A method for extracting, isolating, and purifying ascomycin from mycelium

Abstract

This invention discloses a method for extracting, separating, and purifying ascomycin from mycelia. The method combines extraction, macroporous resin adsorption, and reverse polymer chromatography to separate ascomycin and its main impurity, demethylascomycin, from mycelia, ultimately yielding ascomycin with a purity of over 98%. This method exhibits high impurity removal rate and excellent separation effect. It has significant advantages in the pharmaceutical field and is suitable for industrial application.

Description

Technical Field

[0001] This invention relates to a method for extracting, separating, and purifying ascomycin from mycelium, belonging to the field of antibiotic extraction and purification technology. Background Technology

[0002] Ascomycin (also known as FK-520, FR-900520) is an ethyl analogue of the immunosuppressant tacrolimus (FK-506). It was isolated in 1962 by Fujisawa Pharmaceutical Co., Ltd. of Japan from soil containing *Streptomyces hygroscopicus* No. KK317. Its molecular formula is C2. 43 H 69 NO 12 It is a white crystalline solid with a melting point of 158-161℃. It is almost insoluble in water, slightly soluble in n-hexane and petroleum ether, and soluble in organic solvents such as methanol, ethanol, acetone, ethyl acetate, and chloroform. Studies have found that ascomycin has a strong immunosuppressive effect and can be used to treat autoimmune diseases such as atopic dermatitis, allergic contact dermatitis, psoriasis, lupus erythematosus, lichen planus, vitiligo, Netherton syndrome, and host-suppressing diseases.

[0003] In recent years, there has been increasing research on ascomycin in China. Most studies employ liquid-liquid extraction and macroporous resin-silica gel column chromatography for the separation and purification of ascomycin. A common problem with these two methods is the use of toxic and harmful solvents, which pose significant risks, the complexity of the procedures, and the low yield. Therefore, further research on the separation, purification, and preparation of ascomycin is necessary.

[0004] Most current research on ascomycin focuses on its fermentation, development of derivatives, antibacterial activity, and clinical applications. The main challenges in the current isolation and extraction of ascomycin are its low yield and high separation costs.

[0005] Ascomycin is generally removed from tacrolimus as an impurity. However, with ongoing research, it has been discovered that ascomycin can also function independently, possessing various biological and pharmacological values. Therefore, considering the complexity of removing demethylascomycin (FK523), a similar component produced during the fermentation process of ascomycin, during extraction and purification, the necessity of improving the purity and yield of ascomycin becomes increasingly prominent. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies in purifying and separating ascomycin-containing mycelia, which involve numerous impurities, high separation difficulty, and complex, time-consuming, and low-purity and low-yield purification methods. This invention provides a method for extracting, separating, and purifying ascomycin from mycelia.

[0007] To achieve the above objectives, the present invention provides a method for extracting, isolating, and purifying ascomycin from mycelium, comprising the following steps:

[0008] Step 1) Extraction: Weigh the mycelium containing ascomycin, grind it into powder, dissolve it in an organic solution, and extract it by ultrasonic disruption 2 to 4 times. After extraction, centrifuge, combine the filtrates, and obtain the sample solution.

[0009] Step 2) Macroporous resin adsorption and desorption: Load the sample solution obtained in step 1) onto the macroporous resin column, collect the effluent, and stop loading when the concentration of the effluent is the same as that of the sample solution (i.e., the resin is saturated). Recover the effluent with the same concentration as the sample solution. Desorb using an organic solvent aqueous solution, collect the desorbed liquid, evaporate to dryness, and perform liquid phase detection.

[0010] Step 3) Reverse polymer packing chromatography: Dissolve the sample obtained in step 2) by rotary evaporation, mix it with reverse polymer packing and rotary evaporation, and dry-load the sample onto the reverse polymer packing column. Use 20% to 90% organic solvent aqueous solution (pH 2 to 7) as the mobile phase for gradient elution. Collect the eluent and rotary evaporation to obtain purified ascomycin.

