First, the equipment
721, 723 visible spectrophotometer, 752 UV-visible spectrophotometer, UV-2401PC UV-visible spectrophotometer, UV-3150PC UV-visible near-infrared spectrophotometer, RF-5301PC fluorescence spectrophotometer, colorimetric solution and standard solution, Filter paper, etc.
Second, the instrument structure
Third, the principle spectroscopy instrument is a simple and easy to use spectrophotometric method for the determination of general-purpose instruments, different types of machines to determine the wavelength range is different, you can choose the instrument to use according to your own needs.
The construction of different types of spectroscopic instruments is quite different, but the basic principles are the same. Depending on the absorbance of the transmitted light by various components in the solution, the absorbance (or transmittance) is proportional to the concentration of the solute within a certain range (Bohr's law). By plotting the exact concentration of the standard against its absorbance (transmittance), we can get a standard curve. The absorbance (transmittance) of the sample to be tested at an unknown concentration is measured, and the concentration is obtained by comparison with a standard curve.
Fourth, the scope of application can be widely used in medical and health, clinical testing, biochemistry, petrochemical, environmental testing, quality control and other aspects for quantitative and qualitative analysis.
Specific to our laboratory, qualitative, quantitative measurement of polysaccharides, proteins, nucleic acids and other substances. At the same time, UV-3150PC UV-visible near-infrared spectrophotometer can also measure the absorption spectrum of solid and liquid samples in the near-infrared region. RF-5301PC fluorescence spectrophotometer can measure the luminous intensity spectrum of fluorescent substances, and their own software can be convenient. Data processing and analysis.
V. Operation steps We use UV-3150PC UV-visible near-infrared spectrophotometer as an example to describe the performance and operation method of the spectrometer.
1. Plug in the power and turn on the power switch on the front of the photometer.
2. Double-click the UVProbe icon on the computer desktop to open the UVProbe software. Click the “Connect†icon in the UVProbe to bring up the self-test screen.
3. If the green light is on in the self-test, click “OK†to enter the system. If the red light is on, turn off the photometer and reconnect. If the self-test has not passed, report the instrument to the teacher immediately.
4. Fully pass and enter the system, the UVProbe menu bar and toolbar appear. UVProbe has four main modules, including a spectral scanning module, a photometric module, a time scanning module, and a report generator. Each mode can be directly clicked on the method setting button setting method.
5. Click the spectrum scan icon to display the spectrum scan interface.
6. Click the Method button and the Method Settings dialog box appears. Here you can set the scanning wavelength range, the maximum range of the instrument is 3200 ~ 190nm. The scan speed can be set, and the medium or medium (Normal) can be selected for scanning. The sampling interval indicates how many nm times the photometer reads an absorbance value. If there is no special requirement, select Auto. You can choose whether to repeat the scan curve, and if it is repeated, set the number of repetitions and interval time.
7. Click on “Sample Preparation†to enter sample information. Including sample quality, volume, dilution factor and measuring optical path. You can also enter additional information about the sample.
8. Click on “Instrument Parameters†to set the measurement method and some parameters of the instrument. In the measurement mode, "Transmittance", "Absorbance", "Energy" and "Reflectance" can be selected, and Reflectance is generally used when measuring a solid sample with an integrating sphere. The slit is set to 5 nm at 3200 to 800 nm and 2 nm at 800 to 190 nm. If the wavelengths including the two ranges are 1500 to 500 nm, the slit is selected to be 5 nm.
9. The lamp and detector can be set according to the sample to be tested. The lamp can be changed in any range from 393 to 282 nm. The detector can be set to a value in the range of 895 to 750 nm. However, due to the mechanical movement of the instrument and the detector, the signal fluctuation is large, and the instrument noise is large. At this time, the scanning curve error is large, so the wavelength should be avoided at the absorption peak position of the sample to avoid affecting the measured value.
10. After all the parameters are set, the two sample holders are not placed with the sample or both are placed with the same blank solution. Click “ba seline†to start the baseline calibration. After the instrument scan is completed, click “λGoToWL†to turn the wavelength. At 750 nm, click on "Auto Zero" to set the wavelength of 750 nm to zero. Click “Start†to scan again. If the maximum value of the curve is less than 0.001, the baseline is considered flat. If the value is larger, the absorbance at 750 nm is set to 0 and the baseline scan is performed again.
11. After the baseline calibration is completed, take out the blank solution of the sample holder, put in the sample solution, and click “Start†to start scanning the curve. After the scan is completed, the save path box and file name box appear, and the scan curve appears after the setting.
