1. introduction

Scattered light turbidity method and transmitted light turbidity method are analytical techniques based on the principle of light scattering phenomenon. Light scattering is a physical phenomenon in which a beam of light changes its direction of propagation (known as deflection) due to interaction with sufficiently small particles of matter. According to Maxwell's electromagnetic theory, the prerequisite for scattering to occur is that the refractive index of suspended particles must be different from that of suspended liquids. The greater the difference, the stronger the scattering. There are two types of light scattering:1）Elastic scattering, where the scattered light and incident light have the same wavelength;2）Non elastic light scattering, where the wavelengths of scattered light and incident light are different. Only the first type of light scattering (elasticity) is related to the scattered light turbidity method and the transmitted light turbidity method.

In the transmitted light turbidity method, the intensity of the transmitted light is measured and measured in the direction of the incident light (i.e0Measure the attenuation of incident light intensity caused by scattering and compare it with the incident light intensity (blank measurement). The measured characteristic is an indirect measurement of the scattering effect of suspended particles, called turbidity. Any absorption of light by suspended samples will result in additional attenuation of light intensity (see<857> Ultraviolet-Visible Spectroscopyand<1857> Ultraviolet-Visible Spectroscopy—Theory and Practice）. Therefore, it is crucial to ensure that the tested material does not absorb light at the measurement wavelength. In fact, the equations for controlling absorption and turbidity measurements are the same (although the values of attenuation constants are different). In the scattered light turbidity method, the measurement is proportional to the direction of incident light propagation90The scattered light intensity at an angle of degrees. Therefore, the turbidity measurement by scattered light turbidity method is a direct measurement of the scattering effect of suspended solids.

2. Terms and definitions

The commonly used terms to describe turbidity method and turbidity method include:

•Turbidity (symbol)S）A measure of the decrease in transmitted incident beam intensity due to the light scattering effect of suspended particles. The amount of suspended matter can be measured by observing transmitted light (turbidity method) or scattered light (turbidity method).

log I₀/I_t= kbc = T

I₀=Incident light intensity

I_t=Transmitting light intensity

k=Molar turbidity coefficient

b=Sample pool path length

c=concentration

T=turbidity

•Turbidity (symbol,T）In the measurement of transmitted light turbidity, turbidity is the intensity of the incident light beam for a given suspension/A measure of reduction in unit length. The International Organization for Standardization defines turbidity as "a decrease in the transparency of a liquid due to the presence of undissolved substances”.

•Turbidity measurement unit: The turbidity unit is represented by a descriptor that indicates the measurement method.

•Scattered light turbidity meter turbidity unit（NTU）When using the scattered light turbidity method to measure turbidity, the turbidity meter is measured in the direction of the incident light propagation90°The angle measurement of scattered light is called the scattered light turbidity unit, and the turbidity unit is called the scattered light turbidity method turbidity unit（NTU）.NTUThe size is defined based on the turbidity produced by the primary standard of fumarate (a suspension made by mixing hydrazine sulfate and hexamethylenetetramine solution in water). Safer polymer microsphere suspensions are now available on the market and are considered acceptable alternatives. However, all of these standards can be traced back to fumarate. The turbidity of the primary standard solution of fumarate is4000 NTU.

Other turbidity units include the units used in the Fumalazine turbidity method（FTU）Units for turbidity method with Formahydrazide（FNU）. These units are equivalent to0-40 NTUWithin the scopeNTU.

3. application

The application of transmitted light turbidity method and scattered light turbidity method technology includes1）Solution and/Or concentration determination of suspension (by determining several cations and anions through precipitation and suspension of precipitates produced under well controlled reaction parameters);2）Measure the turbidity of turbid solutions or suspensions;3）Determination of molecular weight1000The weight average molecular weight and size of polydisperse systems ranging from hundreds of millions;4）Measure the reaction kinetics or immunoprecipitation kinetics of immunoassays (ratio scattering turbidity method);5）Monitor the growth of cells and bacteria;6）Measurement of suspended particle size distribution, particle counting, etc.

The ratio scattering turbidity method is widely used for the analysis of vaccine components and/Or quantification of serum components. It is also used for host cell protein identification in recombinant biopharmaceuticals. When using this technology, by measuring antigens-The changes in light scattering reaction of purified antibody complexes with serum or antigen are used to calculate the effects on immune antibodies-Antigen precipitation reaction or agglutination reaction（Ag）Or antibodies（Ab）The quantity. It is usually considered to covalently link or adsorb antigens onto polymer microspheres to improve scattering efficiency; The technology generated from this is called“Particle enhanced immune analysis”Although this technique is called scattered light turbidity method, both scattered light and transmitted light are usually measured using ratio instruments.

The scattered light method for turbidity measurement is more reliable in the low turbidity range (where the concentration of the scattering medium is relatively low). Within this range, the sample concentration and detector signal intensity were observed (asNTUThere is a linear relationship between them. As the concentration increases, the incident angle of multiple scattering also increases, deviating from the linear response. Maximum support for reliable linear relationshipsNTUValue in1750–2000 NTUWithin the scope. The transmitted light turbidity method is suitable for higher turbidity ranges (concentration of scattering medium). In order to obtain consistent results, all measured variables must be carefully controlled. Where possible, extremely dilute suspensions can be measured.

