Reagents and Material
- PerseeL600 HPLC
- PerseeGWB– 2E Ultra-Pure Water Purifier
- 1.Acetonitrile: HPLCgrade.
- Water: HPLCgrade.
- Nerol: ≥97% pure.
- Linalool: ≥98%pure.
- Citronellol: ≥95% pure.
- RoseOil: 9.99% pure.
- 7. LemonOil
Reference Standard Solution Preparation
Precisely measure and transfer 10uL of nerol, linalool, citronellol into 3 different 1mL volumetric flask separately. Dilute each compound with acetonitrile up to the marker
and mix well.
Sample Solution Preparation
Precisely measure and transfer 1uL of sample into a 10mL volumetric flask. Dilute the sample with acetonitrile up to the marker and mix well. Filtrate the solution with 0.22 um filtering membrane, then collect the filtrated solution as testing solution.
Isocratic Pump is required for this experiment. The mobile phases are acetonitrile and water (v/v = 1:1). Unisol C18 Column (4.6 mm*150mm*5um) is used. The measure wavelength and flow rate are set to be 205nm and 1.0mL/min respectively. The column oven temperature is at 30℃ constantly.
Run the 3 reference standard solution (nerol, linalool, citronellol) separately through HPLC, and record the elution time of each compound. Dilute the standard solution into 5 different concentrations (the range of these concentration must include your unknown sample concentration). Calibration curve can be obtained by running these standard solutions through HPLC. Then set up unknown sample sequence to get the concentration of your unknown sample.
Accurately measure 0.01mL, 0.02mL, 0.04mL, 0. 1mL, 0.2mL, 0.4mL, 0.5mL, 1mL and 2mL of nerol stock solution (or linalool, or citronellol stock solution) into 9 different 10mL volumetric flask separately. Dilute each compound with acetonitrile up to the marker and mix well. Run each solution through HPLC by the same condition. The calibration curve, linearity and R2 value can be calculated by the software.R2 value can be calculated by the software.
Degree of Precision Test
Accurately measure and run 10 uL of reference solution in HPLC by the same condition 6 times. Record the peak area. The Relative Standard Deviation (RSD) can be calculated based the measurement.
Accurately measure and transfer 10uL of unknown sample into a 100mL volumetric flask. Dilute each compound with acetonitrile up to the marker and mix well. Repeat this step 5 times to prepare 5 solutions. Run these solutions through HPLC by the same condition in 3 days. Record the peak area and calculate the Relative Standard Deviation (RSD).
Standard Recovery Rate Test
Measure and transfer certain amount ofunknown sample solution 6 sets with samnevolume and samne concentration Add certainamount of reference standard solution. Run eachsolution through HPLC with the same conditionand record the peak area. Calculate the recoveryrate.
The concentration of unknown sample can befound by the calibration curve. The linearity of standard curve generated by nerol is R2 = 1.000; The linearity of standard curve generated by linalool is R2 = 0.9999; and the linearity of standard curve generated by citronellol is R2 = 1.0000. This mean that the linearity of each result is excellent.
The RSD value calculated by nerol in Degree of Precision Test is 0.39%; The RSD value calculated by linalool in Degree of Precision Test is 0.08%; and the RSD value calculated by citronellol in Degree of Precision Test is 0. 11%. This means that the instrument degree of precision quality is excellent. The repeatability test of nerol, linalool and citronellol shows that the RSD values for all the three compounds are less than 3%, this means that the repeatability by this method is excellent.
The Recovery Rate for nerol, linalool and citronellol are calculated to be between 94% and 105%, the RSD values are less than 5%, which means that the experiment result is reliable.