Eluent-based fraction collector

Abstract

To improve a degree of separation in order to utilize difference in eluant property, instead of conventional fraction collectors which require electric motors for driving.SOLUTION: A degree of separation can be compared and determined by obtaining a chromatogram with a detector and recorder attached to a liquid device. As a result, significance of this device is clarified. By changing the concentration of buffer solution, the degree of separation is improved as compared with the conventional device. That is, the purity is improved by the re-sorting technique. In order to prove merit that the product is much cheaper than a conventional fraction collector, the device is manufactured by the inventor to calculate its cost. The developed device is lightweight and can be introduced to a site.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Development of new equipment related to chromatography among measuring and analytical equipment. There are many chromatographs available on the market, and the present invention is directed to a fraction collector, the main purpose of which is fraction collection. [Background technology]

[0002] Many precision chromatography instruments are manufactured by specialized manufacturers and supplied to research institutes around the world. In recent years, the use of chromatography has expanded beyond precision analysis to include the separation and purification of components. As a method for fractionating the separated components, new fraction collectors and techniques are used that are suitable for chromatography. Most research institutes use commercially available instruments, and even if they make their own, they are simply adaptations of commercially available products. Current fraction collectors (commercially available) include motorized devices and are expensive. They are expensive for lab instructors and researchers, and there are limited places where they can be purchased. In addition, it is difficult to further increase the purity of the fractionated components, but this device technology makes it possible to increase the purity of the fractionated components. A feature of the separation mechanism is that it utilizes the specific gravity of the eluent as a fractionation technique. (Note that chromatograph is the device, chromatography is the overall method, and chromatogram is the record of emission data, and the fractionation device is called a fraction collector.) Summary of the Invention [Problem to be solved by the invention]

[0003] Conventional fraction collectors require electric motors for driving, but this device The technology is based on the specific gravity difference of the separation solution. However, the degree of separation became an issue because only the This degree of separation can be determined by obtaining a chromatogram using a detector and recorder attached to the liquid crystal device. It was necessary to compare this chromatogram with the visual chromatogram (Figure 1) of the fraction collector (glass tube) using the density difference separation of this invention. In order to make the visual chromatogram clearer, the experiment was carried out several times with different concentrations of buffer solution as the eluent. The following issues were raised: 1. Change the concentration of the buffer solution to increase the degree of resolution and record the chromatogram using a detector and recorder. To be able to compare it with. 2. When separating components, acquire separation techniques that prevent the components that have been separated from mixing together. 3. Investigating the effect of eluent concentration on gradient elution to improve separation. 4. In order to make the fraction collector more pure than conventional fraction collectors, it is necessary to separate the visual chromatogram into three concentration levels. 5. To prove that the assembly costs are significantly lower than those of conventional fraction collectors in chromatography, we will build this device ourselves and calculate the costs. [Means for solving the problem]

[0004] 1. Change the concentration of the buffer solution to increase the degree of resolution, and record the chromatogram using a detector and recorder. As a solution to make it possible to compare the two, it is important to make the component peaks appear when the concentration of the separation solution changes, and we conducted experiments to see if this effect can be seen within a certain range of eluent concentrations. 2. When separating components, you will perfect your separation techniques to learn how to prevent the components that have already been separated from mixing together. 3. To improve the degree of separation, it is important to utilize gradient elution, and an analytical experiment was conducted to determine the concentration gradient of two eluents (comparing UV detector and visual glass tube separation). In order to create a fractionation device that can improve purity more than conventional fraction collectors, it is necessary to fractionate the visual chromatogram into three levels of concentration. As a solution to this, the separated components are fractionated and divided into upper, middle, and lower layers, and the concentration of each layer is measured using an HPLC-UV detector. 4. In order to create a calculation method to confirm that the purity is higher than that of conventional fraction collectors, samples were taken from the separation zone in the preparative glass tube of the present invention at equal distances above and below the center, and a comparison calculation was made with the chromatogram from the HPLC-UV detector. 5. In order to prove that the assembly cost is significantly lower than that of conventional chromatography fraction collectors, we procured the parts for this device, built it ourselves, and calculated the cost of prototyping this device. The glass tubes for fractionation were prototyped with different diameters, small, medium, and large. They were reconfirmed by visual separation and judged using the methods 4 and 5 above. Effect of the Invention

