Ultraviolet spectrophotometer is a kind of spectrophotometer. It is widely used in laboratories, veterinary drugs, feed and other industries. It is a kind of laboratory general-purpose instrument, mainly used for qualitative and quantitative analysis, along with the chromatographic instrument industry. The rapid advancement and continuous changes in national standards have gradually evolved from single-wavelength UV-visible spectrophotometers to the dual-beam UV-Vis spectrophotometers that are now required for GMP certification. The spectrophotometer or the principle of the dual-wavelength UV-Vis spectrophotometer we are talking about today is what we need to know in the research and application of laboratory instruments.
A spectrophotometer is an instrument that quantitatively and qualitatively analyzes a substance by spectrophotometry. The instrument is a necessary inspection equipment for laboratories, scientific research institutions, medical, agricultural, food plants, drinking water plants and other institutions. It has become a routine instrument in modern molecular biology laboratories. Often used for nucleic acid, protein quantification and quantification of bacterial growth concentrations. The basic principle of the spectrophotometer is to use a light source that can generate multiple wavelengths, through a series of spectroscopic devices, to produce a specific wavelength of light source, after the light source passes through the test sample, part of the light source is absorbed, calculate the absorbance of the sample, and thus transform The concentration of the sample. The absorbance of the sample is proportional to the concentration of the sample. When monochromatic light radiation passes through the solution of the substance to be tested, the amount absorbed by the substance is proportional to the concentration of the substance and the thickness of the liquid layer (length of the optical path), and the relationship is as follows: A = -lg (I / I.) =-lgT=kLc where: A is absorbance; I. Is the incident monochromatic light intensity; I is the transmitted monochromatic light intensity; T is the transmittance of the substance; k is the molar absorption coefficient; L is the optical path of the analyte, ie the side length of the cuvette; c is the substance concentration. The wavelength of selective absorption of light by a substance, and the corresponding absorption coefficient, is the physical constant of the substance. When the absorption coefficient of a pure substance under certain conditions is known, the test product can be formulated into a solution under the same conditions, and the absorbance can be determined, and the content of the substance in the test sample can be calculated from the above formula. In the visible light region, in addition to the absorption of light by certain substances, many substances are not absorbed by themselves, but can be added to a color developing reagent under certain conditions or processed to make color and then measured, so it is also called colorimetric analysis. Since there are many factors affecting the color depth when color development, and the instrument with poor purity of monochromatic light is often used, the standard or reference material is used for simultaneous measurement. The quantification of nucleic acid quantitative nucleic acids is the most frequently used function of the spectrophotometer. Oligonucleotides, single-stranded, double-stranded DNA, and RNA can be quantified in buffer. The absorption peak of the highest absorption peak of the nucleic acid is 260 nm. The molecular composition of each nucleic acid is different, so the conversion factor is different. To quantify different types of nucleic acids, select the corresponding coefficients in advance. For example, the absorbance of 1 OD corresponds to 50 μg/ml of dsDNA, 37 μg/ml of ssDNA, 40 μg/ml of RNA, and 30 μg/ml of Olig. The absorbance after the test is converted by the above coefficients to obtain the corresponding sample concentration. Before testing, select the correct procedure, enter the volume of the stock solution and diluent, and then test the blank and sample solution. However, the experiment was not always smooth. Unstable readings may be the biggest headache for the experimenter. Instruments with higher sensitivity show greater drift in absorbance. In fact, the design principle and working principle of the spectrophotometer allow the absorbance to vary within a certain range, that is, the instrument has certain accuracy and precision. For example, the accuracy of the Eppendorf Biophotometer is ≤1.0% (1A). The results of such multiple tests vary between a mean of 1.0% and are normal. In addition, it is also necessary to consider the physicochemical properties of the nucleic acid itself and the pH of the buffer in which the nucleic acid is dissolved, the ion concentration, etc.: When the ion concentration is too high during the test, the reading shifts, so it is recommended to use a certain pH value and a low ion concentration. Buffers, such as TE, greatly stabilize readings. The dilution concentration of the sample is also a factor that cannot be ignored: due to the inevitable presence of some fine particles, especially nucleic acid samples, in the sample. The presence of these small particles interferes with the test results. In order to minimize the effect of the particles on the test results, the absorbance of the nucleic acid is required to be at least greater than 0.1 A, and the absorbance is preferably between 0.1 and 1.5 A. Within this range, the interference of the particles is relatively small and the results are stable. This means that the concentration of the sample should not be too low or too high (beyond the test range of the photometer). Finally, the operating factors, such as mixing should be sufficient, otherwise the absorbance value is too low, even negative values; the mixture can not exist bubbles, the blank liquid has no suspended matter, otherwise the reading drifts sharply; the same cuvette must be used to test the blank and sample Otherwise, the concentration difference is too large; the conversion factor and the sample concentration unit are selected uniformly; the crust cup with window wear cannot be used; the sample body except the nucleic acid concentration, the spectrophotometer also displays several very important ratios indicating the purity of the sample, such as A260 The ratio of /A280 is used to evaluate the purity of the sample because the absorption peak of the protein is 280 nm. A pure sample with a ratio greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is below 1.8 or 2.0, it indicates the presence of protein or phenolic substances. A230 indicates that some contaminants exist in the sample, such as carbohydrates, peptides, phenols, etc., and the ratio of the purer nucleic acid A260/A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. For pure samples, A320 is generally 0. The direct quantification (UV method) spectrophotometer principle of the knowledge protein of the UV-Vis spectrophotometer demonstrates that the method is to directly test the protein at a wavelength of 280 nm. Select the Warburg formula, the photometer can directly display the concentration of the sample, or select the appropriate conversion method to convert the absorbance to the sample concentration. The protein determination process is very simple, first test the blank and then test the protein directly. Due to the presence of some impurities in the buffer, it is generally necessary to eliminate the "background" information of 320 nm and set this function to "on". Similar to the test nucleic acid, the absorbance of A280 is required to be at least greater than 0.1 A, and the optimum linear range is between 1.0 and 1.5. When the Warburg formula was used to display the sample concentration in the UV-Vis spectrophotometer model experiment, the reading was "drifted". This is a normal phenomenon. In fact, as long as the absorbance of A280 is observed to vary by no more than 1%, the results are very stable. The reason for the drift is because the absorbance value of the Warburg formula is converted into a concentration, multiplied by a certain coefficient, as long as the absorbance value is slightly changed, the concentration is amplified, and the result is unstable. A direct protein quantification method for testing relatively pure, relatively single-component proteins. Compared with the colorimetric method, the ultraviolet direct quantitative method is fast and easy to operate; but it is easily interfered by parallel substances, such as DNA interference; in addition, the sensitivity is low, and the protein concentration is required to be high. Colorimetric Proteins Proteins are usually compounds of a variety of proteins. Colorimetric assays are based on protein constituents: amino acids (such as tyrosine, serine) react with additional chromogenic groups or dyes to produce colored materials. The concentration of the colored substance is directly related to the number of amino acids reacted by the protein, thereby reacting the protein concentration. Colorimetric methods generally include BCA, Bradford, Lowry and other methods. Lowry method: based on the earliest Biuret reaction and improved. The protein reacts with Cu2+ to produce a blue reactant. However, the Lowry method is more sensitive than Biuret. The disadvantage is that several different reagents need to be added sequentially; the reaction takes a long time; it is susceptible to non-protein substances; proteins containing substances such as EDTA, Tritonx-100, ammonia sulfate are not suitable for this method. Bicinchoninine acid assay: This is a newer, more sensitive protein test. The protein to be analyzed reacts with Cu2+ in an alkaline solution to produce Cu+, which forms a chelate with BCA to form a purple compound with an absorption peak at a wavelength of 562 nm. This compound has a strong linear relationship with protein concentration, and the compound formed after the reaction is very stable. Compared with the Lowry method, the operation is simple and the sensitivity is high. However, similar to the Lowry method, it is susceptible to interference between proteins and detergents.