[0011] In some embodiments of the present invention, the organic solution in step 1) is selected from at least one of aqueous solutions of acetone, methanol, ethanol, and butanol, preferably an aqueous solution of methanol or ethanol, with a concentration of 20-80% (V / V); the extraction time for each extraction is 1-6 hours, and the centrifugation conditions are: centrifugation at a speed of 4000-6000 rpm for 10-60 minutes.

[0012] In some embodiments of the present invention, the column packing process of the macroporous resin column in step 2) includes: firstly, using macroporous adsorption resin to wet-load the column with 5-8 BV of organic solvent at a flow rate of 1-6 BV / h; then, replacing the ethanol with deionized water at a flow rate of 1-6 BV / h.

[0013] In some embodiments of the present invention, the macroporous adsorption resin selected for the macroporous resin column in step 2) is selected from polymethacrylate type, polyacrylate type or polystyrene / divinylbenzene chromatography type resin, preferably one of HZ-806, HZ-816, HZ-818, HZ-826 and HZ-828.

[0014] In some embodiments of the present invention, the sample loading flow rate in step 2) is 2-6 BV / h, preferably 4 BV / h; the desorption solution is 50-90% (V / V) alcohol-water solution, preferably 50-75% (V / V) alcohol-water solution; and the desorption flow rate is 0.5-5 BV / h, preferably 1-3 BV / h.

[0015] In some embodiments of the present invention, the column packing process of step 3) of the reverse polymer packed column includes: pre-washing the reverse polymer packing with acetone, then replacing the acetone with deionized water, filtering and drying it for later use; and then dry loading the packing onto the column to complete the column packing.

[0016] In some embodiments of the present invention, the organic solvent in step 3) is selected from at least one of ethanol, acetonitrile, acetone and methanol, and the gradient elution process is as follows: first, prewashing is performed with an aqueous solution of 20% to 50% of the above organic solvent (pH 2 to 7), and then elution is performed with an aqueous solution of 50% to 90% of the above organic solvent (pH 2 to 7); the elution flow rate is 0.5 to 5 BV / h, preferably 1 to 3 BV / h.

[0017] Preferably, the organic solvent in step 3) is an aqueous acetonitrile solution, and the gradient elution process is as follows: first, pre-wash with a 30% to 50% aqueous acetonitrile solution (pH 2 to 4), and then elute with a 50% to 60% aqueous acetonitrile solution (pH 2 to 4).

[0018] In some embodiments of the present invention, the sample loading amount in step 3) is 0.1 to 5% of the sample mass / the mass of the column packing material, preferably 1 to 3% (sample mass / mass of the column packing material).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) This invention separates and purifies ascomycin from mycelia containing ascomycin by ultrasonic extraction, macroporous resin adsorption, and reverse polymer packed column separation. It can effectively separate and remove the main impurity demethylascomycin, and the obtained ascomycin has an HPLC purity of over 98% and a high yield.

[0021] 2) Through extensive experimentation, this invention has found that using a reverse polymer packed column and a suitable elution system results in high sample purity and high recovery rate, which greatly simplifies the process and improves separation efficiency.

[0022] 3) Compared with the prior art, the method of the present invention can significantly shorten the extraction time and improve efficiency. It has many advantages such as simple process, increased environmental protection (significant reduction of toxic reagents) and low cost. It is easy to industrialize and provides a feasible and efficient separation process for the production of ascomycin. Attached Figure Description

[0023] Figure 1 HPLC chromatogram of ascomycin in Streptomyces hygroscopicus;

[0024] Figure 2 The image shows the HPLC chromatogram of the ascomycin isolated and purified in Example 1. Detailed Implementation

[0025] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0026] Unless otherwise specified, the raw materials, reagents, instruments, and equipment involved in the following examples can all be obtained through conventional commercial channels. Specifically, *Streptomyces hydrophila* was provided by Shanghai Baipuda Biotechnology Co., Ltd., and the macroporous resins HZ-806, HZ-816, HZ-818, HZ-826, and HZ-828, as well as the reverse polymer filler (brand name: MONO HZ30), were all purchased from Shanghai Huazhen Technology Co., Ltd.