[0005] 1. The concentration used for the eluent is phosphate buffer. 0.020~0.040 We have determined that separation is best in the molar range. The procedure for determining this range is simple and can be performed in standard chemical experiments. 2. In order to increase the purity of the fractionated parts, the glass stopcock at the bottom was narrowed and the amount of dripping was reduced so that the necessary parts could be extracted, and the fractionated liquids could be separated with almost no mixing. This operation is not complicated either. 3. To improve the degree of separation, it is important to use gradient elution. As a result of analytical experiments to determine the concentration gradient of two eluents, good results were obtained with a mixture ratio of water-soluble buffer and organic solvent methanol in the range of 12% to 58%. If the concentration of the buffer is increased too much, precipitation will occur during gradient elution. This is because there is too much difference in solubility. This time, good results were obtained with a mixture of food coloring materials, but it is expected that similar good results can be obtained with other components, such as nucleotides and vitamins, by determining the eluent and making full use of the gradient elution method. (This can be determined by comparing the chromatogram from the UV detector with the glass tube with the naked eye.) 4. In order to make a fractionation device that can increase purity more than conventional fraction collectors, visual chromatograms were fractionated into three levels of concentration and judged by experimental calculation methods, and the fraction collector of the present invention had a higher purity. This success contributes to the preparation of samples for precision analysis that requires higher purity, such as molecular structure analysis by HPLC-MSASS. Separation was performed in the fractionation glass tube of the present invention to create a calculation method to confirm the increase in purity. Samples were taken from equal distances above and below the center of the band, and a comparison was made with the HPLC-UV detector. It was clear that the purity was higher in the center of the peak. 5. It has been proven that separation and precision are improved compared to conventional fraction collectors without the need for an expensive fraction collector device. Current fraction collectors require electrical energy to operate the drive unit, but this device only requires connection to a glass column or synthetic resin column. Because it is less expensive than conventional devices, it is expected to become much more popular among various research institutions. The equipment currently costs around 320,000 to 350,000 yen, but the equipment of the present invention is much cheaper, costing roughly 10,000 to 20,000 yen. In addition, with conventional fraction collectors, it was necessary to change the type of fraction collection time and pump flow rate to determine the amount of fraction, but with the new method, a wide range of fraction collection can be freely performed by changing the size and length of the fraction collection glass tube. This led to the development of a portable fraction collector that can be carried to each device. Another fraction collection technique that can further increase purity is to attach a septum to the column. [Brief description of the drawings]

[0006] [Figure 1] Actual photo of fraction [Diagram 2] Schematic diagram of the new fraction collector [Diagram 3] Chromatogram and separation zone in glass tube [Figure 4] Photograph of the median strip in Example 1 [Diagram 5] Separation strip of Example 3 [Figure 6]Schematic diagram of a glass tube with a septumThe experimental flow is almost the same as that of a normal liquid chromatograph, with piping connected to a fraction collector after the detector.Normal chromatography glass tubes can be used for the fractionation glass tube, but to improve accuracy, it is better to put a rubber stopper on the top of the glass tube and provide an air hole, and the tap at the bottom should be one that can handle minute amounts.Figures 1 and 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The chromatography may be carried out by any commercially available device, and the piping for the fraction collector may be the same as that used for ordinary eluents. The glass tube for the fraction collector can be a general-purpose chromatography tube, but since there is no need for an attached filter at the bottom, it is better to use a regular glass tube with a tap at the bottom and a fine tap that can adjust the flow rate. It is even better to use a silicon rubber stopper at the top and open an air hole and piping hole. The equipment used is shown in the specific drawings and floor plans. All of the equipment is not special and is available commercially for educational purposes, as it is found in most chemistry laboratories. Specific equipment and procedures are described. 1. Connect two gradient eluents (two eluents with different specific gravities) to a pump in a normal chromatograph, and connect a pipe to a fraction collector at the detector outlet. Then start the experiment. 2. Close the bottom cock of the glass tube and gradually collect the eluate in the glass tube. As the elution progresses, the components separate and separate layers are formed. After the analysis is completed, stop the liquid delivery pump. Then open the bottom cock and gradually collect the separated layers. (It is best to use minute drops for fractionation.) 3. Check the separation accuracy by comparing the chromatogram on the recorder with the separation layer in the preparative glass tube. EXAMPLES

