1. Definition and application of spectrophotometry 1.1 Definition: Spectrophotometry is the use of the material's unique absorption spectrum to identify substances or determine its content analysis and detection technology.

1.2 Features: sensitive, accurate, fast and simple, in the complex component system, without the need to separate, that can detect a very small amount of material contained therein.

1.3 Application: One of the widely used methods in biology research, which is widely used for the rapid quantitative detection of sugar, protein, nucleic acid, and enzymes.

2. Basic structure and working principle of spectrophotometer 2.1 Classification of spectrophotometer 2.2 Working principle of spectrophotometer 2.3 Basic structure of spectrophotometer 2.4 Measurement error of spectrophotometry 2.5 Color reaction and its influencing factors 2.1 Spectrophotometer Classifiers Spectrophotometers Classification Infrared spectrophotometers: Infrared light measuring range greater than 760nm visible light spectrophotometer: Measurement of visible light in the wavelength range from 400 to 760nm Ultraviolet spectrophotometer: Measurement of UV in the wavelength range 200 to 400nm Light Zone 2.2 Spectrophotometer Working Principle The light visible to the human eye only occupies a small part of the electromagnetic spectrum (400 to 760 nm).

It is a kind of electromagnetic wave with a relatively large frequency. According to the frequency, the electromagnetic wave is arranged in a row from the radio wave with the smallest frequency to the gamma-ray with the largest frequency, which is the spectrum of the electromagnetic wave, as shown in the following figure.

2.2.1 spectrophotometer spectral range includes the visible range of the wavelength range of 400 ~ 760nm and the ultraviolet range of the wavelength range of 200 ~ 400nm. Different light sources have their own specific emission spectrum, so different light emitters can be used as Light source of the instrument.

Tungsten Emission Spectrum: A spectrum of 400 to 760 nm wavelength emitted by a tungsten light source. After light is refracted by a prism, continuous chromatography consisting of red, orange, yellow, green, blue, tantalum and violet can be obtained; this chromatogram can be used as Visible light spectrophotometer light source.

Hydrogen lamp emission spectrum: Hydrogen lamp can emit 185 ~ 400nm wavelength spectrum, can be used as a UV light source.

2.2.2 Absorption Spectrum of Substances (1)

If a solution of a certain substance is placed between the light source and the prism, the spectrum displayed on the screen at this time is no longer the spectrum of the light source, and it appears several dark lines, that is, some wavelengths of light in the light emission spectrum of the light source. The absorption of the solution disappears, and the spectrum absorbed by the solution is called the absorption spectrum of the solution.

Absorption spectra of different substances are different. Therefore, according to the absorption spectrum, the substances contained in the solution can be identified.

2.2.2 Absorption Spectrum of Substances (2)

When light passes through a solution of a certain substance, the intensity of transmitted light is weakened because a part of the light is reflected or scattered on the surface of the solution, and part of the light is absorbed by the substances that make up the solution, and only a part of the light passes through the solution.

Incident light = reflected light scattered light absorbed light transmitted light If we use distilled water (or the solvent that makes up this solution) as a "blank" to correct the loss of incident light caused by reflections, dispersion, etc., then:

Incident light = absorbed light Ten transmitted light 2.2.3 Relationship between absorbance (A) and transmittance (T) of the material Let I0 be the intensity of the incident light after the blank correction; I is the intensity of the transmitted light.

According to the experiment I=I0?10-εcl

In the formula, c denotes the molar concentration of the absorbing material; l denotes the optical path of the absorbing material and is expressed in cm; ε denotes the molar extinction coefficient of the absorbing material, which represents the absorption property of the material for light, and the ε value of different substances is different. /I0=10-εcl

Let T (transmittance) = I/I0T = 10 - εcl

If T is plotted against the concentration of the absorbing material, the curve in Figure 1-5-2 is obtained.

From the above formula can be obtained 1g (1/T) = εcl

Lg (l/T) is the absorbance of the substance (A) A = 1g (1/T)

2.2.4 Lambert-Beer law (E=εcl)

The formula above shows that the absorbance of a substance is proportional to the concentration of the absorbing substance and the thickness of the liquid layer. This is the basic law of light absorption - the Lambert-Beer law.