[0027] Example 1

[0028] Streptomyces hydrophila were ground into powder, dissolved in 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, extracted 3 times, and centrifuged at 5000 rpm for 15 min to obtain the extract. The extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then flowed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. 2 The sample was washed with a 37% (v / v) ethanol aqueous solution. It was then desorbed using a 5 BV 70% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness to obtain an oily sample. The macroporous resin was regenerated using 5 BV ethanol at a flow rate not exceeding 5 BV / h. The reverse polymer packing was activated with 10-15 BV acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water. The packing was dried and loaded onto the column using a dry method. 2% (sample mass / packing material mass) of the sample was mixed with 2 g of packing material and evaporated to dryness. A gradient elution method was used: pre-washing with a 17 BV 40% acetonitrile aqueous solution (adjusted to pH 3 with phosphoric acid, the same below), followed by elution with a 55% acetonitrile aqueous solution (pH 3) at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness. The purity of the ascomycin obtained was 99.15%, and the yield was 92.77%. Figure 1 HPLC chromatogram of ascomycin in Streptomyces hygroscopicus; Figure 2 This is the HPLC chromatogram of the purified ascomycin isolated in this embodiment.

[0029] Example 2

[0030] Streptomyces hydrophila were ground into powder, dissolved in 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, extracted 3 times, and centrifuged at 5000 rpm for 15 min to obtain the extract. The extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then flowed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. 2 The sample was washed with a 37% (v / v) ethanol aqueous solution. It was then desorbed using a 5 BV 70% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness to obtain an oily sample. The macroporous resin was regenerated using 5 BV ethanol at a flow rate not exceeding 5 BV / h. The reverse polymer packing was activated with 10-15 BV acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water. The packing was dried and loaded onto the column using a dry method. 3% (sample mass / packing material mass) of the sample was mixed with 2 g of packing material and evaporated to dryness. A gradient elution method was used: pre-washing with a 17 BV 40% acetonitrile aqueous solution (pH 3), followed by elution with a 55% acetonitrile aqueous solution (pH 3) at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness. The purity of the ascomycin obtained was 98.92%, and the yield was 90.23%.

[0031] Example 3

[0032] Streptomyces hydrophila were ground into powder, dissolved in 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, extracted 3 times, and centrifuged at 5000 rpm for 15 min to obtain the extract. The extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then flowed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. 2 The sample was washed with a 37% (v / v) ethanol aqueous solution. It was then desorbed using a 5 BV 70% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness to obtain an oily sample. The macroporous resin was regenerated using 5 BV ethanol at a flow rate not exceeding 5 BV / h. The reverse polymer packing was activated with 10-15 BV acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water. The packing was dried and loaded onto the column using a dry method. 1% (sample mass / packing material mass) of the sample was mixed with 2 g of packing material and evaporated to dryness. A gradient elution method was used: pre-washing with a 17 BV 40% acetonitrile aqueous solution (pH 3), followed by elution with a 55% acetonitrile aqueous solution (pH 3) at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness. The purity of the ascomycin obtained was 98.58%, and the yield was 90.78%.

[0033] Example 4

[0034] Streptomyces hydrophila were ground into powder, dissolved in 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, extracted 3 times, and centrifuged at 5000 rpm for 15 min to obtain the extract. The extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then flowed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. 2 The sample was washed with a 37% (v / v) ethanol aqueous solution. It was then desorbed using a 5% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness to obtain an oily sample. The macroporous resin was regenerated using 5 BV ethanol at a flow rate not exceeding 5 BV / h. The reverse polymer packing was activated with 10-15 BV acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water. The packing was dried and loaded onto the column using a dry method. 3% (sample mass / packing material mass) of the sample was mixed with 2 g of packing material and evaporated to dryness. A gradient elution method was used: pre-washing with a 17 BV 20% acetonitrile aqueous solution (pH 6), followed by elution with a 90% acetonitrile aqueous solution (pH 6) at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness. The purity of the ascomycin obtained was 88.35%, and the yield was 86.32%.

