This experiment is from the official website of College of Chemistry, Qingdao University of Science and Technology.
Operation method
Determination of nitrophenols by reversed-phase liquid chromatography
Principle
Reversed-phase ion-pair chromatography is a method of chromatographic separation using a hydrophobic stationary phase and a polar buffer containing a low concentration of counterions as the mobile phase. It can be used for the separation of ionic or dissociable compounds and is tending to replace ion exchange chromatography as a very dynamic component of liquid chromatography. The most easily accepted mechanism is that the dissociated solute forms hydrophobic ion pairs with antiparticles in the mobile phase, which are then separated according to the laws of reversed-phase chromatography. The size and concentration of the counterions, the concentration of the organic modifier and the pH are all important factors in controlling the separation. Nitrophenols are dissociable organic acids. Tetrabutylammonium ion is used as the counter ion in the separation, and under weak alkaline conditions, nitrophenols flow out in the order of decreasing pKa value. The internal standard method is a widely used method for chromatographic quantification. Before quantification by chromatography, it is necessary to select suitable internal standards and accurately determine the calibration factors. For the determination of nitrophenols, 2,4-dichlorophenol was used as the internal standard. To determine the calibration factor fi, a certain amount of standard of component i was mixed with a certain amount of internal standard, and the chromatographic separation was performed, and the peak areas were measured as Ai and As, respectively, with W representing the weight. For quantitative analysis, a certain amount of internal standard S is added to the sample to be measured, and chromatographic separation is carried out, and the measured peak areas are Ai′ and As′, respectively; then: W'i, W's′ are the amounts of component i and internal standard S in the sample to be measured, respectively. Move I. Conditions Caveat 1、Measure the area of the peaks in Chromatogram 1 and Chromatogram 2. 2、According to the data of chromatogram 1, calculate the relative correction factor of each component. 3、According to the data of chromatogram 2, calculate the content of each component in the sample, g/mL. For more product details, please visit Aladdin Scientific website.
Stationary phase: YWG-C18; Mobile phase: methanol/water 55:45, containing 20 mmol/L tetrabutylammonium bromide, and pH adjusted between 7.5-8.5 with KH2PO4/K2HPO4. Flow rate: 1 mL/min; Detection: UV-254 nm, 0.02 AUFS; Temperature: room temperature; Paper speed: 5 mm/min.
Standard Test: p-Nitrophenol; 2,4-Dinitrophenol; 2,5-Dinitrophenol 2,6-Dinitrophenol; 2,4,6-Trinitrophenol; Internal Standard: 2,4-Dichlorophenol
II. [Operating Procedure]
1. Preparation of standard solution and internal standard solution
(1) Accurately weigh 10 mg of o-nitrophenol, 16 mg of p-nitrophenol, 10 mg of 2,4-dinitrophenol, 6 mg of 2,5-dinitrophenol, 10 mg of 2,6-dinitrophenol, and 20 mg of 2,4,6 trinitrophenol, dissolve in 50 mL of methanol. Dissolve in 50 mL methanol, transfer to 100 mL volumetric flask, dilute with water to the scale and shake well.
(2) Weigh 0.3 g of 2,4-dichlorophenol accurately and prepare 100 mL of methanol/water solution.
2. Determination of calibration factor Fi 13
Mix 1 mL of internal standard solution with 1 mL of standard solution. After the instrument is stabilized, inject 10 μL of the mixture and record the chromatogram 1.
3. Measurement of samples
Mix the sample to be analyzed with the internal standard solution. Inject 10 μL and record the chromatogram 2. Based on the retention value, determine which components are contained in the sample.