Spectrophotometer, also known as spectrometer, is a scientific instrument that decomposes complex light into spectral lines.The measurement range generally includes the visible light area with a wavelength range of 380~780 nm and the ultraviolet light area with a wavelength range of 200~380 nm.Different light sources have their own emission spectra, so different illuminants can be used as the light source of the instrument.Emission spectrum of tungsten lamp: spectral light of 380 ～ 780nm emitted by tungsten lamp light source passes throughprismAfter refraction, a continuous chromatography consisting of red, orange, yellow, green, blue, indigo and purple can be obtained;This chromatography can be used asVisible spectrophotometerThe light source of.
SpectrophotometryIt refers to the absorbance of light at a specific wavelength or within a certain wavelength range to conduct qualitative or quantitative analysis of the substance.The commonly used wavelength range is: (1) 200 ～ 380nm ultraviolet light area, (2) 380 ～ 780nm ultraviolet light areavisible light(3) 2.5 ～ 25 μ m (4000cm<- 1>～ 400cm<- 1>according to wave number) infrared light area.The instruments used are ultraviolet spectrophotometerVisible spectrophotometer(orColorimeter）、Infrared spectrophotometerorAtomic absorption spectrophotometer。To ensure the measuredPrecisionandAccuracy, allinstrumentAccording to the countryMetrological verification regulationOr as specified in this appendix, calibration and verification shall be carried out regularly.
Instrument composition
Spectrophotometer has become a conventional instrument in modern molecular biology laboratory.Commonly used fornucleic acid, protein quantification and bacterial growth concentration quantification.
The instrument is mainly composed of light source, monochromatorSample Room, detector, signal processor and display andstorage system form.
Spectral range
It includes the visible light area with a wavelength range of 400-760 nm and the ultraviolet light area with a wavelength range of 200-400 nm.Different light sources have their own emission spectra, so different illuminants can be used as the light source of the instrument.
Emission spectrum of tungsten lamp: spectral light of 400-760nm wavelength emitted by tungsten lamp light source passes throughprismAfter refraction, continuous chromatography consisting of red orange, yellow green, indigo and purple can be obtained;The chromatogram can be used as the light source of the visible spectrophotometer.
Emission spectrum of hydrogen lamp (or deuterium lamp): the hydrogen lamp can emit a spectrum of 185~400 nm wavelength, which can be used as the light source of the ultraviolet photometer.
Absorption spectrum of the substance:
If a solution of some substance is placed between the light source and the prism, the spectrum displayed on the screen is no longer the spectrum of the light source, but there are several dark lines, that is, the light of some wavelengths in the emission spectrum of the light source disappears due to the absorption of the solution. This spectrum absorbed by the solution is called the absorption spectrum of the solution.
The absorption spectra of different substances are different, so the substances contained in the solution can be identified according to the absorption spectra.
When the light passes through the solution of a certain substance, the intensity of the transmitted light decreases, because some of the light is reflected or dispersed on the surface of the solution, and some of the light is absorbed by the substance that makes up the solution, only some of the light can pass through the solution.
If we use distilled water (or the solvent constituting this solution) as the "blank" to correct the loss of incident light caused by reflection, dispersion and other factors, then:
Incident light=absorbed light+transmitted light
principle
The spectrophotometer uses a light source that can generate multiple wavelengths. Through a series of spectroscopic devices, a light source of specific wavelength is generated. After the light passes through the test sample, part of the light is absorbed, and the absorbance value of the sample is calculated, which is converted into the concentration of the sample.The absorbance value of the sample is proportional to the concentration of the sample.
