Ultraviolet-visible spectrophotometry is a method for measuring the absorbance of substances in the wavelength range of 190-800nm for identification, impurity inspection and quantitative determination. When light passes through the solution of the substance to be tested, the degree of absorption of light by the substance varies with the wavelength of the light. Therefore, by measuring the absorbance of the substance at different wavelengths, and drawing the relationship between the absorbance and the wavelength, the absorption spectrum of the substance to be measured can be obtained. From the absorption spectrum, the maximum absorption wavelength λmax and the minimum absorption wavelength λmin can be determined. The absorption spectrum of a substance has characteristics related to its structure. Therefore, the substance can be identified by comparing the spectrum of the sample with the control spectrum or the spectrum of the reference substance in a specific wavelength range, or by determining the maximum absorption wavelength, or by measuring the absorption ratio at two specific wavelengths. When used for quantification, measure the absorbance of a certain concentration of sample solution at the maximum absorption wavelength and compare it with the absorbance of a certain concentration of control solution or use the absorption coefficient method to calculate the concentration of the sample solution.

Calibration and verification of the instrument

1. Wavelength Due to the influence of environmental factors on the mechanical parts, the wavelength of the instrument often changes slightly. Therefore, in addition to the comprehensive calibration of the instrument used regularly, the measurement wavelength should be calibrated before the measurement. The stronger spectral lines in common mercury lamps are 237.83nm, 253.65nm, 275.28nm, 296.73nm, 313.16nm, 334.15nm, 365.02nm, 404.66nm, 435.83nm, 546.07nm and 576.96nm; or use the 486.02 of the deuterium lamp in the instrument The holmium glass has sharp absorption peaks at the wavelengths of 279.4nm, 287.5nm, 333.7nm, 360.9nm, 418.5nm, 460.0nm, 484.5nm, 536.2nm and 637.5nm, and can also be used for wavelength correction. It is used, but due to different sources or slight changes over time, pay attention when using it; in recent years, holmium perchlorate solution is often used to calibrate dual-beam instruments, and 10% perchloric acid solution is used as a solvent to prepare Holmium oxide (Ho2O3) 4% solution, the absorption peak wavelength of the solution is 241.13nm, 278.10nm, 287.18nm, 333.44nm, 345.47nm, 361.31nm, 416.28nm, 451.30nm, 485.29nm, 536.64nm and 640.52nm.

The allowable error of the wavelength of the instrument is: ±1nm in the ultraviolet region, ±2nm near 500nm.

• The accuracy of the absorbance can be verified with a sulfuric acid solution of potassium dichromate. Take about 60mg of standard potassium dichromate dried to constant weight at 120℃, accurately weigh it, dissolve it with 0.005mol/L sulfuric acid solution and dilute to 1000ml, measure and calculate its absorption coefficient at the specified wavelength, and compare it with the specified The absorption coefficient comparison should meet the requirements in the table.

Wavelength/nm 235 (minimum) 257 (maximum) 313 (minimum) 350 (maximum)

Wavelength/nm                               235(Min.)      257(Max)      313(Min.)    350(Max)

absorption coefficient()specified value         124.5          144.0         48.6          106.6

absorption coefficient（）tolerance range    123.0～126.0   142.8～146.2  47.0～50.3    105.5～108.5

• Stray light inspection can be prepared into an aqueous solution with the reagents and concentrations listed in the following table, placed in a 1cm quartz absorption cell, and the light transmittance measured at the specified wavelength should meet the requirements in the table.

  Reagent        concentration/%（g/ml）        Measuring wavelength/nm        luminousness /%

sodium iodide         1.00                          220                           <0.8

sodium nitrite         5.00                          340                           <0.8

Requirements for solvents

Organic solvents containing heteroatoms usually have strong end absorption. Therefore, when used as a solvent, their use range cannot be smaller than the cut-off wavelength. For example, the cut-off wavelength of methanol and ethanol is 205nm. In addition, when the solvent is not pure, interference absorption may also increase. Therefore, before measuring the test product, you should first check whether the solvent used meets the requirements near the wavelength used by the test product, that is, put the solvent in a 1cm quartz absorption cell and use air as a blank (that is, no substance is placed in the blank optical path) Its absorbance. The absorbance of the solvent and absorption cell shall not exceed 0.40 in the range of 220-240nm, 0.20 in the range of 241-250nm, 0.10 in the range of 251-300nm, and 0.05 when it is above 300nm.

