instrument is installed smoothly and that the power supply voltage and frequency match the equipment requirements (such as 60-100psi for compressed air pressure "AIR" gauge and 5-10psi for "N ₂" gauge).
Check if the solvent storage bottle and gas pipeline are tightly connected to prevent leakage.
Preparation of materials and reagents
Prepare high-purity amino acids, activation reagents (such as HBTU), solvents (DMF, DCM), and resins to ensure no impurities.
Weigh the reagents according to the synthesis plan and mix them strictly in proportion (such as using a 3-fold excess of amino acids for coupling reactions).
Add the resin to the reactor, swell it with DCM, dry it, and wash it with DMF to remove impurities.
Parameter setting and calibration
Enter the peptide sequence and set the reaction conditions (temperature, time, solvent ratio).
Example: Fmoc deprotection (20% piperidine/DMF, 5 minutes x 2 times); Coupling reaction (3-fold excess of amino acids, 1 hour).
Run the device self-test program, calibrate parameters such as temperature, pressure, and liquid sampling needle to ensure sensor accuracy.
Step 2: Synthesis Process Operation - Core Step Analysis
Start the synthesis program
Turn on the power and compressed air switch of the synthesizer, and start the preset program.
The instrument automatically completes deprotection (such as Fmoc group removal), coupling (amino acid cross-linking), washing cycle, and monitors the reaction temperature, pressure, and pH value throughout the process.
Key operational points
Deprotection: Use alkaline solvents (such as 20% piperidine/DMF) to remove the amino protecting group, repeat twice to ensure thorough removal.
Coupling reaction: Inject the activated amino acid solution into the reaction chamber, add DMF to the appropriate concentration, and stir for 1 hour to form peptide bonds.
Washing: Wash with DMF after each reaction step to remove unreacted substances and by-products, reducing impurity accumulation.
Real time monitoring and adjustment
Monitor the reaction process through UV absorbance or mass spectrometry analysis to ensure that each step proceeds as expected.
If uneven reaction solution is found, adjust the stirrer speed; If the temperature is abnormal, check the position of the heating plate.
Step 3: Post synthesis processing and maintenance - ensuring long-term stability
Product collection and purification
Add lysis buffer (such as TFA/water/triisopropylsilane=95:2.5:2.5), stir at room temperature for 2 hours, and lyse the peptide from the resin.
Filter to remove the resin, concentrate the lysate, precipitate the crude peptide with ether, and obtain the solid product by centrifugation and washing.
Purification using high-performance liquid chromatography (HPLC) to remove impurities and obtain high-purity peptides (>95%).
Peptide synthesizerEquipment cleaning and maintenance
Instant cleaning: After the synthesis is completed, immediately clean the reaction vessel, needle, pump head, and pipeline with ethanol or deionized water to avoid residual reagents corroding the equipment.
Regular maintenance: replace worn parts (such as seals and needles) monthly, calibrate sensor accuracy; Lubricate the mechanical parts every quarter to maintain smooth operation.
Long term storage: If the equipment is idle, clean it thoroughly and disassemble it to store it in a dry, ventilated place to prevent rusting or moisture.
Troubleshooting and Recording
Common problems: Pump head leakage (replace sealing ring), high temperature of reaction vessel (adjust heating plate position), equipment cannot be turned on (check power supply and control board).
Record and report: Detailed record of each step's operating parameters (reagent dosage, reaction time, temperature) and product purity, generate experimental reports, and provide a basis for subsequent optimization.