Laboratory liquid chromatography instrumentCalibration of metrology involves multiple aspects, and the following are detailed steps and precautions:

1、 Environmental conditions

Before performing the calibration of liquid chromatography, it is necessary to ensure that the environmental conditions meet the following requirements:

The room temperature should be maintained at (15-30) ℃, and the temperature change during calibration should not exceed 3 ℃ (for differential refractive index detectors, the room temperature change should not exceed 2 ℃).

The indoor relative humidity should be maintained at (20-85)%.

Flammable, explosive, and highly corrosive items are not allowed to be stored in the room.

There is no obvious mechanical vibration and electromagnetic interference, and the ventilation is good.

2、 Standard materials and calibration equipment

National certified standard materials should be used, and calibration equipment should be ensured to pass metrological verification. Common reference materials include:

Naphthalene methanol solution standard substance, with a fixed value of 1.00 × 10 ^ -4g/mL and an expanded uncertainty of less than 4%.

Cholesterol methanol solution standard substance, with a standard value of 200 μ g/mL and an expanded uncertainty of less than 2.0%.

Other standard substances selected based on specific calibration requirements.

Calibration equipment may include pipettes, volumetric flasks, thermometers, etc.

3、 Calibration steps

1. Calibration of infusion system

Pump pressure resistance: Connect all parts of the instrument, use methanol (or pure water) as the mobile phase, and set the flow rate to 0.2mL/min. Start the instrument and maintain stable pressure for 10 minutes, check for leaks at all pipeline interfaces. Remove the chromatography column, block the outlet end of the pump, and maintain the pressure at 90% of the maximum allowable value for 5 minutes to confirm no leakage.

Pump flow setting error and flow stability: Set the flow rate as required, start the instrument and wait for the pressure to stabilize. Collect the mobile phase at the outlet with a clean volumetric flask, time and weigh, and calculate the measured flow rate. Repeat calibration 3 times to ensure accuracy.

Gradient error: Set up a gradient elution program and plot and calculate the gradient change curve using solvent A (such as water) and solvent B (such as an aqueous solution containing 0.1% acetone).

2. Correction of Temperature Setpoint Error and Stability of Column Box

Place the digital thermometer probe in the same position as the chromatographic column thermostat.

Select the set temperature (such as 35 ℃ and 45 ℃), record the reading after the temperature stabilizes, and record it every 10 minutes for a total of 7 times.

Calculate the difference between the average value and the set value as the error of the column temperature set value, and the difference between the maximum and minimum values of the 7 readings as the stability of the column temperature.

3. Detector calibration

Taking UV visible light detector and diode array detector as examples, connect the various parts of the instrument and select appropriate chromatographic columns and mobile phases.

Set corresponding measurement parameters, such as wavelength, sensitivity, etc.

After stabilizing the baseline, inject a certain volume of standard substance solution (such as 1.0 × 10 ^ -4g/mL naphthalene methanol solution), continuously measure and record the retention time and peak area of the chromatographic peak.

4. Calibration of injection volume accuracy (if applicable)

Standard calibration method, weighing method, quantitative ring calibration method, or comparison method with standard injector can be used.

Standard calibration method: Inject a standard solution of known concentration multiple times, record the chromatographic peak response value, and calculate the accuracy of the injection volume.

Weighing method: Calculate the actual volume of solvent absorbed by weighing and compare it with the set volume.

Quantitative ring calibration method: Check the volume accuracy of the quantitative ring by observing the shape of chromatographic peaks and response values to determine.

Comparison method with standard injector: Use a standard injector for comparative analysis, calculate the deviation of the injection volume, and make adjustments.

4、 Calibration records and reports

During the calibration process, detailed records of various data and observation results should be kept.

After the calibration is completed, a calibration report should be prepared, including the calibration date, environmental conditions, calibration equipment, calibration steps, data recording, and analysis results.

The calibration report should be reviewed and signed by professionals for confirmation.

5、 Precautions

During the calibration process, the operating procedures and safety standards should be strictly followed.

When using standard substances, ensure that they are within their expiration date and stored properly.

Calibration equipment should be regularly maintained and calibrated to ensure its accuracy.

The calibration of laboratory liquid chromatography is a complex and delicate process that requires professionals to strictly follow operating procedures to ensure the accuracy and reliability of the instrument.