2.3 The basic structure of the spectrophotometer No matter which type of spectrophotometer includes: light source, monochromator, absorption cell, detector and measuring instrument. The order of the components of the spectrophotometer is as follows:

The basic components of the 5 basic parts spectrophotometer (1):

Light source: There are two types of light sources commonly used in spectrophotometers: tungsten light or hydrogen light. In the visible light region, near ultraviolet light region and near infrared light region, tungsten light is commonly used as light source; in the ultraviolet light region, hydrogen arc lamps are mostly used.

Monochromator: A device that splits mixed light waves into single-wavelength light. It is used as a dispersive element in spectrophotometers.

Absorption cuvettes, cuvettes, and cuvettes are generally made of glass, quartz, or fused silica and are used to hold the solution being measured. When working in areas below 350 nm in the UV region, a quartz cell must be used or Fused quartz pool.

Spectrophotometer basic components (2):

The cell (cell) must be perpendicular to the direction of the beam. In addition, the thickness of each cuvette should be exactly the same to avoid errors. Fingerprints on the cuvette, oil, or deposits on the walls can significantly affect Its light transmission, so be sure to thoroughly clean before use.

Commonly used photocells, photocells and photomultipliers.

Measuring devices - commonly used ultraviolet and visible spectrophotometers There are three kinds of measuring devices, namely, ammeters, recorders and digital readouts. Modern instruments often have automatic recorders that automatically detect absorption curves.

Detector Prism and Grating Prism: When light waves pass through the prism, the refractive index of light of different wavelengths is different; thus, the light of different wavelengths can be separated. The absorption of ultraviolet rays by the glass is strong, so the glass prism is mostly used for the visible spectrophotometer. The quartz prism can be used. In the entire UV light spread, so widely used in UV spectrophotometer.

Diffraction Grating: A number of parallel lines are carved on the surface of quartz or glass (about 15,000 to 30,000 inches per inch). Since the reticle is opaque, the long wave deflection angle is large due to interference and diffraction of light. The short wave deflection angle is small, thus forming a spectrum.

Prism monochromator device schematic Before the light source shines on the prism (or grating), it first passes through an incident slit, and then through the parallel light mirror, it becomes a parallel beam and hits the prism. The light passing through the prism is passed through another condenser lens. A clear spectrogram can be obtained in the focal plane of the condenser. If the slit is placed at the focal line, the prism is rotated to move the spectrum, and the desired monochromatic light can be emitted from the exit slit. The whole device is called " Monochromator

Detector—Photovoltaic cell A round or rectangular sheet made of 3 layers of material inside a special tweezers. The first layer is a metal with good conductivity. This is the negative electrode of the photocell. The middle layer is a very thin layer. Is a semiconductor selenium, the third layer is iron, which is the anode of the photocell. When the photocell is exposed to light, the surface of the semiconductor selenium escapes electrons, these electrons only move to the negative direction, and do not move to the positive pole, so in the upper and lower two metal sheets A potential difference is generated between the lines and the current detector is generated when the lines are connected. Photocell Photocell The photocell consists of a semi-cylindrical cathode and a filament anode encapsulated in a vacuum transparent envelope. A photoemissive material, which emits electrons upon irradiation with light. When a potential is applied between the two electrodes, the emitted electrons flow to the filament anode to generate a photocurrent. For the same radiation intensity, the current it generates is approximately the photocell. 1/4 of the generated current. Since the photocell has a very high resistance, the generated current is easily amplified.

Detector---Photomultiplier tube Photomultiplier tube is superior to ordinary phototube, it can enlarge the number of electrons emitted from the first time to millions of times. When electrons hit the anode, it can cause more Many electrons are emitted from the surface. These ejected electrons are attracted by the second anode and again generate more electrons.

After repeating this process nine times, each photon can form 106 to 107 electrons. These electrons are finally collected on the anode. The resulting doubling current can be further amplified and measured.