[0008] Fractionation of food additive color mixture "Experimental conditions" (Solution 1) 20 mmol phosphate buffer, 5% methanol solution, pH 6.25 (2nd solution) 1% acetic acid, 95% methanol aqueous solution Linear gradient elution: 24 minutes from solution 1 to solution 2 Flow rate 1.0ml ColumnODS column (apparatus / equipment) Conventional liquid chromatograph and preparative glass tubes (inner diameter 10mm, total length 200mm) "sample" Contains 5 types of food coloring A table of resolution (Rt) for the five dyes was prepared. [Table 1] Compared to the chromatogram from the detector, there is almost no difference in the component separation of this device, and the data is sufficiently reflected, with the components being equivalent and pure, but there is also a slight improvement in separation. Most of the improvement in separation is seen in the separation zone. EXAMPLES

[0009] Differences in resolution due to differences in linear gradient "Experimental conditions" (Solution 1) 20 mmol phosphate buffer, 5% methanol solution, pH 6.25 (2nd solution) 1% acetic acid, 95% methanol aqueous solution Gradient elution method A: 5% 1st solution → 80% 1st solution (25 min) Gradient elution method B: 12% 1st solution → 95% 1st solution (25 min) Flow rate 1.0ml ColumnODS column (apparatus / equipment) A conventional liquid chromatograph and a preparative glass tube (inner diameter 10 mm, total length 200 mm) [Table 2] Gradient elution method II has a slightly improved degree of separation. Method B shows that the concentration gradient is suitable for the components in this experiment. The gradient elution settings are also important for other components, and by selecting appropriate elution conditions, a layer with good separation can be obtained, and the degree of purification of the components can be increased. Figure 4. EXAMPLES

[0010] Resolution due to difference in buffer concentration "Experimental conditions" 1, low concentration (Solution 1) 15mmol phosphate buffer, 5% methanol solution, pH 6.25 (2nd solution) 1% acetic acid, 95% methanol aqueous solution Gradient elution method A: 5% 1st solution → 80% 1st solution (25 min) Gradient elution method B: 12% 1st solution → 95% 1st solution (25 min) 2, high concentration (Solution 1) 40mmol phosphate buffer, 5% methanol solution, pH 6.25 (2nd solution) 1% acetic acid, 95% methanol aqueous solution gradient elution method A 1st solution 5% → 1st solution 80% (25 min) gradient elution method Flow rate 1.0ml Column ODS column (apparatus / equipment) A conventional liquid chromatograph and a preparative glass tube (inner diameter 10 mm, total length 200 mm) [Table 3] The higher the concentration of the gradient eluent, the better the resolution. The higher the concentration gradient, the better the separation of the separation zones. This shows that this technique is suitable for this experiment. There is a large difference between B and C, and between C and D. For other components as well, the eluent settings for gradient elution are important, and by selecting appropriate elution conditions, layers with good separation can be obtained. [Industrial Applicability]

[0011] Liquid chromatography is an important analytical tool indispensable for scientific research and is used in research institutions around the world. The fraction collector of the present invention is an essential tool for extracting useful components from a mixture. After separating the useful components, they are used for further research, such as bioactivity measurements and molecular structure analysis. As shown in the drawing, conventional devices are electric and expensive, but the cost of this device is significantly cheaper and it is expected that it will become widespread in laboratories around the world. Furthermore, because separation is based on the density difference of the eluent, it is possible to re-fraction and to perform more precise fractionation than was possible with conventional devices. Therefore, the post-dissolution type fraction collector of this invention can be effectively used in industry immediately. This device and technology are suitable for precision analysis and the separation and purification of useful substances. It can also be used as a portable fraction collector that can be carried to each device. By attaching a septum to the glass tube, it is possible to extract the sample more accurately, which is useful for more precise analysis. [Explanation of symbols]

[0012] A: Sample separation zone B: Sample separation zone C: Sample separation zone D: Sample separation zone E: Sample separation zone S... Start (1) Syringe septum

Claims

1. A fraction collector comprising a glass tube and an outlet opening at the bottom of the glass tube, said fraction collector being used to drip liquid from a chromatograph into said glass tube, said dripped liquid being separated in the downward direction due to differences in the downward speed of each component caused by their specific gravity, and said separated components being sequentially collected in a separated state from said outlet.

2. 2. The fraction collector according to claim 1, wherein the glass tube further comprises a septum, and the components separated in the glass tube can be extracted through the septum.

3. A method for collecting a separated liquid, comprising the steps of: dropping a separation liquid, in which a sample has been separated into its components by chromatography, into a glass tube; stacking the dropped separation liquids in the glass tube without mixing with each other; further promoting separation of the stacked separation liquids due to differences in specific gravity; and collecting each of the separation liquids whose separation has been promoted in a separated state.

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