12. After the curve scan is completed, click the right mouse button on the spectrum to display the shortcut menu. Click on "AutoScale" but the software automatically adjusts the image coordinates to fit the scanned data. Click on the “Cross Hair>Display†mouse to display the cross line. Use the mouse to place the cross line on a point on the curve to display the wavelength and absorbance value of the point.
13. Click on "Label" to add a label to the spectrum where you can enter text to mark the spectrum. Click “Legend†to display the data save map. If the spectrum data is checked in the front box, the curve will be displayed on the left overlay.
14. If you select “Customize†from the shortcut menu, you can customize the curve display color and define the value of the axis.
15. After the curve is obtained, a series of processing can be performed on the spectrum.
(1) Click the “DataPrint†icon to display the spectrum curve directly as a value.
(2) Click the “Manipulate†icon to perform addition, subtraction, multiplication and division, difference spectrum, blank subtraction, reciprocal spectrum, logarithmic spectrum and so on.
(3) Click the “PeakPoint†button to display the values ​​of “Crest†and “Valleyâ€.
(4) Click the “PointPick†icon, enter the wavelength value to be displayed, and immediately display the absorbance value at the input wavelength.
(5) Click “PeakArea†to set the peak threshold, which shows the peak area larger than this threshold.
(6) Click on the “Sewingbox†icon to crop and stitch the spectrum.
16. After the spectral curve scan is completed, the data is actually saved directly in the memory and is not saved to the hard disk. If the window is closed, the computer will prompt “Some Spectrum data has not been saved! Do you still want to exit and lostunsaved change? (And the spectral data has not been saved yet, are you leaving without saving the data?) "Select No" to return to the main interface to save all the data.
Six, matters needing attention
1. The photometer lamp source has a limited life. If it is not measured for a long time, it should be disconnected by UVProbe software (click “Disconnectâ€), then turn off the photometer power supply.
2. Use spectrophotometer to ensure that the sample chamber is absolutely clean. Carefully put the sample into the sample. Before putting it into the cuvette, wipe the outer surface of the cuvette with filter paper and mirror paper. Do not contaminate the sample cell and the photometer. surface.
3. Do not open the sample chamber cover during the self-test and scanning of the instrument.
4. The software does not automatically save the data. All data must be saved by clicking “Save†or “SaveAsâ€. Otherwise the data will be lost.
5, carefully fill in the use of valuable equipment records.
721, 723 visible spectrophotometer, 752 UV-visible spectrophotometer, UV-2401PC UV-visible spectrophotometer, UV-3150PC UV-visible near-infrared spectrophotometer, RF-5301PC fluorescence spectrophotometer, colorimetric solution and standard solution, Filter paper, etc.
Second, the instrument structure
The construction of different types of spectroscopic instruments is quite different, but the basic principles are the same. Depending on the absorbance of the transmitted light by various components in the solution, the absorbance (or transmittance) is proportional to the concentration of the solute within a certain range (Bohr's law). By plotting the exact concentration of the standard against its absorbance (transmittance), we can get a standard curve. The absorbance (transmittance) of the sample to be tested at an unknown concentration is measured, and the concentration is obtained by comparison with a standard curve.
Fourth, the scope of application can be widely used in medical and health, clinical testing, biochemistry, petrochemical, environmental testing, quality control and other aspects for quantitative and qualitative analysis.
Specific to our laboratory, qualitative, quantitative measurement of polysaccharides, proteins, nucleic acids and other substances. At the same time, UV-3150PC UV-visible near-infrared spectrophotometer can also measure the absorption spectrum of solid and liquid samples in the near-infrared region. RF-5301PC fluorescence spectrophotometer can measure the luminous intensity spectrum of fluorescent substances, and their own software can be convenient. Data processing and analysis.
V. Operation steps We use UV-3150PC UV-visible near-infrared spectrophotometer as an example to describe the performance and operation method of the spectrometer.
1. Plug in the power and turn on the power switch on the front of the photometer.
2. Double-click the UVProbe icon on the computer desktop to open the UVProbe software. Click the “Connect†icon in the UVProbe to bring up the self-test screen.
3. If the green light is on in the self-test, click “OK†to enter the system. If the red light is on, turn off the photometer and reconnect. If the self-test has not passed, report the instrument to the teacher immediately.
4. Fully pass and enter the system, the UVProbe menu bar and toolbar appear. UVProbe has four main modules, including a spectral scanning module, a photometric module, a time scanning module, and a report generator. Each mode can be directly clicked on the method setting button setting method.