When the monochromatic light radiation passes through the solution of the measured substance, the amount absorbed by the substance is proportional to the concentration of the substance and the thickness of the liquid layer (the length of the optical path), and the relationship is as follows:
A=-lg(I/Izero)=-lgT=kLc
Where: A is the absorbance;
IzeroIs the intensity of incident monochromatic light;
I is the intensity of transmitted monochromatic light;
L is the optical path of the analyte, that is, the side length of the cuvette;
C is the concentration of the substance;
The selective absorption wavelength of a substance to light and the corresponding absorption coefficient are the physical constants of the substance.When the absorption coefficient of a pure substance under certain conditions is known, the test article can be prepared into a solution under the same conditions, and its absorption can be measured, then the content of the substance in the test article can be calculated from the above formula.In the visible light area, except that some substances absorb light, many substances do not absorb light themselves, but can be added with color developing reagents under certain conditions or treated to develop color before measurement, so it is also calledColorimetric analysis。Because there are many factors that affect the color depth during color rendering, and monochromatic light is often usedpurityPoor instrument, so the standard or reference substance shall be used for simultaneous operation during measurement.
Instrument characteristics
The unique double light path and double beam optical system makes the instrument have higher resolution, lower stray light, stronger stability and reliability, and more accurate analysis;
The 320 * 240 matrix high brightness 6 "LCD display is adopted, with clear display and complete information;
The unique design of long optical path makes the resolution of the instrument higher, especially suitable for the powerful data processing function of micro test, so that the test results can be fully applied, and users can edit more easily and quickly;
The suspension optical system design is adopted, and the overall optical path is independently fixed on the 16mm thick aluminum deformation free base. The deformation of the base plate and the external vibration have no impact on the optical system, thus greatly improving the stability and reliability of the instrument;
Synchronous sine mechanism is adopted, with high wavelength accuracy and good repeatability;
Adopt ARM system;
The six band spectral bandwidth of 0.1/0.2/0.5/1.0/2.0/4.0 is automatically selectable to meet the measurement needs of different users;
The main components are imported, which makes the instrument less stray light, more stable and more reliable;
More powerful functions, the host can independently complete photometric measurement, quantitative measurement, spectral scanning, dynamics, DNA/protein testing, multi wavelength testing, data printing and other functions;
Fully considering the usage habits of different users, this series of instruments are equipped with spectrum scanning software of Meta Analysis Company as standard. When operating online, in addition to all the testing functions of the host, it can also achieve more powerful data processing functions, and make data storage unlimited.
Instrument index
Model UV-9000 Wavelength range 190-900nm;The spectral bandwidth is 0.1/0.2/0.5/1.0/2.0/4.0nm, with six selectable wavelengths, and the accuracy is ± 0.1nm (D2 656.1nm), ± 0.3nm;The wavelength repeatability of the whole area is ≤ 0.1nm.
Photometric accuracy: ± 0.2% T
Photometric repeatability: ≤ 0.1% T
Stray light: ≤ 0.01% T
Stability: ± 0.0004A/h (at 500nm)
Baseline straightness: ± 0.001A
Noise level: ± 0.0004A/h
Photometric range: 0-200% T
Power supply: AC 220V/50Hz or 110V/60Hz
Weight: 30kg
Technical parameters:
Wavelength range: 320~1000nm
Spectral bandwidth: 4nm
Stray light: ≤ 0.5% (at 360nm)
Wavelength accuracy: better than ± 2nm
Transmittance accuracy: ≤± 1nmT
Common use
Quantification of nucleic acid
The quantification of nucleic acid is the most frequently used function of spectrophotometer.Oligonucleotides, single stranded DNA, double stranded DNA, and RNA that can be quantitatively dissolved in buffer solution.The absorption wavelength of the highest absorption peak of nucleic acid is 260 nm.The molecular composition of each nucleic acid is different, so itsConversion factorDifferent.To quantify different types of nucleic acids, the corresponding coefficients should be selected in advance.For example, the absorbance value of 1OD is equivalent to 50 μ g/ml dsDNA, 37 μ g/ml ssDNA, 40 μ g/ml RNA and 30 μ g/ml Olig respectively.The absorbance value after the test is converted by the above coefficient to obtain the corresponding sample concentration.Before the test, select the correct program, input the volume of stock solution and diluent, and then test the blank solution and sample solution.However, the experiment was not smooth.Unstable readings may be the most troublesome problem for the experimenter.The higher the sensitivity of the instrument, the greater the absorption value drift.
