Determination of Settlement Coefficient of Centrifuge

The determination of sedimentation coefficient is the most important use of centrifuge analysis. Usually only a few tens of milligrams or even tens of micrograms of sample are required to prepare a solution of 1 to 2 milliliters, which is loaded into an analysis cell and centrifuged for a few hours to obtain a series of centrifugal sedimentation charts. According to the sedimentation diagram, it can be used for qualitative analysis of the components contained in the sample, as well as for the determination of the sedimentation coefficient and estimated molecular size of each component, for sample purity verification and non-uniformity determination, based on the relative content of the components.

1. Principle

The principle of measuring the sedimentation coefficient is to determine the sedimentation velocity of the sample particles under a constant centrifugal force field. Because the sample particles are so small that their sedimentation motion cannot be directly seen, the interface movement speed of the sample particles during centrifugation is regarded as the average sedimentation speed of the sample particles. Schlieren and absorption optics are commonly used to record interface settlement maps. In the sedimentation diagram, the sample interface generally shows a symmetrical peak, and the highest point of the peak represents the position of the interface. Usually the measurement accuracy of the settlement coefficient is ± 2%, but if the surface boundary pattern shows an asymmetric peak shape, or it is desired that the measurement accuracy of the settlement coefficient reaches ± 1% or less, it is not enough to use the highest point of the peak as the interface position At this time, the second-order distance method should be used to calculate the interface position.

2. Examples of determination of settlement coefficient

â‘´Sample: bovine serum albumin

⑵Sample solution and centrifugation: bovine serum albumin was dissolved in 0.14M NaCl and 0.01M phosphate buffer at a concentration of 0.6%, pH 7.0. Using a 12mm thick dual-slot analysis cell, add solvent while adding the solution, about 0.3ml each. The balance weight of the analysis cell and the balance cell is to make the balance cell less than 0.5g lighter than the analysis cell, and then put them into the analysis rotor respectively. The analysis rotor is installed to analyze the reason of centrifugation, close the rotation cavity and evacuate. Turn on the Schlieren light source, choose a working speed of 60,000 rpm, and centrifuge at room temperature. After the rotating chamber reaches a vacuum, the machine starts to accelerate and the centrifugal pattern can be seen in the observation window. After reaching the working speed, it is constant speed centrifugation. After seeing the tip of the sample peak, you can take photos at 6-minute intervals and take a total of 6 photos. Turn off the camera after taking the photos, remove the sample liquid, and clean the rotor and analysis cell. The photographs of Schlieren light path settlement graphics were obtained after the photographs were developed and developed with high contrast.

⑶ Subsidence image measurement: Schlieren's subsidence map can be measured with a specific length meter, reading microscope, or projector. The measured lateral range is required to be 6cm, and the measurement accuracy should be better than 10μm. Usually the reading microscope can meet the requirements. The projector can enlarge the image on the screen, it is easier to read and measure, and the eyes are not easy to fatigue. When measuring, put the negative film of the settlement image on the measuring instrument so that the vertical line of the liquid surface coincides with the vertical line in the measuring instrument, and then use the cross mark to measure the internal reference hole, liquid surface, interface peak tip, and external reference hole in sequence The position of each image is read at least three times and the average value is taken. Each image is measured in the same way in turn, and the data is listed in Table 5.

(4) Calculation of settlement coefficient S: After measuring the settlement graph, first check the x2-x1 values ​​of each graph and the sixth graph. The difference between the x2-x1 values ​​of the two graphs should be less than the measurement error of the interface peak position. The measured difference between x2-x1 is 0.002, indicating that the liquid level changes to 0.002 mm within 30 minutes of centrifugation. The movement distance of the interface peak in 30 minutes is x1-x3 in the sixth figure minus x1-x3 in the first figure, which is 3.208mm, and the peak position measurement means that it is allowed to be 0.03. The inland liquid level variation of 0.002mm is much smaller than the peak position measurement error of 0.03mm, indicating that no leakage occurred during centrifugation.

Press a = (x4-x1) /1.7 to calculate the magnification of the optical path to the analysis cell, then press (x3-x1) / a to calculate the actual distance of the interface peak from the inner reference hole, and then press x = 5.65 + (x3- x1) / a calculate the actual distance of the interface peak from the center of rotation, and then calculate the logx plant by the logarithm table. The calculated data are also listed in Table 5.