UV-Vis Spectrophotometer Basic Knowledge Q&A Bradford Method: The principle of this method is that the protein reacts with Coomassie brilliant blue to produce a colored compound absorption peak of 595 nm. Its biggest feature is that it has good sensitivity, which is twice as high as Lowry and BCA; it is simpler and faster; only one reagent is needed; the compound can be stable for 1 hour, which is convenient for the result; The reducing agent (such as DTT, mercaptoethanol) which interferes with Lowry, BCA reaction is compatible. But it is still sensitive to detergents. The main disadvantage is that different standards can lead to large differences in the results of the same sample, which is incomparable. Some researchers who have first-time colorimetric assays may be inconsistent with the results of various colorimetric methods, and are confused. Which method should I believe? Since the groups reacted by the various methods and the chromogenic groups are different, the concentration of the sample obtained by the same sample is incomparable at the same time using several methods. For example, Keller et al. tested the protein in human milk. As a result, the concentration measured by Lowry and BCA was significantly higher than that of Bradford, and the difference was significant. Even if the same sample was measured, the standard samples selected by the same colorimetric method were inconsistent, and the concentrations after the test were inconsistent. If the protein in the cell homogenate is tested with Lowry, BSA is used as a standard at a concentration of 1.34 mg/ml, and a globulin is used as a standard at a concentration of 2.64 mg/ml. Therefore, before selecting the colorimetric method, it is preferable to refer to the chemical composition of the sample to be tested, and to find a standard protein having a similar chemical composition as a standard. In addition, colorimetric methods for quantifying proteins often have problems in that the absorbance of the sample is too low, resulting in a large difference between the measured sample concentration and the actual concentration. The key issue is that the color of the cuvette after the reaction, the important part of the 1011 spectrophotometer, has a certain half-life, so each colorimetric method lists the reaction test time, all samples (including standard samples), Must be tested during this time. When the time is too long, the obtained absorbance value becomes small, and the converted concentration value decreases. In addition, the reaction temperature, pH value of the solution, etc. are all important reasons for the experiment. In addition, it is very important to use plastic colorimetry. Avoid using quartz or glass cuvettes because the color of the reaction will stain the quartz or glass, resulting in inaccurate sample absorbance. Ultraviolet-visible spectrophotometer Encyclopedia common sense Bacterial cell density (OD 600) laboratory to determine the bacterial growth density and growth period, based on experience and visual assessment of bacterial growth density. In the case of more demanding experiments, it is necessary to accurately determine the bacterial cell density using a spectrophotometer. OD600 is the standard method for tracking microbial growth in liquid cultures. The culture solution containing no bacterial solution was used as a blank solution, and then the culture-containing culture solution after the culture was quantitatively determined. In order to ensure proper operation, the cell count must be performed with a microscope for each microorganism and each instrument to make a calibration curve. Occasionally, there is a negative value of the OD value of the bacterial liquid in the experiment, because the color developing medium is used, that is, after the bacteria are cultured for a while, it reacts with the medium to cause a color change reaction. In addition, it should be noted that the sample tested cannot be centrifuged to maintain the bacterial suspension. An important component of the spectrophotometer - the cuvette cuvette is roughly divided into quartz cups, glasses and plastic cups according to the material. Depending on the measurement volume, there are cuvettes and capillary cuvettes. Generally, the test nucleic acid and the ultraviolet quantitative protein are both quartz cups or glasses, but are not suitable for colorimetric determination. Because the dye in the reaction (such as Coomassie Brilliant Blue) can color quartz and glass, a disposable plastic cup must be used. Plastic cups are generally not suitable for testing samples in the ultraviolet range. Due to the different sample sizes tested, general spectrophotometer manufacturers offer cuvettes of different volumes to meet different needs of users. At present, there is a plastic cup which can be used for nucleic acid, ultraviolet protein quantification or protein colorimetric determination. The sample dosage is only 50 μl, and the cuvette can be individually packaged to recover the sample. For example, the Eppendorf UVette® plastic cuvette is an innovation in the current cuvette market. With the development of life sciences and related disciplines, higher requirements are placed on experimental research in such sciences. Spectrophotometers will be indispensable instruments in molecular biology laboratories, and will become necessary for microbiology, food, pharmaceutical and other related laboratories. One of the equipment. The basic principle of the spectrophotometer The principle of measuring the absorption spectrum of a substance by using ultraviolet light, visible light, infrared light and laser, and using the absorption spectrum to qualitatively and quantitatively analyze the substance and analyze the structure of the substance, which is called spectrophotometry or spectrophotometry. Technology, the instrument used is called spectrophotometer. This spectrophotometer has high sensitivity, fast measurement speed and wide application range. Among them, UV/visible spectrophotometry is one of the essential basic methods in biochemistry research. . 1. Spectroscopy: Light is an electromagnetic wave, which can be represented by the wavelength "λ". The electromagnetic spectrum is composed of spectra of continuous wavelengths of different nature. For biochemistry, the most important wavelength regions are visible light and ultraviolet light. The wavelength of the quoted light of the UV-Vis spectrophotometer is the distance between two adjacent peaks. The propagation of light is composed of mutually perpendicular electric field components "E" and magnetic field components "H". λ=C/νλ—wavelength C—speed of light ν—frequency, the number of waves passing through a fixed point per unit time. Light can be thought of as consisting of particles with energy. The original energy "E" of these particles is calculated by the following formula: E = h · νH - Planck's constant ( 6.624 × 10-27 erg · sec) ν - the frequency ultraviolet region can be divided into ultraviolet (near ultraviolet) And vacuum ultraviolet (far ultraviolet). Since the absorption cell (also known as the sample cell, cuvette, etc.) and the optical element and oxygen can absorb light having a wavelength of less than 190 nm, conventional ultraviolet measurement is concentrated in the near-ultraviolet region, that is, 200 nm to 400 nm. The visible light region is from 400 nm to 800 nm. The molecules of the constituent materials are all in a certain energy state and move continuously. The motion of the molecules can be divided into translational motion, rotation, vibration and movement of electrons in the molecule. Each motion state is at a certain energy level, so the energy of the molecule It can be written as: E=E0+E+E++E+E E0 is the intrinsic energy that does not change with molecular motion. The translational energy E is only a function of temperature, so the energy change related to the spectrum is molecular. Rotating energy, vibrational energy, and the electron energy of a molecule. Each energy of a molecule has a series of energy levels. The energy level is not arbitrary, but has a characteristic of quantization. Usually, the molecule is in the ground state. When it absorbs a certain energy transition to the excited state, an absorption spectrum is generated. Molecular rotation, vibration, and transitions in the electronic energy levels produce rotational, vibration, and electronic spectra accordingly. According to the principle of quantum mechanics, the molecular energy state changes in a hopping manner according to a certain law. When the substance absorbs light under the irradiation of incident light, the increase of energy is discontinuous. The material can only absorb light of a certain energy and absorb light. The frequency and the energy difference between the two energy levels are in accordance with the following relationship: E = E2 - E1 = hE1, E2 represent the energy of the initial energy state and the final energy state, respectively, and the energy difference between the initial energy state and the final energy state Larger, the higher the frequency of the absorbed light (ie, the shorter the wavelength), and vice versa, the lower the frequency of the absorbed light (ie, the longer the wavelength). Since the absorption is discontinuous, a series of absorption dark bands appear in a certain portion of the light. Because the energy of molecular rotation, vibration, and electron energy level transitions is quite different, their absorption spectra appear in different spectral regions. The molecular rotation level difference is small, â–³E<0.05 eV (ev), and the absorption of the molecular rotation spectrum appears in the far infrared or microwave region. The difference between the longitudinal and the vibrational energy levels is large, E=0.05~1.0 ev, and the vibration spectrum appears in the mid-infrared region. The level of the electron energy level is larger, E = 1 ~ 20 ev, so the spectrum obtained by the electronic transition appears in the visible, ultraviolet or shorter wavelength region. The principle of spectrophotometer shows that the absorption spectrum of visible light and ultraviolet light is formed by the absorption of optical radiation energy by the loosely connected valence electrons in the molecule, that is, the molecule changes from the ground state to the excited state, and the electron is composed of a low energy level. The orbit (ie, the keyed orbit) absorbs light energy transitions to high-energy orbits (called anti-bond orbits). The three electrons associated with the absorption spectrum are: (1) a covalent bond (ie, a single bond) formed by electrons of two atoms along their symmetry directions, called the sigma bond, and the electrons forming the bond are called σ electrons, such as C-C, C. -H key. (2) A valence bond (ie, a double bond) formed parallel to two atomic orbitals, called a Ï€ bond, and an electron forming a Ï€ bond is called a Ï€ electron, such as a C=C bond. (3) An electron that does not share a bond, called n electron. The order of energy required for various electronic transitions is: n→π*<π→π*≤ n→σ*<π→σ*<σ→π*<σ→σ* The UV