In the experimental discipline, data plays a vital role, and obtaining scientific and effective data is the quality that every experimenter should have. Based on the working experience of our laboratory in recent ten years and the experience of other laboratories, this paper mainly lists the matters needing attention in the determination of optical rotation, nuclear magnetic resonance and enantiomeric excess (ee value). The principle is: collect the necessary data as soon as possible, otherwise it will take a lot of unnecessary time and energy to supplement the data in the future. If known, it should be compared with the literature data. For unknown compounds, nuclear magnetic resonance and carbon spectrum, high-resolution mass spectrometry or elemental analysis, melting point (if it is a solid), optical rotation (if it is a chiral compound) and infrared spectrum should be collected. For known compounds, NMR spectra, melting point (if solid) and optical rotation (if chiral) should be collected. After the test, the data should be recorded in the experimental notebook in time and accurately (don't copy it on a small piece of paper first, then on the experimental notebook, the more steps, the greater the possibility of making mistakes) for reference, and write papers and references in future research work.

Part I: Matters needing attention in optical rotation determination

1. After the product is obtained, its optical rotation (in this case, specific rotation) should be determined as soon as possible, and it is not advisable to leave it for a long time before measuring it. Because standing for a long time may lead to deterioration of the product, the quality of the bottle may change without calibrating the balance again, resulting in inaccurate optical rotation value.

2. Before determining the optical rotation, if it is a known object, it is best to find out the optical rotation value reported in the literature. Because the values in many literatures are inconsistent, we should try to choose the data from authoritative papers, including concentration, temperature, solvent and optical rotation. When measuring the optical rotation of the product, it can be compared with the literature value, so as to know fairly well.

3. The measured product must be emptied before measurement to ensure the accuracy of weighing quality.

4. It is best to use the newly opened solvent to prepare the product solution to be tested. Because the unsealed solvent may be contaminated when used, there will be impurities, which will lead to inaccurate measurement data.

5. The concentration of the prepared test solution should be moderate, neither too high nor too low, and the general concentration is 0.2- 1.0( 10.

Mg/ml-100

Mg/mL) is better.

6. When preparing the solution to be tested, all products should be dissolved in it. If it is turbid after dissolution, the solvent with good solubility should be considered; If there are small particles after dissolution, it proves that there are impurities in the product. The product must be treated first to remove insoluble impurities, and then determined.

7. Before determining the optical rotation, it is best to use a substance with known optical rotation value as a standard to calibrate the optical rotation instrument used to ensure the accuracy of the measurement data of the optical rotation instrument.

8. After turning on the polarimeter, it will take some time to measure. Because the normal start-up time of sodium lamp used in polarimeter is at least 20.

Minutes, then the luminescence can be stable, and then the determination will be more accurate.

9. When measuring the glass at both ends of the smooth surface of the lamp tube, don't touch it by hand, so as to avoid the glass being polluted, affecting the light path and making the measurement inaccurate. If it is not clean, wipe it with a soft cloth or special wiping paper instead of ordinary paper, so as not to damage the glass surface and affect the optical path.

10. Before putting the measuring tube containing the solution to be measured into the sample chamber, make sure that there are no bubbles in the optical path, otherwise it will have a great influence on the optical rotation value of the sample.

1 1. When measuring, the position and direction of the measuring tube should be fixed as far as possible, and marked properly, so as to reduce the stress error of the measuring tube and the cover glass and make the measurement data more accurate.

12. When measuring optical rotation, be sure to record the temperature in time. Because temperature has a certain influence on the optical rotation of matter, the optical rotation measured at different temperatures may be different.

13. When measuring optical rotation, the solvent used must be recorded in time. Because in different solvents, due to the different conditions of association, solvation and dissociation, the specific rotation will change, and even change the optical rotation direction. Chloramphenicol with the structure of 1, 3- propanediol shows antibacterial activity only in isomer (1r, 2r)-(-), which is dextrorotatory in absolute ethanol, with specific rotation of+18.5 ~+21.5, and levorotatory in ethyl acetate.

14. After the measurement, clean and dry the measuring tube, and then put it back. The data should be written in a notebook immediately, and it is best to write the optical rotation data in the literature in a notebook and compare it with the obtained data.

Part II: Matters needing attention in nuclear magnetic detection.

1. The nuclear magnetic resonance vibration should be measured immediately after the product is obtained, but not after long-term storage, because long-term storage may lead to product deterioration. In an extreme case, the product will be isomerized after long-term storage, such as trans isomer becoming cis isomer, or rearrangement reaction, resulting in inaccurate NMR data.

2. The inner diameter of the nuclear magnetic tube used for general laboratory test is 5.

Mm (3 mm sample tube is used for trace samples) The sample volume dissolved by deuterated reagent is about 3.5 cm high, and it is 0.5 in nuclear magnetic tube.

ML, too little solvent will affect the shimming, and too much solvent will cause waste.