[0035] Example 5

[0036] Streptomyces hydrophila were ground into powder, dissolved in 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, extracted 3 times, and centrifuged at 5000 rpm for 15 min to obtain the extract. The extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then flowed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. 2 The sample was washed with a 37% (v / v) ethanol aqueous solution. It was then desorbed using a 5 BV 90% (v / v) ethanol aqueous solution at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness to obtain an oily sample. The macroporous resin was regenerated using 5 BV ethanol at a flow rate not exceeding 5 BV / h. The reverse polymer packing was activated with 10-15 BV acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water. The packing was dried and loaded onto the column using a dry method. 3% (sample mass / packing material mass) of the sample was mixed with 2 g of packing material and evaporated to dryness. A gradient elution method was used: pre-washing with a 17 BV 50% acetonitrile aqueous solution (pH2), followed by elution with a 60% acetonitrile aqueous solution (pH2) at a flow rate of 2 BV / h. The eluent was collected and evaporated to dryness. The purity of the ascomycin obtained was 86.83%, and the yield was 85.28%.

[0037] Comparative Example 1

[0038] Streptomyces hygroscopicus was ground into powder, dissolved in a 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, and extracted three times. The extracts were obtained by centrifugation at 5000 rpm for 15 min, and the extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then passed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. The sample was washed with 2 BV of 37% (V / V) ethanol aqueous solution; it was then desorbed with 5 BV of 70% (V / V) ethanol aqueous solution at a flow rate of 2 BV / h, and the eluent was collected and evaporated to obtain an oily sample; the macroporous resin was regenerated with 5 BV of ethanol at a flow rate not exceeding 5 BV / h; the reverse polymer packing was activated with 10-15 BV of acetone until the effluent did not turn white upon contact with water, and then the acetone was replaced with deionized water; the packing was dried and loaded onto the column using a dry method, and the sample was dissolved and loaded using a wet method with a loading amount of 0.5% (sample mass / packing material mass); the sample was eluted with 5 BV of 80% ethanol aqueous solution at a flow rate of 0.5 BV / h, and the eluent was collected and evaporated to obtain an ascomycin with a purity of 88.46% and a yield of 70.41%.

[0039] Comparative Example 2

[0040] Streptomyces hygroscopicus was ground into powder, dissolved in a 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, and extracted three times. The extracts were obtained by centrifugation at 5000 rpm for 15 min, and the extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then passed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. V / h; Wash with 2 BV of 37% (V / V) ethanol aqueous solution; Desorb with 5 BV of 70% (V / V) ethanol aqueous solution at a flow rate of 2 BV / h, collect the eluent and evaporate to dryness to obtain an oily sample; Regenerate the macroporous resin with 5 BV of ethanol at a flow rate not exceeding 5 BV / h; Activate the reverse polymer packing with 10-15 BV of acetone until the eluent does not turn white upon contact with water, then replace the acetone with deionized water; Dry-load the packing with dry method, and wet-load the sample by dissolving it with 3% (sample mass / packing material mass); Elute with 5 BV of 40% acetonitrile aqueous solution at a flow rate of 2 BV / h, collect the eluent and evaporate to dryness, the purity of the obtained ascomycin is 94.24%, and the yield is 89.62%.