absorption spectrum is mainly due to double bond electrons. , in particular, the excitation of Ï€ electrons in conjugated double bonds and excitation of unshared electron pairs. Therefore, the light absorbing properties of various substance molecules on ultraviolet light depend on the number of double bonds of the molecule and the conjugate of unshared electron pairs. The following table shows the relationship between the type of electronic transition and the wavelength of ultraviolet absorption (nm). Table of examples of electronic transition types UV absorption wavelength range σ→σ* C-H 100～150 nmπ→π*(non-conjugated) C=O <200 nmÏ€ →π*(Conjugation)=C-C=200～300 nmn→π* C=O～300 nmπ→π* Transition: The energy required for such transition is small, and the absorption wavelength is 200-300 nm in the ultraviolet region. Saturated hydrocarbons, conjugated olefins and aromatic hydrocarbons can undergo such transitions. Amino acids, proteins and nucleic acids contain a large number of conjugated double bonds. Therefore, the UV absorption measurement of 200-300 nm has a very wide range of applications in biochemical experimental techniques. UV-Vis spectrophotometer common sense If you gradually change the wavelength of incident light that illuminates a substance, and determine the degree of absorption of light by various substances (absorbance "A" or optical density "OD") or transmission (transparency) T"), taking the wavelength λ as the abscissa, "A" or "T" as the ordinate, and drawing a continuous "A~λ" or "T~λ" curve, which is the absorption spectrum curve of the substance. Absorbance ADC Bλmax λmin Wavelength (nm) From the above figure, the characteristics of the absorption spectrum can be seen: (1) The maximum absorption peak at the "A" on the curve, the wavelength corresponding to it is called the maximum absorption wavelength, expressed as λmax. (2) There is a valley at the "B" on the curve, which is called the minimum absorption. The wavelength corresponding to it, the corresponding wavelength, is called the minimum absorption wavelength, and is expressed by λmin. (3) There is a small peak "C" next to the maximum absorption peak on the curve, which is called the shoulder peak. UV-visible spectrophotometer small common sense (4) at the end of the absorption curve at the shortest wavelength, "D" at the curve, the absorption is quite strong, but not peak shape, here called terminal absorption. Λmax is the characteristic wavelength absorbed by electron-level transitions in compounds. Different substances have different maximum absorption peaks, so it is extremely important for identifying compounds. In the absorption spectrum, the shape of λmax, λmin, shoulder, and the entire absorption spectrum is determined by the nature of the substance, and its characteristics vary with the structure of the substance, so it is the basis for qualitative properties. Ultraviolet-visible spectrophotometer knowledge introduces the curve of the ultraviolet absorption spectrum of a substance, which can be compared with the known standard ultraviolet spectrum. The control conditions, such as solvent and concentration, should be noted. The commonly used standard purple absorption spectrum is the "Sadtler" UV standard map set prepared by Sadler Research and Experiments. By the end of the 1970s, 28,585 compounds have been collected, and there are also UV spectra of drugs and non-polar solvents. Figure 2000 more than one. Since the compound has less ultraviolet absorption peaks and wide peak shapes, unlike the infrared spectrum, which is a lot of fingerprint peaks, when qualitatively identifying the compounds by ultraviolet absorption spectroscopy, it should be noted that the compounds have the same UV spectrum should be identical; The same UV spectrum is not necessarily the same as the compound. It may be that only some of the same chromophores or groups are present, so it should be combined with the infrared spectrum when it is identified. Since the electronic transition also causes the rotation and vibrational spectra of the molecule, it is impossible to completely separate the electronic transition from the molecular vibration and the transition of the rotation. Therefore, our common ultraviolet absorption spectrum is composed of one or several wide absorption bands. Composition. UV-visible spectrophotometers commonly used in the ultraviolet spectrum of the term chromophore, chromophore, color enhancement and color reduction effects. Chromophore: Any group that is connected to a saturated hydrocarbon to cause an electronic transition such as n→π*, π→π*, n→σ* is called a chromophore. For example: C=C, C=O and other chromophores. UV-Vis Spectrophotometer Basics Chromophore: The chromophore is a group with non-covalent bonds (such as OH, NH2, SH, etc.). These groups have no absorption at wavelengths >200 nm. When they are attached to the chromophore, they cause the absorption band of the chromophore to move toward long waves, called red shift (or light color effect), while red shifting while absorbing the band The strength increases. If the chromophore is linked to the chromophore, an n→π* transition is generated, causing the absorption wavelength to shift to a short wave, called the blue shift (or dark effect). Hyperchromic effect: The denaturation or degradation of nucleic acid, so that the absorption of ultraviolet light by DNA or RNA solution is significantly increased, that is, the ε value (absorbance coefficient or extinction coefficient) is significantly increased. This phenomenon is called color enhancement effect. This effect is due to a change in the electronic interaction between the bases, usually measured at 260 nm. Hypochromic effect: Under certain conditions, the denatured nucleic acid can be renatured. At this time, the ε value is significantly reduced, and the ε value of the original nucleic acid molecule is restored to a lower level, that is, the DNA or RNA solution at this time. The absorption of ultraviolet light is significantly reduced. This phenomenon is called the color reduction effect. This effect is also caused by changes in the electronic interaction between the bases, usually measured at 260 nm. 2. Law of Light Absorption: Lambert-Beer Law of Light Absorption: A=-lgT=εb cA——Absorbance, also known as optical density “ODâ€. T - transmittance, T = I / I. , I. - For the intensity of light impinging on the absorption cell, I - is the light intensity that passes through the absorption cell. UV-visible spectrophotometer small knowledge ε - molar absorptivity or molar absorptivity (L · mol -1 · cm -1). UV-visible spectrophotometer brand b - sample optical path (cm), usually using 1.0 cm of absorption, b = 1 cm. UV-visible spectrophotometer knowledge and introduction C - sample concentration (mol / L). It can be seen from the above formula that the absorbance A is proportional to the absorption coefficient "ε" of the substance and the concentration "C" of the substance. UV-Vis spectrophotometer common sense reveals the molar absorption coefficient: is a measure of the ability of a substance to absorb light of a certain wavelength. The larger ε, the stronger the ability to absorb light, and the higher the sensitivity of the corresponding spectrophotometric method. The larger the value of ε, the greater the probability of electronic transition, usually ε=10～105: ε>104 is considered to be strong absorption; ε=103～104 is strong absorption; ε<102 is weak absorption, then spectrophotometry Not sensitive. Since the minimum absorbance A=0.001 is usually detected using a spectrophotometer, when b=1cm and ε=105, the minimum concentration of the detectable solution is C=10-8 mol/L. UV absorption spectrophotometer basic knowledge commonly used absorption coefficient and a percentage of absorption coefficient, that is, at a certain wavelength, the solution concentration is 1% (W / V), the liquid layer thickness b = 1cm absorbance, to E1% Λmax is expressed. C - percent concentration (W / V). L——Liquid layer thickness, absorption cup diameter. A - absorbance. The values ​​of ε and E1%λmax at the maximum absorption wavelength λmax can be converted by the following formula: ε=E1%λmax×molecular weight/10 absorbance “A†has an important property that it has additive: A=ε1C1b1+ε2C2b2+ε3C3b3+...=mixture The total absorbance is equal to the arithmetic sum of the absorbance at each wavelength of each component in the solution. This is the basis for quantitative analysis of multi-component spectrophotometry. If the absorption coefficient ε of each solute in the solution is the same, the magnitude of the absorbance of each solute is proportional to the solute concentration. For example, the ion exchange column chromatography separation of nucleotides can be used to calculate the recovery by absorbance: m = C · V, ∵, ∴ m - the amount of solute C - solute concentration V - solution volume A - absorbance ε - Absorbance coefficient b - absorption cell optical diameter ∴ recovery (100%) (assuming ε is equal to the ε of each nucleotide in the above formula) Example 1: uracil nucleotide solution is measured with a 1cm quartz absorption cell 260nm The absorbance at the position was 0.650, and the absorbance of the pure solvent was measured by the same absorption cell to be 0.070. The molar concentration of the uracil solution was calculated, and the molar absorptivity was known to be 8.2 × 103 M-1 cm -1 (M = mol / L). ∵ A= εbC ∵ A=(absorbance of solvent plus sample)-(absorbance of solvent)∴ A=0.650-0.070=0.580 ∵ b=1cm∴ C==7.1×10-5 mol / L Example 2: 1% ( The absorbance of the W/V, 10 mg / ml tyrosinase solution was 24.9 (1 cm absorption cell, 280 nm), and the concentration of the tyrosinase solution of A280 = 0.250 was calculated. Since the percent absorption coefficients of the two enzyme solutions "E1% 1 cm, 280 nm" are the same, the concentration can be calculated by the proportional method. ∵ ∴ ∴ C Unknown = 0.01% = 0.1mg / ml Spectrophotometer composition and structure 1. Composition: Various models of UV / visible spectrophotometer, regardless of the type, basically consist of five parts: 1) a light source; (2) a monochromator (including an optical system that produces parallel light and directs light toward the detector); (3) a sample chamber; (4) a receiving detection amplification system; (5) a display or recorder. Domestic spectrophotometers have been greatly developed in recent years, and various grades of spectrophotometers have been updated and upgraded. The visible light series are: 721, 722, 723 and other models, and the ultraviolet/visible series are: 751, 752, 753. Models 754, 756, etc. The main production plants are Shanghai Spectrum Instruments. The