3. The amount of samples required for hydrogen spectrum test is less, about 3-7 mg, and the amount of samples required for carbon spectrum test is more, generally more than 20.

Mg, when the concentration is too low, the noise is large and the baseline is uneven; If the concentration is too high, the peak distribution is not good, please master it properly; When testing 13C, high concentration can shorten the testing time, with low noise, straight baseline and beautiful spectrogram. If 1H and 13C spectra are performed at the same time, it is necessary to ensure that the number of samples in the pipeline is greater than 20.

mg .

4. In order to ensure the quality of the spectrogram, the nuclear magnetic tube must be cleaned, the higher the purity of the sample, the better, and the residual solvent must be removed, otherwise it will seriously affect the analysis of the spectrogram. The sample has good solubility in deuterated reagent (the sample sender should choose a suitable solvent in advance), and the dissolved solution is even and transparent. If there are solid particles, they must be filtered first, otherwise the instrument cannot detect them, and the sample must not contain paramagnetic material.

5. The nuclear magnetic tube should be cleaned in time. First of all, the solvent with the best solubility to the sample should be added for washing, and acetone should generally be used. After washing for 2-3 times, clean it with a nuclear magnetic tube brush (cotton is tied at the end), and then clean it with acetone. It is best not to put the nuclear magnetic tubes flat in the drying oven, but to put several nuclear magnetic tubes vertically together, so as to ensure that the nuclear magnetic tubes will not deform at high temperature.

6. In some samples, the energy barrier of the conversion of two conformational isomers is high at room temperature, and the equilibrium cannot be reached quickly. At this time, some broad peaks will appear on the NMR spectrum, or even disappear or not appear. At this time, if the temperature of nuclear magnetic resonance is increased, the isomers of the two conformational isomers can reach equilibrium quickly (see J.

Org。 Chemistry. July, 2005, 1679), so as to obtain the normal nuclear magnetic resonance spectrum.

Part III: Matters needing attention in the test of enantiomeric excess (ee value)

When determining the enantiomeric excess (ee value) by HPLC, a small number of samples are needed, but racemic samples are needed as control, otherwise the peaks of impurities and samples can not be distinguished (especially when ee value is high). At the same time, we should also pay attention to the following aspects:

1. The required sample size on the chromatogram is 80-200.

MAU is better, too few noise peaks are easy to cause interference, too many may stay on the column, and the signal peak intensity should not exceed 500 mAU.

To prevent the chromatographic column from clogging. Before the test, you can use a capillary to spot samples on the silica gel plate and use an ultraviolet lamp to develop color, and then observe whether the concentration is appropriate according to experience.

2. The purity of the sample should be high, otherwise the impurities may completely or partially coincide with the target peak, resulting in inaccurate measurement data and inability to obtain a beautiful chromatogram; In addition, if the sample contains more impurities, especially powdery impurities, it will block the chiral column. Therefore, when preparing a sample, the sample should be completely dissolved or filtered with a filter to obtain a clear solution.

3. Residual solvent in the sample should be removed before preparation to prevent beautiful chromatogram from being obtained due to high solvent peak. It is best to choose the same solvent as the mobile phase or a solvent with better solubility in the mobile phase.

4. When preparing the sample, after the sample is completely dissolved, take one or two drops and transfer them to a special liquid measuring bottle, and then add solvent to dilute them to a suitable concentration. For solid samples, the samples must be completely dissolved before preparation, because in solid samples, the ee values of different parts may be different (for example, racemic compounds, some parts may be 100%).

Ee, the other part may be 0%

Ee), which may lead to poor data repeatability. Therefore, for solid samples, it is necessary to dissolve all the samples, take a proper amount in the sample bottle, and then dilute it to the required concentration.

5. Generally, samples need to be prepared on site, which is easy to deteriorate after being left for a long time.

6. When using chiral column to determine HPLC data, it should be noted that the column pressure should not exceed 50.

Bars. For example, when isopropanol/n-hexane is used as the mobile phase, when the solvent volume ratio is 90/ 10, the flow rate generally does not exceed 1.0 mL/min, and when it is 80/20, the flow rate generally does not exceed 0.8 mL/min.

When more polar fluidity is selected, the flow rate should be reduced accordingly so that the column pressure does not exceed 50.

Bar.

7. When measuring samples, pay attention to the volume change of mobile phase. When it is less than 20% of the total volume, it should be added in time to prevent gas from entering the system.

8. The measured data should be recorded in the experimental record book and the special HPLC data record book in time.

The above are the matters needing attention in the determination of optical rotation, nuclear magnetic resonance and ee value summarized from experience. To obtain scientific experimental data, please refer to the above experience. The principle is: collect all necessary data as soon as possible. For unknown compounds, 1 H NMR, high resolution mass spectrometry or elemental analysis, melting point (if any), optical rotation (if any) and infrared spectrum should be collected; For known compounds, NMR hydrogen and carbon spectra, melting point and optical rotation (if any) should be collected. Please correct me if there are any deficiencies or imperfections.