In fact, the design principle and working principle of the spectrophotometer allow the absorbance value to change within a certain range, that is, the instrument has certain accuracy and precision.For example, the accuracy of Eppendorf Biophotometer is ≤ 1.0% (1A).It is normal for the results of such multiple tests to vary between 1.0% of the average value.In addition, it is also necessary to consider the physical and chemical properties of nucleic acid itself, pH value of buffer solution for dissolving nucleic acid, ion concentration, etc.: when testing, too high ion concentration will also cause reading drift, such as TE, which can greatly stabilize the reading.The dilution concentration of the sample is also a factor that cannot be ignored: because there are inevitably some small particles in the sample, especially nucleic acid samples.The presence of these small particles interferes with the test results.In order to minimize the impact of particles on the test results, it is required that the nucleic acid absorbance value should be at least greater than 0.1A, and the absorbance value should preferably be 0.1-1.5A.Within this range, there shall be no bubbles in the mixed solution and no suspended solids in the blank solution, otherwise the reading will drift violently;The same cuvette must be used to test the blank solution and sample, otherwise the concentration difference is too large;The conversion factor is consistent with the sample concentration unit;The cuvette with worn window cannot be used;The volume of the sample must reach the minimum volume required by the cuvette.
In addition to the nucleic acid concentration, the spectrophotometer displays several very important ratios at the same time to indicate the purity of the sample, such as the A260/A280 ratio, which is used to evaluate the purity of the sample, because the absorption peak of the protein is 280 nm.For pure samples, the ratio is greater than 1.8 (DNA) or 2.0 (RNA).If the ratio is lower than 1.8 or 2.0, it indicates the presence of protein or phenolic substances.A230 indicates that there are some pollutants in the sample, such as carbohydrate, polypeptide, phenol, etc. The ratio of relatively pure nucleic acid A260/A230 is greater than 2.0.A320 Test the turbidity and other interference factors of the solution.For pure samples, A320 is generally 0.
Direct quantification of protein (UV method)
This method is to directly test the protein at the wavelength of 280 nm.If Warburg formula is selected, the photometer can directly display the sample concentration, or select the corresponding conversion method to convert the absorbance value to the sample concentration.Protein determinationThe process is very simple. First test the blank solution, then directly test the protein.Since there are some impurities in the buffer solution, the "background" information of 320nm should be removed and this function should be set to "ON".Similar to the nucleic acid test, the absorbance value of A280 is required to be at least greater than 0.1A, and the optimal linear range is between 1.0-1.5.When Warburg formula is selected to display the sample concentration in the experiment, it is found that the reading is "drifting".This is a normal phenomenon.In fact, as long as the absorbance value of A280 is observed to change within 1%, the result is very stable.The reason for the drift is that the absorbance value of Warburg formula is converted into concentration and multiplied by a certain coefficient. As long as the absorbance value is slightly changed, the concentration will be amplified, which makes the result very unstable.The direct quantitative method of protein is suitable for testing pure and relatively single protein.Compared with colorimetry, UV direct quantitative method is fast and easy to operate;But it is vulnerable to the interference of parallel substances, such as DNA;In addition, the sensitivity is low and the concentration of protein is required to be high.
Colorimetric protein quantification
Protein is usually a mixture of multiple proteins. The basis of colorimetric determination is protein composition: amino acids (such as tyrosine and serine) react with added chromogenic groups or dyes to produce colored substances.The concentration of colored substances is directly related to the number of amino acids in protein reaction, thus reflecting the concentration of protein.
Colorimetric method
Generally, there are BCA, Bradford, Lowry and other methods.
Lowry method: Based on the earliest Biuret reaction and improved.Protein reacts with Cu2 to produce blue reactants.However, compared with Biuret, Lowry method is more sensitive.The disadvantage is that several different reaction reagents need to be added in sequence;The reaction takes a long time;Susceptible to non protein substances;Proteins containing EDTA, Triton x-100, ammonia sulfonate and other substances are not suitable for this method.