UNICON860 violet/visible spectrophotometer produced by KONTRON, Switzerland is a high-end spectrophotometer with dual beam, fast automatic scanning and screen display. The dual-beam spectrophotometer is characterized in that a continuous spectrum from a light source is split by a concave holographic grating, and a monochromatic light is obtained from the exit slit, and is decomposed into a "sample" by a rotating mirror driven by a motor for about 25 weeks/second. The “reference†beam is sequentially irradiated onto the photomultiplier tube through the reference cell and the sample absorption cell. Since the two optical paths are measured almost simultaneously, the reference signal is continuously compared with the standard voltage, so that the reference signal is constant. Therefore, the instrument can be automatically eliminated by the influence of light source, monochromator, external stray light, photomultiplier tube and power supply voltage. The fastest wavelength scanning speed is 1200nm/min, with five measurement functions and five data processing functions. UV-Vis spectrophotometer 100 questions and 200 spectrophotometer samples and reference light path, respectively, have their own temperature-controlled photodiode detector, thus eliminating the rotating mirror driven by the motor, greatly improving the stability of the instrument and Each detection index has a wavelength range of 190 nm to 1100 nm, an absorbance measurement range of 0 to 3 A, and a variable slit width of 1 nm, 2 nm, and 5 nm. When the instrument is controlled by a microcomputer, the scanning speed is up to 6000 nm/min, and the width is wide. The sample chamber can be equipped with various accessories, and the instrument has excellent performance and is suitable for teaching and scientific research. The instructions for use of the instrument are detailed in the appendix. 2. Construction: (1) Light source: The ideal light source is: 1 can provide continuous radiation; 2 light intensity is large enough; 3 the spectral intensity does not change significantly with wavelength in the whole spectral region; 4 spectral range is wide; 5 long service life, The price is low. The light source for the visible and near-infrared regions is a tungsten lamp. The most common one is the Halogenlamp, which is filled with halogen to increase the life of the tungsten lamp. The applicable wavelength range is 320 to 1100 nm. Since the fluctuation of the energy output is four times the voltage fluctuation, the power supply voltage must be stable. For the ultraviolet light region, it is a deuterium lamp. The applicable wavelength range is 195-400 nm. Due to the limited life of xenon lamps, the life of domestic xenon lamps is only about 500 hours. Pay attention to saving lamps. UV-Vis Spectrophotometer Common Sense (2) Monochromator: The monochromator is the heart part of the spectrophotometer. Its function is to decompose the mixed light from the light source into monochromatic light and change the wavelength at will. Its main components and functions are: 1 incident slit - to limit the entry of stray light. 2 Dispersive elements - prisms or gratings - are the core components that break down the mixed light into monochromatic light. 3 Collimating mirror - converts the beam from the entrance slit into equal light and focuses the equal light from the dispersive element on the exit slit. 4 exit slits - only the light of the rated wavelength is emitted from the monochromator. Rotating the prism or grating wavelength disc can change the wavelength of the exiting beam of the monochromator; adjusting the width of the incident slit gap can change the bandwidth of the outgoing beam and the purity of the monochromatic light. Gratings: Gratings have transmission gratings and reflection gratings. The practical applications are reflection gratings, which can be divided into two types: planar reflection gratings (known as reflection gratings or scintillation gratings) and concave reflection gratings. Concave reflective gratings can be used as dispersive elements. And the collimating mirror acts to focus the dispersed beam on the exit slit to obtain a sharp line spectrum. There are two kinds of grating engraving methods: machine-engraved grating: it is obtained by pressing a 0.5~1 aluminum reflective layer on a hard glass with a diamond knife. The engraving work is extremely large, generally only 10 lines per minute can be engraved, and a 600 line/mm grating with a width of 100 mm is required to be 100 hours. Up to 3600 lines/mm. Due to its long manufacturing cycle and high cost, only a small number of mother gratings can be produced. However, most of the practical applications are replica gratings, that is, silicone oil is coated on the mother grating, and then a layer of aluminum is coated and bonded with epoxy resin. , you get the copy raster. The disadvantage of the engraved grating is that the "ghost line" appears when the slot is slightly defective, that is, the illegible false line on both sides of the strong line of the spectrum. The basics of UV-Vis spectrophotometers explain holographic gratings: high-precision gratings engraved with holographic methods. Even with high-intensity coherent monochromatic light, such as laser, recording interference fringes with high-resolution photosensitive material - photoresist, exposure for 1 hour, chemical treatment of the light-receiving part, and vacuum coating (plating) Aluminum) to obtain a holographic reflection grating. This kind of grating has almost no cycle error between the slots, and there is almost no "ghost line" with little stray light. The maximum slot density can reach 6500 lines/mm, and the maximum diameter can reach 400mm. The more the lines are, the higher the resolution is. The most commonly used holographic grating is 1200~1500 lines/mm. Slit, spectral bandwidth and resolution: The width of the exit slit is usually expressed in two ways: one is the actual width of the slit, expressed in millimeters (mm), and the other is the spectral bandwidth, which means The spectral width of the beam exiting the beam, expressed in nanometers nm. For example, the width of the exit slit is 6 nm, not to say that the width of the exit slit is 6 nm, but the light emitted from the slit has a spectral bandwidth of 6 nm. Pure monochromatic light is only an ideal situation. The "monochromatic light" that can be obtained by a spectrophotometer is actually only a band with a certain wavelength range. The wider the slit, the wider the wavelength range is included. UV-visible spectrophotometer expertise For monochromatic light purity, the narrower the slit, the better, but the weaker the intensity of the light, so the slit can not be unlimited, the minimum width of the slit spectrum effective bandwidth "b'" - is the wavelength interval between two points at half the peak of the light energy detected by the detector, as shown below: light intensity Pb' - spectral effective frequency bandwidth (nm) b - slit width ( Mm) 1/2h - line dispersion rate b' spectral effective frequency bandwidth b'dλ - wavelength difference 1/2hdS - distance (mm) separated by two wavelengths λ ray (dλ) on the exit slit plane It can be seen that b' is proportional to b and inversely proportional to the line dispersion rate. The larger the line dispersion rate, the smaller the effective band width that can be obtained. Resolution: It is the minimum wavelength interval that the instrument can distinguish between two adjacent peaks. It is the ability of the instrument to distinguish two adjacent lines. UV-visible spectrophotometer knowledge, for example, if the sodium double line can be separated: the high resolution is up to: R=105. Therefore, the smaller the slit width b, the smaller the spectral bandwidth b' and the higher the resolution. What is the knowledge of the UV-Vis spectrophotometer? It can be distinguished: When the peaks defined by the two lines are in the same position as the valley, the two peaks are considered to be just distinguishable. Since the grating splitting has a linear dispersion, the measurement of the light of various wavelengths with only one slit width has the same resolution, that is, the slit width does not have to be adjusted frequently. As long as the light intensity meets the requirements, the slit width should be as small as possible to increase the resolution. (3) Sample room: includes a cell holder, an absorption cell (ie, a cuvette), and various replaceable accessories. The pool frame has a common pool frame and a constant temperature pool frame, and the constant temperature pool frame has a water thermostatic cell holder and an electric constant temperature cell holder. The water thermostatic pool frame needs to be circulated into the circulating water to maintain the temperature of the cell holder with the circulating water thermostat. The temperature control accuracy is 0.1 °C. The electric constant temperature cell holder is very expensive, and the temperature control precision can reach 0.05 °C. The absorption tank has two kinds of optical glass and quartz glass. Since the optical glass absorbs ultraviolet light, the optical glass can only be used for visible light, and the applicable wavelength range is 400 nm to 2000 nm. Quartz glass is the most commonly used absorption cell for transmitting ultraviolet, visible and infrared light. The wavelength range is from 180nm to 3000nm. The shape of the absorption tank is rectangular, square and cylindrical, the optical path can be from 0.1cm to 10cm, the most commonly used is 1cm pool (volume 3ml), the optical path is extremely precise, and the transparent glass surface is strictly perpendicular to the light path.的石英æ¯ä¸Šæ–¹åˆ»æœ‰ç®å¤´â€œâ†’â€ï¼Œæ ‡æ˜Žæ¯å使用时的é€å…‰æ–¹å‘，åæ–¹å‘使用会有å差。有å„ç§ç”¨é€”的石英å¸æ”¶æ± ï¼šå¦‚æ¶²ä½“æ± ã€æ°”ä½“æ± ã€å¾®é‡æ± （容积5μl～1ml）ã€æµåŠ¨æ± （测é‡è¿žç»æµåŠ¨çš„æ ·å“）ã€é•¿å…‰å¾„æ± ï¼ˆæµ‹é‡ç¨€æº¶æ¶²ç”¨ï¼‰ã€å¯è£…æ‹†æ± ï¼ˆä¾¿äºŽæ¸…æ´—ï¼‰ç‰ã€‚石英æ¯é€šå¸¸è¿˜é…有玻璃或塑料盖，用以防æ¢æ ·å“挥å‘和氧化，以åŠæ¯å†…æ ·å“的快速混åˆã€‚å¸æ”¶æ± 使用注æ„事项：①è¦å½»åº•æ¸…洗，尤其是盛过蛋白质ç‰æº¶æ¶²ï¼Œå¹²åŽå½¢æˆä¸€å±‚膜，ä¸æ˜“洗去，通常æ¯åä¸ç”¨æ—¶å¯æ”¾åœ¨1ï¼…æ´—æ´å‡€æ¶²ä¸æµ¸æ³¡ï¼ŒåŽ»æ±¡æ•ˆæžœå¥½ï¼Œä½¿ç”¨æ—¶ç”¨æ°´å†²æ´—干净，è¦æ±‚æ¯å£ä¸æŒ‚æ°´ç ，还å¯ä»¥ç”¨ç»¸å¸ƒä¸çº¿æˆ–软塑料制作一个å°åˆ·å清洗æ¯å。 â‘¡ 严ç¦ç”¨æ‰‹æŒ‡è§¦æ‘¸é€å…‰é¢ï¼Œå› 指纹ä¸æ˜“洗净。严ç¦ç”¨ç¡¬çº¸å’Œå¸ƒæ“¦æ‹é€å…‰é¢ï¼Œåªèƒ½ä½¿ç”¨é•œå¤´çº¸å’Œç»¸å¸ƒã€‚ â‘¢ 严ç¦åŠ çƒçƒ˜çƒ¤ã€‚急用干的æ¯å时，å¯ç”¨é…’ç²¾è¡æ´—åŽç”¨å†·é£Žå¹å¹²ã€‚决ä¸å¯ç”¨è¶…声波清洗器清洗。 â‘£å¸æ”¶æ± çš„æ ¡æ£ï¼šè¦å›ºå®šå‚比æ¯å’Œæ ·å“æ¯ï¼Œå¯åœ¨æ¯çš„毛玻璃é¢ä¸Šå†™ä¸Šè®°å·ã€‚用盛有å‚比液的å‚比æ¯å’Œæ ·å“æ¯æµ‹å®šå¸å…‰åº¦â€œA0â€ï¼Œæ ·å“æ¯æ¢ä¸Šæ ·å“液åŽæµ‹å®šçš„å¸å…‰åº¦ä¸ºâ€œA1â€ï¼Œåˆ™æ ¡æ£åŽçš„实际å¸å…‰åº¦A为：A= A1ï¼A0高档的分光光度计有自动置零系统，å¯å°†äºŒä¸ªæ¯åçš„å差置零。