4. instrumentation and meters

The instruments used for measuring transmitted light turbidity and scattered light turbidity are called transmitted light turbidity meter and scattered light turbidity meter, respectively. Usually, these instruments include a mercury lamp with a filter (for strong green or blue lines), a shutter, a set of neutral filters with known transmittance, and a sensitive photomultiplier tube that can be installed in the direction of incident light propagation0°or90°The position of the corner, or on an arm, can rotate around the solution unit and be set by a dial outside the opaque shell−135°to0°to+135°From any angle. There are various shapes of solution pools, such as those used for measurement90°Scattered squares;45°、90°and135°Scattering is a semi octagonal shape; Cylindrical scattering can be applied to all angles (see figure)1）.

image1.Representative turbidity meter. Attention, detector2Can be installed on a movable arm.

Turbidity can also be measured using a standard photoelectric filter photometer or spectrophotometer, preferably illuminated in the blue part of the spectrum. The scattered light turbidity method requires an instrument equipped with a photoelectric tube to receive scattered light, rather than transmitted light. Due to the same geometric structure used in fluorescence meters, it can be used as a turbidity meter by appropriately selecting a filter. The ratio turbidity meter is combined with90°Scattered light turbidity method and transmitted light turbidity method. It contains phototubes, which receive and measure the sample90°The scattered light from the angle, as well as the forward scattered light in front of the receiving and measuring sample. It also measures the light that directly passes through the sample. pass90°The linearity can be obtained by calculating the ratio of the measured values of angular scattered light, forward scattered light, and transmitted light. The advantage of using a ratio turbidity measurement system is that the measurement of stray light becomes negligible. In addition, the turbidity measurement of colored suspensions is only carried out using a transmission turbidity meter or turbidity meter (with ratio mode), as this program compensates for the light attenuation caused by the color of the suspension. Usually, the light source in these instruments is a tungsten lamp2700 KWorking at the filament temperature, most of the light intensity is about550 nmOther suitable light sources can also be used. Usually, detectors are▲silicon diode▲And photomultiplier tubes. Another method to eliminate color effects is to use infrared light-emitting diodes as light sources, with a maximum emission center of approximately860 nmThe spectral bandwidth is60 nm.When laser light sources are also used, especially in turbidity measuring instruments, this technique is commonly referred to as“Laser turbidity measurement”The advantage of using a laser scattering turbidity meter is that the signal-to-noise ratio is significantly improved at very low detection levels. Usually, the light source operates at a wavelength of660 nmThe laser diode. The high power density of laser beams results in higher scattering intensity for smaller particles. Combined with an optical trap that absorbs unscattered light, this system can significantly reduce stray light. When a program in a monograph instructs the use of a scattered light turbidity meter or a transmitted light turbidity meter, instruments operating in ratio mode can be used.

5. Fumalazine turbidity standard

Fumalazine is currentlyThe known main turbidity standards. All other standards are secondary and must be traced back to fumarate.The main standards are in ▲IUPAC Compendium of Chemical Terminology▲（ERR 1-May-2019）第 2 The gold book is defined as a standard prepared by users using clearly defined methods and conditions from traceable materials.

Fumalazine suspension has many characteristics to ensure its suitability as the primary standard. It can be consistently and accurately prepared from reagent grade starting materials. This suspension is composed of polymers of different lengths and random configurations, with the shapes and sizes of the polymers ranging from less than0.1 μmTo greater than10 μmDon't wait. Although the distribution of polymer chain length has been shown to vary depending on preparation, the overall turbidity results can be well reproduced.

5.1 Preparation of the Formazin StandardsPreparation of Fumalazine Standard Solution

•硫酸肼溶液：Translate into English1.000 g ACSGrade hydrazine sulfate（N₂H₄·H₂SO₄）Dissolve in100 mLofADilute to the mark with particle free water in a volumetric flask. Let the solution stand still4-6Hour.

•Primary Formalazine Standard Solution:Translate into English2.50 gAnalytical grade hexamethylenetetramine[(CH₂)₆N₄]Dissolve in25.0 mLFill in particle free water100 mLFlask. useAclass25 mLPipette addition25.0 mLHydrazine sulfate solution and mix thoroughly. Before use, allow the preparation to25±1Let it stand at a temperature of ° C48Hour. The resulting suspension can operate stably2A month.

•Fumalazine reserve standard suspension1：use15 mL AClass of pipettes, will15 mLTransfer the primary standard solution of formalin to1 LDilute to the mark with particle free water in a volumetric flask and mix. The turbidity of the obtained suspension is60 NTU.

•Fumalazine reserve standard suspension2：use50 mL AClass of pipettes, will50 mLTransfer the primary standard solution of formalin to200 mLDilute to the mark with particle free water in a volumetric flask and mix. The turbidity of the obtained suspension is1000 NTU.

•Fumalazine reference suspension:according to the table1, in100 mLMix each portion of the standard suspension of fumarate and particle free water in a volumetric flask to prepare a reference suspension of fumarate.