BCA (Bicinchonine acid assay) methodThis is a new and more sensitive protein test method.The protein to be analyzed reacts with Cu2 in alkaline solution to produce Cu, which forms a chelate with BCA to form a purple compound, with the absorption peak at 562nm.The linear relationship between this compound and protein concentration is strong, and the compound formed after reaction is very stable.Compared with Lowry method, it is easy to operate and has high sensitivity.But similar to Lowry method, it is vulnerable to interference between proteins and detergent.
Bradford method: The principle of this method is that the protein reacts with Coomassie brilliant blue to produce a 595nm absorption peak of colored compounds.Its biggest feature is its good sensitivity, which is twice as high as that of Lowry and BCA;The operation is simpler and faster;Only one reaction reagent is required;The compound can be stable for 1 hour to facilitate the results;It is also compatible with a series of reducing agents (such as DTT, mercaptoethanol) that interfere with Lowry and BCA reactions.But it is still sensitive to detergent.The main disadvantage is that different standards will lead to large differences in the results of the same sample, which is incomparable.
Some researchers who are exposed to colorimetry for the first time may be puzzled by the inconsistency of the results measured by various colorimetric methods. Which method should they believe?Because the reaction groups and chromogenic groups of various methods are different, the sample concentration obtained by using several methods at the same time for the same sample is not comparable.For example, Keller et al. tested the protein in human milk and found that the concentration measured by Lowry and BCA was significantly higher than Bradford, with significant difference.Even if the same sample is determined by the same colorimetric methodStandard sampleInconsistent, and the concentration after test is also inconsistent.If Lowry is used to test the protein in the cell homogenate, BSA is used as the standard with a concentration of 1.34mg/ml, and a-globulin is used as the standard with a concentration of 2.64mg/ml.Therefore, before choosing the colorimetric method, it is better to refer to the chemical composition of the sample to be tested and look for the standard protein with similar chemical composition as the standard.In addition, the problem often occurs when protein is quantified by colorimetry is that the absorbance value of the sample is too low, resulting in a large gap between the measured sample concentration and the actual concentration.The key problem is that the color of cuvette, an important accessory of 1011 spectrophotometer after reaction, has a certain half-life, so each colorimetric method lists the reaction test time, and all samples (including standard samples) must be tested within this time.If the time is too long, the absorbance value obtained becomes smaller and the converted concentration value decreases.In addition, the reaction temperature and solution PH value are important factors affecting the experiment.In addition, it is very important to use the plastic colorimetry.Avoid usingquartzOr glass cuvette, because the color after reaction will make quartz or glass colored, resulting in inaccurate absorbance value of the sample.
Bacterial cell density (OD 600)
The laboratory determines the growth density and growth period of bacteria, and mostly infers the growth density of bacteria based on experience and visual inspection.In the experiment with high requirements, it is necessary to use a spectrophotometer to accurately determine the bacterial cell density.OD600 is a standard method for tracking microbial growth in liquid cultures.Take the culture medium without bacterial solution as the blank solution, and then quantitatively culture the culture medium containing bacteria.In order to ensure correct operation, it is necessary to count cells with a microscope for each microorganism and each instrument, and make a correction curve.Occasionally in the experiment, the OD value of the bacterial solution appears negative because of the use of a chromogenic culture medium, that is, after a period of bacterial culture, the bacteria react with the culture medium and change color.In addition, it should be noted that the tested samples cannot be centrifuged to keep bacteria suspendedstate。
Important accessory of spectrophotometer -- cuvette
The cuvette can be roughly divided into quartz cup, glass cup and plastic cup according to the material.According to different measurement volumes, there are cuvettes and capillary cuvettes.Generally, quartz cup or glass cup is used to test nucleic acid and UV quantitative protein, but it is not suitable for colorimetric determination.Because the dyes in the reaction (such as Coomassie Brilliant Blue) can color quartz and glass, disposable plastic cups must be used.The plastic cup is generally not suitable for testing samples in the ultraviolet range.