其他é‡è¦é™„ä»¶ï¼šé«˜æ¡£åˆ†å…‰å…‰åº¦è®¡çš„æ ·å“室还å¯ä»¥æ›´æ¢å„ç§é‡è¦é™„件，用于å„ç§ç‰¹æ®Šé‡æµ‹ã€‚如æ¢ä¸Šâ€œç§¯åˆ†çƒâ€ï¼Œå¯ç”¨æ¥æ£€æµ‹å¾®å¼±é€å…‰å’Œä¸é€å…‰çš„æ ·å“。æ¢ä¸Šâ€œå‡èƒ¶æ‰«æ装置â€ï¼Œå¯ç”¨äºŽç”µæ³³å‡èƒ¶èƒ¶æ¡ä¸Šæ ·å“带的扫æ测é‡ã€‚ â‘· 检测器：检测器是一ç§å…‰ç”µè½¬æ¢è®¾å¤‡ï¼Œå³æŠŠå…‰å¼ºåº¦ä»¥ç”µè®¯å·æ˜¾ç¤ºå‡ºæ¥ï¼Œå¸¸ç”¨çš„检测器有光电管，光电å€å¢žç®¡å’Œå…‰ç”µäºŒæžç®¡ç‰ä¸‰ç§ã€‚当下必备的紫外å¯è§åˆ†å…‰å…‰åº¦è®¡çŸ¥è¯†â‘ 光电管：光电管å¯æ£€æµ‹10微微安（10-11A）的光电æµï¼Œç®¡å†…抽真空充入惰性气体，常用国产真空光电管有GD-5å…°æ•å…‰ç”µç®¡ï¼ˆé€‚用波长为210～625nm）；GD-6红æ•å…‰ç”µç®¡ï¼ˆé€‚用波长为625～1000nm）。 751型分光光度计å³ä½¿ç”¨è¿™ä¸¤åªå…‰ç”µç®¡ã€‚ ②光电å€å¢žç®¡ï¼šå®ƒæ˜¯æ£€æµ‹å¼±å…‰çš„最çµæ•æœ€å¸¸ç”¨çš„光电元件，其çµæ•åº¦æ¯”光电管高200多å€ï¼Œå…‰ç”µå由阴æžåˆ°é˜³æžé‡å¤å‘å°„9次以上，æ¯ä¸€ä¸ªå…‰ç”µå最åŽå¯äº§ç”Ÿ106～107个电åï¼Œå› æ¤æ€»æ”¾å¤§å€æ•°å¯è¾¾106～107å€ï¼Œå…‰ç”µå€å¢žç®¡çš„å“应时间æžçŸï¼Œèƒ½æ£€æµ‹10-8～10-9秒级的脉冲光。其çµæ•åº¦ä¸Žå…‰ç”µç®¡ä¸€æ ·å—到暗电æµçš„é™åˆ¶ï¼Œæš—电æµä¸»è¦æ¥è‡ªé˜´æžå‘å°„çš„çƒç”µå和电æžé—´çš„æ¼ç”µã€‚ ③光电二æžç®¡ï¼šå…¶åŽŸç†æ˜¯è¿™ç§ç¡…二æžç®¡å—紫外～近红外è¾å°„照射时，其导电性增强的大å°ä¸Žå…‰å¼ºæˆ–æ£æ¯”。近年æ¥åˆ†å…‰å…‰åº¦è®¡ä½¿ç”¨å…‰ç”µäºŒæžç®¡ä½œæ£€æµ‹å™¨åœ¨å¢žåŠ ，虽然其çµæ•åº¦è¿˜èµ¶ä¸ä¸Šå…‰ç”µå€å¢žç®¡ï¼Œä½†å®ƒçš„ç¨³å®šæ€§æ›´å¥½ï¼Œä½¿ç”¨å¯¿å‘½æ›´é•¿ï¼Œä»·æ ¼ä¾¿å®œï¼Œå› è€Œè®¸å¤šè‘—åå“牌的高档分光光度计都在使用它作检测器。尤其值得注æ„的是由于计算机技术的飞速å‘展，使用光电二æžç®¡çš„二æžç®¡é˜µåˆ—分光光度计有了很大的å‘展。二æžç®¡æ•°ç›®å·²è¾¾1024个，大大æ高了分辨率。这ç§æ–°åž‹åˆ†å…‰å…‰åº¦è®¡çš„特点是“åŽåˆ†å…‰â€ï¼Œå³æ°˜ç¯å‘å°„çš„å…‰ç»é€é•œèšç„¦åŽç©¿è¿‡æ ·å“å¸æ”¶æ± ，ç»å…¨æ¯å…‰æ …色散åŽè¢«äºŒæžç®¡é˜µåˆ—çš„å„个二æžç®¡æŽ¥æ”¶ï¼Œä¿¡å·ç”±è®¡ç®—机进行处ç†å’Œå˜å‚¨ï¼Œå› 而扫æ速度æžå¿«ï¼Œçº¦10mså°±å¯å®Œæˆå…¨æ³¢æ®µæ‰«æ，绘出å¸å…‰åº¦ã€æ³¢é•¿å’Œæ—¶é—´çš„三维立体色谱图，å¯ä»¥æœ€æ–¹ä¾¿å¿«é€Ÿåœ°å¾—到任一波长的å¸æ”¶æ•°æ®ï¼Œå®ƒæœ€é€‚宜用于动力å¦æµ‹å®šï¼Œä¹Ÿæ˜¯é«˜æ•ˆæ¶²ç›¸è‰²è°±ä»ªæœ€ç†æƒ³çš„检测器。关于紫外å¯è§åˆ†å…‰å…‰åº¦è®¡çš„一些å°å¸¸è¯†â‘¸ 显示装置：低档分光光度计现在已都使用数å—显示，有的还连有打å°æœºã€‚现代高性能的紫外分光光度计å‡å¯ä»¥è¿žæŽ¥å¾®æœºï¼Œè€Œä¸”有的主机还使用带液晶或CRTè§å±æ˜¾ç¤ºçš„微处ç†æœºå’Œæ‰“å°ç»˜å›¾æœºï¼Œæœ‰çš„è¿˜å¸¦æœ‰æ ‡å‡†è½¯é©±ï¼Œå˜å–æ•°æ®æ›´åŠ 方便（例如ä¸å›½ä»ªå™¨è‰²è°±ç½‘供应的紫外å¯è§åˆ†å…‰å…‰åº¦è®¡723Nå‡çº§æœº
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A spectrophotometer is an instrument that quantitatively and qualitatively analyzes a substance by spectrophotometry. The instrument is a necessary inspection equipment for laboratories, scientific research institutions, medical, agricultural, food plants, drinking water plants and other institutions. It has become a routine instrument in modern molecular biology laboratories. Often used for nucleic acid, protein quantification and quantification of bacterial growth concentrations. The basic principle of the spectrophotometer is to use a light source that can generate multiple wavelengths, through a series of spectroscopic devices, to produce a specific wavelength of light source, after the light source passes through the test sample, part of the light source is absorbed, calculate the absorbance of the sample, and thus transform The concentration of the sample. The absorbance of the sample is proportional to the concentration of the sample. When monochromatic light radiation passes through the solution of the substance to be tested, the amount absorbed by the substance is proportional to the concentration of the substance and the thickness of the liquid layer (length of the optical path), and the relationship is as follows: A = -lg (I / I.) =-lgT=kLc where: A is absorbance; I. Is the incident monochromatic light intensity; I is the transmitted monochromatic light intensity; T is the transmittance of the substance; k is the molar absorption coefficient; L is the optical path of the analyte, ie the side length of the cuvette; c is the substance concentration. The wavelength of selective absorption of light by a substance, and the corresponding absorption coefficient, is the physical constant of the substance. When the absorption coefficient of a pure substance under certain conditions is known, the test product can be formulated into a solution under the same conditions, and the absorbance can be determined, and the content of the substance in the test sample can be calculated from the above formula. In the visible light region, in addition to the absorption of light by certain substances, many substances are not absorbed by themselves, but can be added to a color developing reagent under certain conditions or processed to make color and then measured, so it is also called colorimetric analysis. Since there are many factors affecting the color depth when color development, and the instrument with poor purity of monochromatic light is often used, the standard or reference material is used for simultaneous measurement. The quantification of nucleic acid quantitative nucleic acids is the most frequently used function of the spectrophotometer. Oligonucleotides, single-stranded, double-stranded DNA, and RNA can be quantified in buffer. The absorption peak of the highest absorption peak of the nucleic acid is 260 nm. The molecular composition of each nucleic acid is different, so the conversion factor is different. To quantify different types of nucleic acids, select the corresponding coefficients in advance. For example, the absorbance of 1 OD corresponds to 50 μg/ml of dsDNA, 37 μg/ml of ssDNA, 40 μg/ml of RNA, and 30 μg/ml of Olig. The absorbance after the test is converted by the above coefficients to obtain the corresponding sample concentration. Before testing, select the correct procedure, enter the volume of the stock solution and diluent, and then test the blank and sample solution. However, the experiment was not always smooth. Unstable readings may be the biggest headache for the experimenter. Instruments with higher sensitivity show greater drift in absorbance. In fact, the design principle and working principle of the spectrophotometer allow the absorbance to vary within a certain range, that is, the instrument has certain accuracy and precision. For example, the accuracy of the Eppendorf Biophotometer is ≤1.0% (1A). The results of such multiple tests vary between a mean of 1.0% and are normal. In addition, it is also necessary to consider the physicochemical properties of the nucleic acid itself and the pH of the buffer in which the nucleic acid is dissolved, the ion concentration, etc.: When the ion concentration is too high during the test, the reading shifts, so it is recommended to use a certain pH value and a low ion concentration. Buffers, such as TE, greatly stabilize readings. The dilution concentration of the sample is also a factor that cannot be ignored: due to the inevitable presence of some fine particles, especially nucleic acid samples, in the sample. The presence of these small particles interferes with the test results. In order to minimize the effect of the particles on the test results, the absorbance of the nucleic acid is required to be at least greater than 0.1 A, and the absorbance is preferably between 0.1 and 1.5 A. Within this range, the interference of the particles is relatively small and the results are stable. This means that the concentration of the sample should not be too low or too high (beyond the test range of the photometer). Finally, the operating factors, such as mixing should be sufficient, otherwise the absorbance value is too low, even negative values; the mixture can not exist bubbles, the blank liquid has no suspended matter, otherwise the reading drifts sharply; the same cuvette must be used to test the blank and sample Otherwise, the concentration difference is too large; the conversion factor and the sample concentration unit are selected uniformly; the crust cup with window wear cannot be used; the sample body except the nucleic acid concentration, the spectrophotometer also displays several very important ratios indicating the purity of the sample, such as A260 The ratio of /A280 is used to evaluate the purity of the sample because the absorption peak of the protein is 280 nm. A pure sample with a ratio greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is below 1.8 or 2.0, it indicates the presence of protein or phenolic substances. A230 indicates that some contaminants exist in the sample, such as carbohydrates, peptides, phenols, etc., and the ratio of the purer nucleic acid A260/A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. For pure samples, A320 is generally 0. The direct quantification (UV method) spectrophotometer principle of the knowledge protein of the UV-Vis spectrophotometer demonstrates that the method is to directly test the protein at a wavelength of 280 nm. Select the Warburg formula, the photometer can directly display the concentration of the sample, or select the appropriate conversion method to convert the absorbance to the sample concentration. The protein determination process is very simple, first test the blank and then test the protein directly. Due to the presence of some impurities in the buffer, it is generally necessary to eliminate the "background" information of 320 nm and set this function to "on". Similar to the test nucleic acid, the absorbance of A280 is required to be at least greater than 0.1 A, and the optimum linear range is between 1.0 and 1.5. When the Warburg formula was used to display the sample concentration in the UV-Vis spectrophotometer model experiment, the reading was "drifted". This is a normal phenomenon. In fact, as long as the absorbance of A280 is observed to vary by no more than 1%, the results are very stable. The reason for the drift is because the absorbance value of the Warburg formula is converted into a concentration, multiplied by a certain coefficient, as long as the absorbance value is slightly changed, the concentration is amplified, and the result is unstable. A direct protein quantification method for testing relatively pure, relatively single-component proteins. Compared with the colorimetric method, the ultraviolet direct quantitative method is fast and easy to operate; but it is easily interfered by parallel substances, such as DNA interference; in addition, the sensitivity is low, and the protein concentration is required to be high. Colorimetric Proteins Proteins are usually compounds of a variety of proteins. Colorimetric assays are based on protein constituents: amino acids (such as tyrosine, serine) react with additional chromogenic groups or dyes to produce colored materials. The concentration of the colored substance is directly related to the number of amino acids reacted by the protein, thereby reacting the protein concentration. Colorimetric methods generally include BCA, Bradford, Lowry and other methods. Lowry method: based on the earliest Biuret reaction and improved. The protein reacts with Cu2+ to produce a blue reactant. However, the Lowry method is more sensitive than Biuret. The disadvantage is that several different reagents need to be added sequentially; the reaction takes a long time; it is susceptible to non-protein substances; proteins containing substances such as EDTA, Tritonx-100, ammonia sulfate are not suitable for this method. Bicinchoninine acid assay: This is a newer, more sensitive protein test. The protein to be analyzed reacts with Cu2+ in an alkaline solution to produce Cu+, which forms a chelate with BCA to form a purple compound with an absorption peak at a wavelength of 562 nm. This compound has a strong linear relationship with protein concentration, and the compound formed after the reaction is very stable. Compared with the Lowry method, the operation is simple and the sensitivity is high. However, similar to the Lowry method, it is susceptible to interference between proteins and detergents.