Determination method

    When determining, unless otherwise specified, the same batch of solvent used to prepare the test solution should be used as a blank control, and a 1cm quartz absorption cell should be used to test the absorbance at several points within the specified absorption peak wavelength ± 2nm, or by the instrument Automatic scanning measurement near the specified wavelength to check whether the absorption peak wavelength position of the test product is correct. Unless otherwise specified, the absorption peak wavelength should be within ±2nm of the wavelength specified under the item, and the wavelength with the highest absorbance shall be used as the measurement wavelength. Generally, the absorbance reading of the test solution should be between 0.3 and 0.7. The width of the slit band of the instrument should be less than one-tenth of the half-height width of the absorption band of the test product, otherwise the measured absorbance will be low; the choice of slit width should be based on reducing the width of the test product The absorbance is no longer subject to increase. Since the absorption cell and the solvent itself may have blank absorption, the blank reading should be subtracted after the absorbance of the test product is measured, or the blank reading should be automatically deducted by the instrument to calculate the content.

When the pH value of the solution has an impact on the measurement result, the pH value of the test solution and the pH value of the reference solution should be adjusted to the same.

    1. Identification and inspection shall be carried out in accordance with the methods specified under each category.

    2. Content determination There are generally the following methods.

    (1) Reference substance comparison method According to the method under each category, prepare the test solution and the reference solution respectively. The amount of the tested component contained in the reference solution should be the specified amount of the tested component in the test solution 100%±10%, the solvents used should also be exactly the same. After measuring the absorbance of the test solution and the reference solution at the specified wavelength, calculate the concentration of the test solution in the test product as follows:

 c_X=（A_X/A_R）c_R

Where c_X is the concentration of the test solution;

    A_X is the absorbance of the test solution;

    c_R is the concentration of the reference solution;

    A_R is the absorbance of the reference solution.

(2) Absorption coefficient method Prepare the test solution according to the method under each species, measure its absorbance at the specified wavelength, and then calculate the content based on the absorption coefficient of the species under the specified conditions. When measuring with this method, the absorption coefficient should usually be greater than 100, and attention should be paid to the calibration and verification of the instrument.

 (3) Calculating spectrophotometric method There are many kinds of calculating spectrophotometric method, and they should be used in accordance with the methods specified under each category. When the absorbance is measured at the abrupt rise or fall of the absorption curve, the small change in wavelength may have a significant impact on the measurement result, so the test conditions of the reference substance and the test substance should be as consistent as possible. Computational spectrophotometry is generally not suitable for content determination.

(4) Colorimetric method The colorimetric test product itself does not have strong absorption in the ultraviolet-visible light region, or although it has absorption in the ultraviolet light region, in order to avoid interference or improve sensitivity, an appropriate color developer can be added to shift the maximum absorption of the reaction product. To the visible light region, this measurement method is called colorimetry.

    When using the colorimetric method, because there are many factors that affect the depth of the color during color development, the test product and the reference product or standard product should be taken at the same time. Unless otherwise specified, the blank used in the colorimetric method refers to the use of the same volume of solvent instead of the reference substance or test solution, and then the same amount of corresponding reagents are added in sequence, and the same method is used. After measuring the absorbance of the reference substance and the test solution at the specified wavelength, calculate the concentration of the test substance according to the method (1) above.

When the relationship between absorbance and concentration does not show a good linearity, you should take several gradients of the reference solution and add the solvent to the same volume. After color development, the absorbance of each solution is measured, and then a standard curve is drawn based on the absorbance and the corresponding concentration. Then check the corresponding concentration on the standard curve according to the absorbance of the test product, and find its content.