Because the amount of samples tested is different, generally spectrophotometer manufacturers provide cuvettes with different volumes to meet different needs of users.There is already a plastic cup in the market that can be used not only for nucleic acid and UV protein quantification, but also for protein colorimetric determination. The sample consumption is only 50 μ l. The cuvette can be recycled in a single sterile package.E.g. Eppendorf UVette®The plastic cuvette is an innovation in the cuvette market.along withlife sciencesAs well as the development of related disciplines, higher requirements are put forward for the experimental research of this kind of science. The spectrophotometer will be an indispensable instrument in the molecular biology laboratory, as well as one of the necessary equipment in microbiology, food, pharmacy and other related laboratories.
With the development of science and technology, the cuvette is no longer necessary for spectrophotometer.Compared with the old type spectrophotometer, the ND1000 spectrophotometer produced by foreign Nanodrop Company (now purchased by Thermo Fisher Company) can be used without diluting the sample and using the cuvette. Only 1-2 μ l sample is needed for each measurement.[1]
Relevant information
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Operation method
1. Turn on the power supply, turn on the instrument switch, open the sample chamber dark box cover, and preheat for 10 minutes.
2. Turn the sensitivity switch to "1" (If the zero adjuster cannot be adjusted to "0", a higher gear is required.)
3. Turn the wavelength selection button according to the required wavelength.
4. Pour the blank solution and determination solution into 3/4 of the cuvette respectively, and useMirror wiping paperWipe off the outer wall, put it into the sample chamber, and align the blank tube with the optical path.
5. Adjust the zero adjuster when the cover of the darkbox is open, so that the pointer of the reading plate points to t=0.
6. Put on the cover of the darkbox, adjust the "100" regulator to make t=100 of the blank tube, pull out the sample slide rod gradually after the pointer is stable, read out the optical density value of the measuring tube respectively, and record it.
7. After color comparison, turn off the power, take out the cuvette and clean it, and wipe the sample room with soft cloth or paper.
matters needing attention
1. The instrument should be placed in a dry room and on a firm and stable workbench when used. The indoor lighting should not be too strong.In hot weather, it is not allowed to use an electric fan to blow directly to the instrument to prevent the bulb filament from lighting instability.
2. Before using the instrument, users should first understand the structure and working principle of the instrument, as well as the functions of each control knob.Before the power on is not pressed, the safety performance of the instrument should be checked. The power connection should be firm and the power on should be good. The starting position of each adjustment knob should be correct, and then press the power on switch.
3. When the instrument is not connected to the power supply, the pointer of the meter must be on the "0" mark. If this is not the case, you can use the correction screw on the meter to adjust.
Routine maintenance
Analytical instrument workers should understand the daily maintenance of the instrument and the simple test methods for the main technical indicators. They often maintain and test the instrument to ensure that the instrument works in the best state.
1、 Temperature and humidity are important factors affecting the performance of the instrument.They can cause corrosion of mechanical parts, reduce the smoothness of the metal mirror surface, and cause errors or performance degradation of the mechanical part of the instrument;The aluminum film of optical components such as grating, reflector, focusing mirror, etc. is rusted, resulting in insufficient light energy, stray light, noise, etc., and even the instrument stops working, thus affecting the service life of the instrument.It shall be corrected regularly during maintenance.The instrument room with constant humidity throughout the year shall be equipped with constant temperature equipment, especially the laboratory located in the south.
2、 The dust and corrosive gas in the environment can also affect the flexibility of the mechanical system, reduce the reliability of various limit switches, keys and optoelectronic couplers, and is one of the reasons for the corrosion of the aluminum film of the necessary components.Therefore, it must be cleaned regularly to ensure the environment and indoor sanitary conditions and prevent dust.
3、 After the instrument is used for a certain period, a certain amount of dust will accumulate inside. It is better to open the instrument cover regularly by the maintenance engineer or under the guidance of the engineer to remove dust inside, and at the same time, retighten the radiators of each heating element, clean the sealing window of the optical box, calibrate the optical path if necessary, clean and lubricate the mechanical part,Finally, restore the original state, and then carry out some necessary tests, adjustments and records.