UV-Vis Spectrophotometer Basic Knowledge Q&A Bradford Method: The principle of this method is that the protein reacts with Coomassie brilliant blue to produce a colored compound absorption peak of 595 nm. Its biggest feature is that it has good sensitivity, which is twice as high as Lowry and BCA; it is simpler and faster; only one reagent is needed; the compound can be stable for 1 hour, which is convenient for the result; The reducing agent (such as DTT, mercaptoethanol) which interferes with Lowry, BCA reaction is compatible. But it is still sensitive to detergents. The main disadvantage is that different standards can lead to large differences in the results of the same sample, which is incomparable. Some researchers who have first-time colorimetric assays may be inconsistent with the results of various colorimetric methods, and are confused. Which method should I believe? Since the groups reacted by the various methods and the chromogenic groups are different, the concentration of the sample obtained by the same sample is incomparable at the same time using several methods. For example, Keller et al. tested the protein in human milk. As a result, the concentration measured by Lowry and BCA was significantly higher than that of Bradford, and the difference was significant. Even if the same sample was measured, the standard samples selected by the same colorimetric method were inconsistent, and the concentrations after the test were inconsistent. If the protein in the cell homogenate is tested with Lowry, BSA is used as a standard at a concentration of 1.34 mg/ml, and a globulin is used as a standard at a concentration of 2.64 mg/ml. Therefore, before selecting the colorimetric method, it is preferable to refer to the chemical composition of the sample to be tested, and to find a standard protein having a similar chemical composition as a standard. In addition, colorimetric methods for quantifying proteins often have problems in that the absorbance of the sample is too low, resulting in a large difference between the measured sample concentration and the actual concentration. The key issue is that the color of the cuvette after the reaction, the important part of the 1011 spectrophotometer, has a certain half-life, so each colorimetric method lists the reaction test time, all samples (including standard samples), Must be tested during this time. When the time is too long, the obtained absorbance value becomes small, and the converted concentration value decreases. In addition, the reaction temperature, pH value of the solution, etc. are all important reasons for the experiment. In addition, it is very important to use plastic colorimetry. Avoid using quartz or glass cuvettes because the color of the reaction will stain the quartz or glass, resulting in inaccurate sample absorbance. Ultraviolet-visible spectrophotometer Encyclopedia common sense Bacterial cell density (OD 600) laboratory to determine the bacterial growth density and growth period, based on experience and visual assessment of bacterial growth density. In the case of more demanding experiments, it is necessary to accurately determine the bacterial cell density using a spectrophotometer. OD600 is the standard method for tracking microbial growth in liquid cultures. The culture solution containing no bacterial solution was used as a blank solution, and then the culture-containing culture solution after the culture was quantitatively determined. In order to ensure proper operation, the cell count must be performed with a microscope for each microorganism and each instrument to make a calibration curve. Occasionally, there is a negative value of the OD value of the bacterial liquid in the experiment, because the color developing medium is used, that is, after the bacteria are cultured for a while, it reacts with the medium to cause a color change reaction. In addition, it should be noted that the sample tested cannot be centrifuged to maintain the bacterial suspension. An important component of the spectrophotometer - the cuvette cuvette is roughly divided into quartz cups, glasses and plastic cups according to the material. Depending on the measurement volume, there are cuvettes and capillary cuvettes. Generally, the test nucleic acid and the ultraviolet quantitative protein are both quartz cups or glasses, but are not suitable for colorimetric determination. Because the dye in the reaction (such as Coomassie Brilliant Blue) can color quartz and glass, a disposable plastic cup must be used. Plastic cups are generally not suitable for testing samples in the ultraviolet range. Due to the different sample sizes tested, general spectrophotometer manufacturers offer cuvettes of different volumes to meet different needs of users. At present, there is a plastic cup which can be used for nucleic acid, ultraviolet protein quantification or protein colorimetric determination. The sample dosage is only 50 μl, and the cuvette can be individually packaged to recover the sample. For example, the Eppendorf UVette® plastic cuvette is an innovation in the current cuvette market. With the development of life sciences and related disciplines, higher requirements are placed on experimental research in such sciences. Spectrophotometers will be indispensable instruments in molecular biology laboratories, and will become necessary for microbiology, food, pharmaceutical and other related laboratories. One of the equipment. The basic principle of the spectrophotometer The principle of measuring the absorption spectrum of a substance by using ultraviolet light, visible light, infrared light and laser, and using the absorption spectrum to qualitatively and quantitatively analyze the substance and analyze the structure of the substance, which is called spectrophotometry or spectrophotometry. Technology, the instrument used is called spectrophotometer. This spectrophotometer has high sensitivity, fast measurement speed and wide application range. Among them, UV/visible spectrophotometry is one of the essential basic methods in biochemistry research. . 1. Spectroscopy: Light is an electromagnetic wave, which can be represented by the wavelength "λ". The electromagnetic spectrum is composed of spectra of continuous wavelengths of different nature. For biochemistry, the most important wavelength regions are visible light and ultraviolet light. The wavelength of the quoted light of the UV-Vis spectrophotometer is the distance between two adjacent peaks. The propagation of light is composed of mutually perpendicular electric field components "E" and magnetic field components "H". λ=C/νλ—wavelength C—speed of light ν—frequency, the number of waves passing through a fixed point per unit time. Light can be thought of as consisting of particles with energy. The original energy "E" of these particles is calculated by the following formula: E = h · νH - Planck's constant ( 6.624 × 10-27 erg · sec) ν - the frequency ultraviolet region can be divided into ultraviolet (near ultraviolet) And vacuum ultraviolet (far ultraviolet). Since the absorption cell (also known as the sample cell, cuvette, etc.) and the optical element and oxygen can absorb light having a wavelength of less than 190 nm, conventional ultraviolet measurement is concentrated in the near-ultraviolet region, that is, 200 nm to 400 nm. The visible light region is from 400 nm to 800 nm. The molecules of the constituent materials are all in a certain energy state and move continuously. The motion of the molecules can be divided into translational motion, rotation, vibration and movement of electrons in the molecule. Each motion state is at a certain energy level, so the energy of the molecule It can be written as: E=E0+E+E++E+E E0 is the intrinsic energy that does not change with molecular motion. The translational energy E is only a function of temperature, so the energy change related to the spectrum is molecular. Rotating energy, vibrational energy, and the electron energy of a molecule. Each energy of a molecule has a series of energy levels. The energy level is not arbitrary, but has a characteristic of quantization. Usually, the molecule is in the ground state. When it absorbs a certain energy transition to the excited state, an absorption spectrum is generated. Molecular rotation, vibration, and transitions in the electronic energy levels produce rotational, vibration, and electronic spectra accordingly. According to the principle of quantum mechanics, the molecular energy state changes in a hopping manner according to a certain law. When the substance absorbs light under the irradiation of incident light, the increase of energy is discontinuous. The material can only absorb light of a certain energy and absorb light. The frequency and the energy difference between the two energy levels are in accordance with the following relationship: E = E2 - E1 = hE1, E2 represent the energy of the initial energy state and the final energy state, respectively, and the energy difference between the initial energy state and the final energy state Larger, the higher the frequency of the absorbed light (ie, the shorter the wavelength), and vice versa, the lower the frequency of the absorbed light (ie, the longer the wavelength). Since the absorption is discontinuous, a series of absorption dark bands appear in a certain portion of the light. Because the energy of molecular rotation, vibration, and electron energy level transitions is quite different, their absorption spectra appear in different spectral regions. The molecular rotation level difference is small, â–³E<0.05 eV (ev), and the absorption of the molecular rotation spectrum appears in the far infrared or microwave region. The difference between the longitudinal and the vibrational energy levels is large, E=0.05~1.0 ev, and the vibration spectrum appears in the mid-infrared region. The level of the electron energy level