5. Click the spectrum scan icon to display the spectrum scan interface.
6. Click the Method button and the Method Settings dialog box appears. Here you can set the scanning wavelength range, the maximum range of the instrument is 3200 ~ 190nm. The scan speed can be set, and the medium or medium (Normal) can be selected for scanning. The sampling interval indicates how many nm times the photometer reads an absorbance value. If there is no special requirement, select Auto. You can choose whether to repeat the scan curve, and if it is repeated, set the number of repetitions and interval time.
7. Click on “Sample Preparation†to enter sample information. Including sample quality, volume, dilution factor and measuring optical path. You can also enter additional information about the sample.
8. Click on “Instrument Parameters†to set the measurement method and some parameters of the instrument. In the measurement mode, "Transmittance", "Absorbance", "Energy" and "Reflectance" can be selected, and Reflectance is generally used when measuring a solid sample with an integrating sphere. The slit is set to 5 nm at 3200 to 800 nm and 2 nm at 800 to 190 nm. If the wavelengths including the two ranges are 1500 to 500 nm, the slit is selected to be 5 nm.
9. The lamp and detector can be set according to the sample to be tested. The lamp can be changed in any range from 393 to 282 nm. The detector can be set to a value in the range of 895 to 750 nm. However, due to the mechanical movement of the instrument and the detector, the signal fluctuation is large, and the instrument noise is large. At this time, the scanning curve error is large, so the wavelength should be avoided at the absorption peak position of the sample to avoid affecting the measured value.
10. After all the parameters are set, the two sample holders are not placed with the sample or both are placed with the same blank solution. Click “ba seline†to start the baseline calibration. After the instrument scan is completed, click “λGoToWL†to turn the wavelength. At 750 nm, click on "Auto Zero" to set the wavelength of 750 nm to zero. Click “Start†to scan again. If the maximum value of the curve is less than 0.001, the baseline is considered flat. If the value is larger, the absorbance at 750 nm is set to 0 and the baseline scan is performed again.
11. After the baseline calibration is completed, take out the blank solution of the sample holder, put in the sample solution, and click “Start†to start scanning the curve. After the scan is completed, the save path box and file name box appear, and the scan curve appears after the setting.
12. After the curve scan is completed, click the right mouse button on the spectrum to display the shortcut menu. Click on "AutoScale" but the software automatically adjusts the image coordinates to fit the scanned data. Click on the “Cross Hair>Display†mouse to display the cross line. Use the mouse to place the cross line on a point on the curve to display the wavelength and absorbance value of the point.
13. Click on "Label" to add a label to the spectrum where you can enter text to mark the spectrum. Click “Legend†to display the data save map. If the spectrum data is checked in the front box, the curve will be displayed on the left overlay.
14. If you select “Customize†from the shortcut menu, you can customize the curve display color and define the value of the axis.
15. After the curve is obtained, a series of processing can be performed on the spectrum.
(1) Click the “DataPrint†icon to display the spectrum curve directly as a value.
(2) Click the “Manipulate†icon to perform addition, subtraction, multiplication and division, difference spectrum, blank subtraction, reciprocal spectrum, logarithmic spectrum and so on.
(3) Click the “PeakPoint†button to display the values ​​of “Crest†and “Valleyâ€.
(4) Click the “PointPick†icon, enter the wavelength value to be displayed, and immediately display the absorbance value at the input wavelength.
(5) Click “PeakArea†to set the peak threshold, which shows the peak area larger than this threshold.
(6) Click on the “Sewingbox†icon to crop and stitch the spectrum.
16. After the spectral curve scan is completed, the data is actually saved directly in the memory and is not saved to the hard disk. If the window is closed, the computer will prompt “Some Spectrum data has not been saved! Do you still want to exit and lostunsaved change? (And the spectral data has not been saved yet, are you leaving without saving the data?) "Select No" to return to the main interface to save all the data.
Six, matters needing attention
1. The photometer lamp source has a limited life. If it is not measured for a long time, it should be disconnected by UVProbe software (click “Disconnectâ€), then turn off the photometer power supply.
2. Use spectrophotometer to ensure that the sample chamber is absolutely clean. Carefully put the sample into the sample. Before putting it into the cuvette, wipe the outer surface of the cuvette with filter paper and mirror paper. Do not contaminate the sample cell and the photometer. surface.
3. Do not open the sample chamber cover during the self-test and scanning of the instrument.
4. The software does not automatically save the data. All data must be saved by clicking “Save†or “SaveAsâ€. Otherwise the data will be lost.
5, carefully fill in the use of valuable equipment records.
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