Maintenance
As a kind of precision instrument, due to various reasons such as working environment and operation method, the technical condition of spectrophotometer will inevitably change, which may affect the performance of the equipment, and even cause equipment failures and accidents.Therefore, analysts must understand the basic principle and operating instructions of the spectrophotometer, find out and eliminate these hidden dangers in time, and maintain the generated faults in time to ensure the normal operation of the instrument and equipment.
1) If the test wavelength is changed significantly, it is necessary to wait for a while and recalibrate the "0" and "100%" points after the heat balance of the lamp.Then measure.
2) When the pointer instrument is not connected to the power supply, the pointer of the ammeter must be on the zero scale.If this is not the case, mechanical zeroing is required.
3) After using the cuvette, please immediately wash it with distilled water and wipe off the traces of water with clean soft gauze to prevent the surface finish from being damaged and affecting the transmittance of the cuvette.
4) The operator shall not easily move the bulb and reflector lamp to avoid affecting the light efficiency.
5) Model 1900 isospectrophotometer, because its photoelectric receiving device isPhotomultiplier tubeIt can be used to detect weak photoelectric signals instead of strong light because of its large amplification factor.Otherwise, signal drift and sensitivity decrease may occur.In view of the above characteristics, care should be taken not to expose the photomultiplier tube to light for a long time when maintaining and using such instruments. Therefore, when preheating, the cover of the cuvette should be opened or the light barrier rod should be used to prevent its performance from drifting due to long-term exposure, which will lead to unstable operation.
6) The amplifier sensitivity must be reset after shifting.
7) The matching of cuvettes.The cuvette must be used together, otherwise the test results will lose meaning.Comparison shall be made before each test.The specific methods are as follows:;Respectively inject the same solution into the two cups to be measured, place the instrument at a certain wavelength, quartz cuvette;Distilled water is filled at 220nm and 700nm, and distilled water is filled at 700nm in the glass cuvette. The transmission ratio of one cell is adjusted to 100%, the transmission ratios of other cells are measured, and the difference between the indicated values and the light transmission direction are recorded. If the difference between the transmission ratios is within ± 0.5%, it can be used together. If it is beyond this range, its impact on the test results should be considered.
Several typical faults easy to occur in the operation of spectrophotometer and their troubleshooting methods:
1) The instrument cannot be zeroed.Possible causes:
a) The light door cannot be completely closed.Solution: Fix the light door assembly and make it completely closed.
b) The transmissivity is "100%".Solution: Readjust the "100%" knob.
c) The instrument is seriously affected with damp.Solution: Open the photocell cassette, dry it with a hair dryer for a while, and replace the desiccant.
d) Circuit fault.Solution: Send it to the repair department to repair the circuit.
2) The instrument cannot be adjusted to "100%".Possible causes:
a) There is not enough light energy.Solution: increase the sensitivity ratio gear, or replace the light source lamp (although the lamp is still on).
b) The cuvette rack is not in place.Solution: Adjust the cuvette rack to make it fall.
c) The photoelectric conversion part is aging.Solution: Replace the parts.
d) Circuit fault.Solution: adjust and repair the circuit.
3) During the measurement, the "100%" point often changes.Possible causes:
a) The position of the cuvette in the cuvette rack is inconsistent, or there are liquid drops on its surface.Solution: Wipe the surface of the cuvette with mirror wiping paper, then place it on the left side of the cuvette, and position it with a positioning clip.
b) Circuit failure (voltage, photoelectric receiving, amplification circuit).Solution: Send for repair.
4) The digital display is unstable.Possible causes:
a) Insufficient warm-up time.Solution: extend the preheating time to about 30 minutes (some instruments will also work unstably when they are in working state for a long time due to aging and other reasons).
b) The desiccant in the photocell fails, causing the microCurrent amplifierBe affected with damp.Solution: Bake the circuit, and replace or bake the desiccant.
c) The environmental vibration is too large, the air velocity near the light source is large, and the external strong light is exposed.Solution: improve the working environment.
d) Photocell, circuit and other reasons.Solution: Send for repair.[2]