is larger, E = 1 ~ 20 ev, so the spectrum obtained by the electronic transition appears in the visible, ultraviolet or shorter wavelength region. The principle of spectrophotometer shows that the absorption spectrum of visible light and ultraviolet light is formed by the absorption of optical radiation energy by the loosely connected valence electrons in the molecule, that is, the molecule changes from the ground state to the excited state, and the electron is composed of a low energy level. The orbit (ie, the keyed orbit) absorbs light energy transitions to high-energy orbits (called anti-bond orbits). The three electrons associated with the absorption spectrum are: (1) a covalent bond (ie, a single bond) formed by electrons of two atoms along their symmetry directions, called the sigma bond, and the electrons forming the bond are called σ electrons, such as C-C, C. -H key. (2) A valence bond (ie, a double bond) formed parallel to two atomic orbitals, called a Ï€ bond, and an electron forming a Ï€ bond is called a Ï€ electron, such as a C=C bond. (3) An electron that does not share a bond, called n electron. The order of energy required for various electronic transitions is: n→π*<π→π*≤ n→σ*<π→σ*<σ→π*<σ→σ* The UV absorption spectrum is mainly due to double bond electrons. , in particular, the excitation of Ï€ electrons in conjugated double bonds and excitation of unshared electron pairs. Therefore, the light absorbing properties of various substance molecules on ultraviolet light depend on the number of double bonds of the molecule and the conjugate of unshared electron pairs. The following table shows the relationship between the type of electronic transition and the wavelength of ultraviolet absorption (nm). Table of examples of electronic transition types UV absorption wavelength range σ→σ* C-H 100～150 nmπ→π*(non-conjugated) C=O <200 nmÏ€ →π*(Conjugation)=C-C=200～300 nmn→π* C=O～300 nmπ→π* Transition: The energy required for such transition is small, and the absorption wavelength is 200-300 nm in the ultraviolet region. Saturated hydrocarbons, conjugated olefins and aromatic hydrocarbons can undergo such transitions. Amino acids, proteins and nucleic acids contain a large number of conjugated double bonds. Therefore, the UV absorption measurement of 200-300 nm has a very wide range of applications in biochemical experimental techniques. UV-Vis spectrophotometer common sense If you gradually change the wavelength of incident light that illuminates a substance, and determine the degree of absorption of light by various substances (absorbance "A" or optical density "OD") or transmission (transparency) T"), taking the wavelength λ as the abscissa, "A" or "T" as the ordinate, and drawing a continuous "A~λ" or "T~λ" curve, which is the absorption spectrum curve of the substance. Absorbance ADC Bλmax λmin Wavelength (nm) From the above figure, the characteristics of the absorption spectrum can be seen: (1) The maximum absorption peak at the "A" on the curve, the wavelength corresponding to it is called the maximum absorption wavelength, expressed as λmax. (2) There is a valley at the "B" on the curve, which is called the minimum absorption. The wavelength corresponding to it, the corresponding wavelength, is called the minimum absorption wavelength, and is expressed by λmin. (3) There is a small peak "C" next to the maximum absorption peak on the curve, which is called the shoulder peak. UV-visible spectrophotometer small common sense (4) at the end of the absorption curve at the shortest wavelength, "D" at the curve, the absorption is quite strong, but not peak shape, here called terminal absorption. Λmax is the characteristic wavelength absorbed by electron-level transitions in compounds. Different substances have different maximum absorption peaks, so it is extremely important for identifying compounds. In the absorption spectrum, the shape of λmax, λmin, shoulder, and the entire absorption spectrum is determined by the nature of the substance, and its characteristics vary with the structure of the substance, so it is the basis for qualitative properties. Ultraviolet-visible spectrophotometer knowledge introduces the curve of the ultraviolet absorption spectrum of a substance, which can be compared with the known standard ultraviolet spectrum. The control conditions, such as solvent and concentration, should be noted. The commonly used standard purple absorption spectrum is the "Sadtler" UV standard map set prepared by Sadler Research and Experiments. By the end of the 1970s, 28,585 compounds have been collected, and there are also UV spectra of drugs and non-polar solvents. Figure 2000 more than one. Since the compound has less ultraviolet absorption peaks and wide peak shapes, unlike the infrared spectrum, which is a lot of fingerprint peaks, when qualitatively identifying the compounds by ultraviolet absorption spectroscopy, it should be noted that the compounds have the same UV spectrum should be identical; The same UV spectrum is not necessarily the same as the compound. It may be that only some of the same chromophores or groups are present, so it should be combined with the infrared spectrum when it is identified. Since the electronic transition also causes the rotation and vibrational spectra of the molecule, it is impossible to completely separate the electronic transition from the molecular vibration and the transition of the rotation. Therefore, our common ultraviolet absorption spectrum is composed of one or several wide absorption bands. Composition. UV-visible spectrophotometers commonly used in the ultraviolet spectrum of the term chromophore, chromophore, color enhancement and color reduction effects. Chromophore: Any group that is connected to a saturated hydrocarbon to cause an electronic transition such as n→π*, π→π*, n→σ* is called a chromophore. For example: C=C, C=O and other chromophores. UV-Vis Spectrophotometer Basics Chromophore: The chromophore is a group with non-covalent bonds (such as OH, NH2, SH, etc.). These groups have no absorption at wavelengths >200 nm. When they are attached to the chromophore, they cause the absorption band of the chromophore to move toward long waves, called red shift (or light color effect), while red shifting while absorbing the band The strength increases. If the chromophore is linked to the chromophore, an n→π* transition is generated, causing the absorption wavelength to shift to a short wave, called the blue shift (or dark effect). Hyperchromic effect: The denaturation or degradation of nucleic acid, so that the absorption of ultraviolet light by DNA or RNA solution is significantly increased, that is, the ε value (absorbance coefficient or extinction coefficient) is significantly increased. This phenomenon is called color enhancement effect. This effect is due to a change in the electronic interaction between the bases, usually measured at 260 nm. Hypochromic effect: Under certain conditions, the denatured nucleic acid can be renatured. At this time, the ε value is significantly reduced, and the ε value of the original nucleic acid molecule is restored to a lower level, that is, the DNA or RNA solution at this time. The absorption of ultraviolet light is significantly reduced. This phenomenon is called the color reduction effect. This effect is also caused by changes in the electronic interaction between the bases, usually measured at 260 nm. 2. Law of Light Absorption: Lambert-Beer Law of Light Absorption: A=-lgT=εb cA——Absorbance, also known as optical density “ODâ€. T - transmittance, T = I / I. , I. - For the intensity of light impinging on the absorption cell, I - is the light intensity that passes through the absorption cell. UV-visible spectrophotometer small knowledge ε - molar absorptivity or molar absorptivity (L · mol -1 · cm -1). UV-visible spectrophotometer brand b - sample optical path (cm), usually using 1.0 cm of absorption, b = 1 cm. UV-visible spectrophotometer knowledge and introduction C - sample concentration (mol / L). It can be seen from the above formula that the absorbance A is proportional to the absorption coefficient "ε" of the substance and the concentration "C" of the substance. UV-Vis spectrophotometer common sense reveals the molar absorption coefficient: is a measure of the ability of a substance to absorb light of a certain wavelength. The larger ε, the stronger the ability to absorb light, and the higher the sensitivity of the corresponding spectrophotometric method. The larger the value of ε, the greater the probability of electronic transition, usually ε=10～105: ε>104 is considered to be strong absorption; ε=103～104 is strong absorption; ε<102 is weak absorption, then spectrophotometry Not sensitive. Since the minimum absorbance A=0.001 is usually detected using a spectrophotometer, when b=1cm and ε=105, the minimum concentration of the detectable solution is C=10-8 mol/L. UV absorption spectrophotometer basic knowledge commonly used absorption coefficient and a percentage of absorption coefficient, that is, at a certain wavelength, the solution concentration is 1% (W / V), the liquid layer thickness b = 1cm absorbance, to E1% Λmax is expressed. C - percent concentration (W / V). L——Liquid layer thickness, absorption cup diameter. A - absorbance. The values ​​of ε and E1%λmax at the maximum absorption wavelength λmax can be converted by the following formula: ε=E1%λmax×molecular weight/10 absorbance “A†has an important property that it has additive: A=ε1C1b1+ε2C2b2+ε3C3b3+...