[0041] Comparative Example 3

[0042] Streptomyces hygroscopicus was ground into powder, dissolved in a 37% (v / v) ethanol aqueous solution, ultrasonically disrupted for 1.5 h, and extracted three times. The extracts were obtained by centrifugation at 5000 rpm for 15 min, and the extracts were combined to obtain the loading solution. Macroporous resin HZ-816 was packed with 5-8 BV ethanol using a wet packing method, and the flow rate was 1-2 BV / h. Deionized water was then passed through the resin layer at a flow rate of 2-3 BV / h to displace the ethanol. The loading solution obtained from the extraction was loaded onto the macroporous resin column until the concentration of ascomycin in the effluent was consistent with the concentration in the loading solution, i.e., when the resin was saturated. The loading flow rate was 4 BV / h. V / h; Wash with 2 BV of 37% (V / V) ethanol aqueous solution; Desorb with 5 BV of 70% (V / V) ethanol aqueous solution at a flow rate of 2 BV / h, collect the eluent and evaporate to dryness to obtain an oily sample; Regenerate the macroporous resin with 5 BV of ethanol at a flow rate not exceeding 5 BV / h; Activate the reverse polymer packing with 10-15 BV of acetone until the effluent does not turn white upon contact with water, then replace the acetone with deionized water; Dry-load the packing with dry method, and wet-load the sample by dissolving it with 2% (sample mass / packing material mass); Elute with 5 BV of 45% acetonitrile aqueous solution at a flow rate of 2.5 BV / h, collect the eluent and test its purity, combine the eluents according to the purity test results and evaporate to dryness, the purity of the obtained ascomycin is 92.19%, and the yield is 78.06%.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A process for the extraction of an isolated mycophenolic acid from mycelium, characterized in that, The method comprises the following steps: Step 1) extraction: the mycelium containing ascomycin is weighed, ground into powder, dissolved in an organic solution, and extracted by ultrasonic crushing for 2-4 times, then centrifuged, and the filtrates are combined to obtain a sample solution; Step 2) macroporous resin adsorption and desorption: the sample solution obtained in step 1) is loaded onto a macroporous resin column, and the effluent is collected; when the concentration of the effluent is consistent with that of the sample solution, i.e. the resin adsorption is saturated, the loading is stopped, and the effluent with the same concentration as the sample solution is recovered; an organic solvent aqueous solution is used for desorption, and the desorption solution is collected, dried, and detected by liquid chromatography; the macroporous adsorption resin selected for the macroporous resin column is selected from poly methacrylate, polyacrylate, or polystyrene / divinylbenzene chromatographic resin; Step 3) reverse phase polymer filler column chromatography: the sample obtained by drying in step 2) is dissolved, mixed with reverse phase polymer filler MONOHZ30, and dried, and then loaded onto a reverse phase polymer filler column by dry method; a gradient elution is performed using a PH 2-4 acetonitrile aqueous solution as the mobile phase, and the desorption solution is collected and dried to obtain purified ascomycin; the gradient elution process is as follows: first, a 30%-50% acetonitrile aqueous solution is used for pre-washing, and then a 50%-60% acetonitrile aqueous solution is used for elution.

2. The method of claim 1, wherein, The organic solution in step 1) is selected from any one of an aqueous solution of acetone, methanol, ethanol, and butanol; the time for each extraction is 1-6 h, and the centrifugation conditions are as follows: centrifugation at a speed of 4000-6000 rpm for 10-60 min.

3. The method of claim 1, wherein, The column loading process of the macroporous resin column in step 2) comprises the following steps: using macroporous adsorption resin, first wet loading is performed using 5-8 BV of an organic solvent at a flow rate of 1-6 BV / h; and then deionized water is used to replace the ethanol at a flow rate of 1-6 BV / h.

4. The method of claim 1, wherein, The macroporous adsorption resin is selected from one of HZ-806, HZ-816, HZ-818, HZ-826, and HZ-828.

5. The method of claim 1, wherein, The loading flow rate in step 2) is 2-6 BV / h; the desorption solution is a 50-90% V / V alcohol aqueous solution; and the desorption flow rate is 0.5-5 BV / h.

6. The method of claim 1, wherein, The column loading process of the reverse phase polymer filler column in step 3) comprises the following steps: the reverse phase polymer filler is pre-washed using acetone until the outflowing acetone does not turn white when it meets water, then deionized water is used to replace the acetone, and the filler is filtered and dried for later use; the filler is loaded onto the column by dry method to complete the column loading.

7. The method of claim 1, wherein, The organic solvent in step 2) is selected from at least one of ethanol, acetonitrile, acetone, and methanol, and the elution flow rate is 0.5-5 BV / h.

8. The method of claim 1, wherein, In step 3), the sample is loaded in a proportion of 0.1-5% of the sample mass to the mass of the column filler.

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