3. Analysis of centrifugal images

When the peak of rapid sedimentation is seen at the beginning of centrifugation, it will reach the bottom of the analysis cell within a few minutes. Generally, it is due to the partial polymerization of the sample to form a rapidly sedimented polymer, such as when the protein sample solution encounters some denaturation factors Will partially denature and precipitate. The centrifugal image of the sample after centrifugation reaches a speed shows a symmetrical peak shape, and the sample is generally considered to be uniform in centrifugation. But the true homogeneity of the sample should be further tested by other methods, such as electrophoresis and chromatography. Some mixed samples also occasionally give a symmetrical peak. The peak shape usually expands with time, which is the result of sample diffusion. But if the peak shape expands quickly, the sample may be polydisperse. If there are several peaks in the centrifugal image, it indicates that there are several components in the sample, and each peak represents the sedimentation interface of the corresponding component, so the sedimentation coefficient value of each component can be determined. The concentration value of the component can be measured according to the area of ​​the peak.

Sometimes the centrifuged image shows an asymmetric peak, which may be caused by the following conditions. â‘ The peak shapes of several components with close sedimentation coefficients overlap, â‘¡The sample is polydisperse, and its molecular weight distribution is uneven. â‘¢Some strong interaction polymers, the sedimentation rate of which depends heavily on the concentration, such as DNA, polysaccharides Molecules, if measured at high concentrations, have an asymmetric distribution of peak shapes in the interface area due to inconsistent settling velocity due to concentration changes. This type of sample even forms a very sharp interface at higher concentrations, which appears as a vertical line in the centrifugal sedimentation diagram, and no peak shape can be seen. The error in determining the settlement coefficient based on this pattern will be large. Usually this kind of sample should be measured at a very dilute concentration. Under this condition, the molecular interaction becomes smaller and the peak shape of the interface expansion can measure the correct sedimentation coefficient.

4. Determination of sample purity and concentration

Analytical centrifuges can determine the purity and concentration of samples, which is also used for analytical centrifugation. Generally, when the centrifugal pattern of a sample shows a symmetrical peak shape, it can be considered that the sample is uniform in centrifugation. However, when making the purity determination, it is necessary to pay attention to the measurement concentration of the sample. The measurement concentration of the sample should be more than one hundred times greater than the sensitivity of the measurement method to make the measurement meaningful. For example, if the Schlieren optical path determines the sensitivity of the sample to be 0.1 mg / ml, then the concentration of the sample when measuring purity should be 90 mg / ml. If the sample concentration of 1 mg / ml is used for the determination, even if the sedimentation pattern shows a single peak, it is still impossible to determine whether its purity is 0% or 99%. If the sample is multi-component, several peaks appear in the centrifugal graph, and the relative concentration value of each component can be obtained according to the ratio of the area of ​​each peak to the total area of ​​all peaks. In the calculation, the radiation dilution effect should be considered, that is, the area value of each component should be corrected to the value at the liquid level according to its location using the radiation dilution formula, and then the relative calculation should be calculated. Also note that such concentration estimates are based on the assumption that the components have the same refractive index ratio. According to the peak shape area combined with the concentration and refractive index ratio of the component and the instrument constant, the absolute concentration value of the group can also be calculated.

5. Band settlement method

The band settlement method is similar to the rate zone centrifugation method. Methods A synthetic interface cell was used. 0.55ml of 1M NaCl solution was added to the cell in advance, then 0.01ml of sample solution was spread at 2000 rpm, and then accelerated to 30,000 rpm, and the sedimentation pattern of the sample was recorded with the absorption light path. Due to the NaNl-like sample, the product band diffuses to form a density gradient zone, which can stabilize the sample zone to settle. If there are multiple components with different settlement coefficients in the sample, they will be zoned settlement according to their respective settlement velocity. Using this method, the purity of the sample can be verified, and the relative content and sedimentation coefficient of the composition can be estimated. It is suitable for the analysis and determination of trace amounts of nucleic acid and virus samples.

The centrifuge is a special instrument that uses centrifugal force to separate and precipitate the mixed liquid (containing solids). Commonly used electric centrifuges in the laboratory include low-speed, high-speed centrifuges and low-speed and high-speed refrigerated centrifuges, as well as various models such as overspeed analysis and preparation of dual-purpose refrigerated centrifuges. Among them, low-speed (including large-capacity) centrifuges and high-speed refrigerated centrifuges are the most widely used and are indispensable and important tools for biochemical laboratory separation and preparation of biological macromolecules. In the course of the experiment, to separate the precipitate from the mother liquor, filtration and centrifugation are often used. But in the following cases, the use of centrifugal method is better

â‘  The precipitate is sticky or the mother liquor is sticky.

â‘¡The precipitated particles are small and easy to penetrate the filter paper.

â‘¢ Too much sediment and loose.

â‘£The amount of precipitation is very small and needs to be quantitatively determined. Or the amount of mother liquor is very small, the loss should be reduced during separation.

⑤ Precipitation and mother liquor must be separated quickly.

â‘¥General colloid solution.

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