=mixture The total absorbance is equal to the arithmetic sum of the absorbance at each wavelength of each component in the solution. This is the basis for quantitative analysis of multi-component spectrophotometry. If the absorption coefficient ε of each solute in the solution is the same, the magnitude of the absorbance of each solute is proportional to the solute concentration. For example, the ion exchange column chromatography separation of nucleotides can be used to calculate the recovery by absorbance: m = C · V, ∵, ∴ m - the amount of solute C - solute concentration V - solution volume A - absorbance ε - Absorbance coefficient b - absorption cell optical diameter ∴ recovery (100%) (assuming ε is equal to the ε of each nucleotide in the above formula) Example 1: uracil nucleotide solution is measured with a 1cm quartz absorption cell 260nm The absorbance at the position was 0.650, and the absorbance of the pure solvent was measured by the same absorption cell to be 0.070. The molar concentration of the uracil solution was calculated, and the molar absorptivity was known to be 8.2 × 103 M-1 cm -1 (M = mol / L). ∵ A= εbC ∵ A=(absorbance of solvent plus sample)-(absorbance of solvent)∴ A=0.650-0.070=0.580 ∵ b=1cm∴ C==7.1×10-5 mol / L Example 2: 1% ( The absorbance of the W/V, 10 mg / ml tyrosinase solution was 24.9 (1 cm absorption cell, 280 nm), and the concentration of the tyrosinase solution of A280 = 0.250 was calculated. Since the percent absorption coefficients of the two enzyme solutions "E1% 1 cm, 280 nm" are the same, the concentration can be calculated by the proportional method. ∵ ∴ ∴ C Unknown = 0.01% = 0.1mg / ml Spectrophotometer composition and structure 1. Composition: Various models of UV / visible spectrophotometer, regardless of the type, basically consist of five parts: 1) a light source; (2) a monochromator (including an optical system that produces parallel light and directs light toward the detector); (3) a sample chamber; (4) a receiving detection amplification system; (5) a display or recorder. Domestic spectrophotometers have been greatly developed in recent years, and various grades of spectrophotometers have been updated and upgraded. The visible light series are: 721, 722, 723 and other models, and the ultraviolet/visible series are: 751, 752, 753. Models 754, 756, etc. The main production plants are Shanghai Spectrum Instruments. The UNICON860 violet/visible spectrophotometer produced by KONTRON, Switzerland is a high-end spectrophotometer with dual beam, fast automatic scanning and screen display. The dual-beam spectrophotometer is characterized in that a continuous spectrum from a light source is split by a concave holographic grating, and a monochromatic light is obtained from the exit slit, and is decomposed into a "sample" by a rotating mirror driven by a motor for about 25 weeks/second. The “reference†beam is sequentially irradiated onto the photomultiplier tube through the reference cell and the sample absorption cell. Since the two optical paths are measured almost simultaneously, the reference signal is continuously compared with the standard voltage, so that the reference signal is constant. Therefore, the instrument can be automatically eliminated by the influence of light source, monochromator, external stray light, photomultiplier tube and power supply voltage. The fastest wavelength scanning speed is 1200nm/min, with five measurement functions and five data processing functions. UV-Vis spectrophotometer 100 questions and 200 spectrophotometer samples and reference light path, respectively, have their own temperature-controlled photodiode detector, thus eliminating the rotating mirror driven by the motor, greatly improving the stability of the instrument and Each detection index has a wavelength range of 190 nm to 1100 nm, an absorbance measurement range of 0 to 3 A, and a variable slit width of 1 nm, 2 nm, and 5 nm. When the instrument is controlled by a microcomputer, the scanning speed is up to 6000 nm/min, and the width is wide. The sample chamber can be equipped with various accessories, and the instrument has excellent performance and is suitable for teaching and scientific research. The instructions for use of the instrument are detailed in the appendix. 2. Construction: (1) Light source: The ideal light source is: 1 can provide continuous radiation; 2 light intensity is large enough; 3 the spectral intensity does not change significantly with wavelength in the whole spectral region; 4 spectral range is wide; 5 long service life, The price is low. The light source for the visible and near-infrared regions is a tungsten lamp. The most common one is the Halogenlamp, which is filled with halogen to increase the life of the tungsten lamp. The applicable wavelength range is 320 to 1100 nm. Since the fluctuation of the energy output is four times the voltage fluctuation, the power supply voltage must be stable. For the ultraviolet light region, it is a deuterium lamp. The applicable wavelength range is 195-400 nm. Due to the limited life of xenon lamps, the life of domestic xenon lamps is only about 500 hours. Pay attention to saving lamps. UV-Vis Spectrophotometer Common Sense (2) Monochromator: The monochromator is the heart part of the spectrophotometer. Its function is to decompose the mixed light from the light source into monochromatic light and change the wavelength at will. Its main components and functions are: 1 incident slit - to limit the entry of stray light. 2 Dispersive elements - prisms or gratings - are the core components that break down the mixed light into monochromatic light. 3 Collimating mirror - converts the beam from the entrance slit into equal light and focuses the equal light from the dispersive element on the exit slit. 4 exit slits - only the light of the rated wavelength is emitted from the monochromator. Rotating the prism or grating wavelength disc can change the wavelength of the exiting beam of the monochromator; adjusting the width of the incident slit gap can change the bandwidth of the outgoing beam and the purity of the monochromatic light. Gratings: Gratings have transmission gratings and reflection gratings. The practical applications are reflection gratings, which can be divided into two types: planar reflection gratings (known as reflection gratings or scintillation gratings) and concave reflection gratings. Concave reflective gratings can be used as dispersive elements. And the collimating mirror acts to focus the dispersed beam on the exit slit to obtain a sharp line spectrum. There are two kinds of grating engraving methods: machine-engraved grating: it is obtained by pressing a 0.5~1 aluminum reflective layer on a hard glass with a diamond knife. The engraving work is extremely large, generally only 10 lines per minute can be engraved, and a 600 line/mm grating with a width of 100 mm is required to be 100 hours. Up to 3600 lines/mm. Due to its long manufacturing cycle and high cost, only a small number of mother gratings can be produced. However, most of the practical applications are replica gratings, that is, silicone oil is coated on the mother grating, and then a layer of aluminum is coated and bonded with epoxy resin. , you get the copy raster. The disadvantage of the engraved grating is that the "ghost line" appears when the slot is slightly defective, that is, the illegible false line on both sides of the strong line of the spectrum. The basics of UV-Vis spectrophotometers explain holographic gratings: high-precision gratings engraved with holographic methods. Even with high-intensity coherent monochromatic light, such as laser, recording interference fringes with high-resolution photosensitive material - photoresist, exposure for 1 hour, chemical treatment of the light-receiving part, and vacuum coating (plating) Aluminum) to obtain a holographic reflection grating. This kind of grating has almost no cycle error between the slots, and there is almost no "ghost line" with little stray light. The maximum slot density can reach 6500 lines/mm, and the maximum diameter can reach 400mm. The more the lines are, the higher the resolution is. The most commonly used holographic grating is 1200~1500 lines/mm. Slit, spectral bandwidth and resolution: The width of the exit slit is usually expressed in two ways: one is the actual width of the slit, expressed in millimeters (mm), and the other is the spectral bandwidth, which means The spectral width of the beam exiting the beam, expressed in nanometers nm. For example, the width of the exit slit is 6 nm, not to say that the width of the exit slit is 6 nm, but the light emitted from the slit has a spectral bandwidth of 6 nm. Pure monochromatic light is only an ideal situation. The "monochromatic light" that can be obtained by a spectrophotometer is actually only a band with a certain wavelength range. The wider the slit, the wider the wavelength range is included. UV-visible spectrophotometer expertise For monochromatic light purity, the narrower the slit, the better, but the weaker the intensity of the light, so the slit can not be unlimited, the minimum width of the slit spectrum effective bandwidth "b'" - is the wavelength interval between two points at half the peak of the light energy detected by the detector, as shown below: light intensity Pb' - spectral effective frequency bandwidth (nm) b - slit width ( Mm) 1/2h - line dispersion rate b' spectral effective frequency bandwidth b'dλ - wavelength difference 1/2hdS - distance (mm) separated by two wavelengths λ ray (dλ) on the exit slit plane It can be seen that b' is proportional to b and inversely proportional to the line dispersion rate. The larger the line dispersion rate, the smaller the effective band width that can be obtained. Resolution: It is the minimum wavelength interval that the instrument can distinguish between two adjacent peaks. It is the ability of the instrument to distinguish two adjacent lines. UV-visible spectrophotometer knowledge, for example, if the sodium double line can be separated: the high resolution is up to: R=105. Therefore, the smaller the slit width b, the smaller the spectral bandwidth b' and the higher the resolution. What is the knowledge of the UV-Vis spectrophotometer? It can be distinguished: When the peaks defined by the two lines are in the same position as the valley, the two peaks are considered to be just distinguishable. Since the grating splitting has a linear dispersion, the measurement of the light of various wavelengths with only one slit width has the same resolution, that is, the slit width does not have to be adjusted frequently. As long as the light intensity meets the requirements, the slit width should be as small as possible to increase the resolution. (3) Sample room: includes a cell holder, an absorption cell (ie, a cuvette), and various replaceable accessories. The pool frame has a common pool frame and a constant temperature pool frame, and the constant temperature pool frame has a water thermostatic cell holder and an electric constant temperature cell holder. The water thermostatic pool frame needs to be circulated into the circulating water to maintain the temperature of the cell holder with the circulating water thermostat. The temperature control accuracy is 0.1 °C. The electric constant temperature cell holder is very expensive, and the temperature control precision can reach 0.05 °C. The absorption tank has two kinds of optical glass and quartz glass. Since the optical glass absorbs ultraviolet light, the optical glass can only be used for visible light, and the applicable wavelength range is 400 nm to 2000 nm. Quartz glass is the most commonly used absorption cell for transmitting ultraviolet, visible and infrared light. The wavelength range is from 180nm to 3000nm. 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