KR20120108816A - Extacting method for humic acid and fulvic acid from humic substances - Google Patents
Extacting method for humic acid and fulvic acid from humic substances Download PDFInfo
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- 239000004021 humic acid Substances 0.000 title claims abstract description 55
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 239000002509 fulvic acid Substances 0.000 title abstract description 42
- PUKLDDOGISCFCP-JSQCKWNTSA-N 21-Deoxycortisone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2=O PUKLDDOGISCFCP-JSQCKWNTSA-N 0.000 title abstract description 41
- FCYKAQOGGFGCMD-UHFFFAOYSA-N Fulvic acid Natural products O1C2=CC(O)=C(O)C(C(O)=O)=C2C(=O)C2=C1CC(C)(O)OC2 FCYKAQOGGFGCMD-UHFFFAOYSA-N 0.000 title abstract description 41
- 229940095100 fulvic acid Drugs 0.000 title abstract description 41
- 239000000126 substance Substances 0.000 title abstract description 23
- 238000000034 method Methods 0.000 title abstract description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 30
- 238000000605 extraction Methods 0.000 claims abstract description 25
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000243 solution Substances 0.000 claims abstract description 17
- 239000002244 precipitate Substances 0.000 claims abstract description 14
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000003513 alkali Substances 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000002198 insoluble material Substances 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- 239000000463 material Substances 0.000 claims description 31
- 239000012528 membrane Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 2
- 238000005070 sampling Methods 0.000 abstract 2
- 239000000843 powder Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 10
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 238000011088 calibration curve Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 150000007524 organic acids Chemical class 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 235000005985 organic acids Nutrition 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000000975 bioactive effect Effects 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000004442 gravimetric analysis Methods 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 238000010306 acid treatment Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- QPJSUIGXIBEQAC-UHFFFAOYSA-N n-(2,4-dichloro-5-propan-2-yloxyphenyl)acetamide Chemical compound CC(C)OC1=CC(NC(C)=O)=C(Cl)C=C1Cl QPJSUIGXIBEQAC-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H6/00—Macromolecular compounds derived from lignin, e.g. tannins, humic acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0292—Treatment of the solvent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/02—Details relating to pores or porosity of the membranes
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Abstract
Description
본 발명은 천연 휴믹 물질로부터 생리 활성 유기산 추출방법, 보다 상세하게는 종래의 추출 방법을 개량한 천연 휴믹 물질로 부터 생리 활성 유기산인 휴믹산과 풀빅산 추출방법에 관한 것이다. The present invention relates to a method for extracting physiologically active organic acids from natural humic substances, and more particularly, to a method for extracting physiologically active organic acids, humic acid and fulvic acid, from a natural humic substance which is an improvement of a conventional extraction method.
천연 휴믹 물질 (Natural Humic substances)에 대한 국제적인 학회인 International Humic Substances Society (IHSS)는 생리활성 유기산인 휴믹산 (humic acid)과 풀빅산 (fulvic acid)의 추출법으로 강염기 (NaOH, pH 10) 처리 후 강산 (HCl, pH 1)에서 휴믹산을 침전시켜 용존하고 있는 풀빅산으로부터 분리하는 방법을 제시하고 있다 (IHSS website: http://www.ihss.gatech.edu/). 이후 휴믹산과 풀빅산의 정제법으로는 흡착크로마토그래피 (adsorption chromatography), 양이온교환수지 크로마토그래피 (cation-exchange chromatography), 투석 (dialysis) 등의 방법을 사용한다 [도 1: IHSS에서 추천하는 토양 원료로부터 다양한 유기산 (humic acid, fulvic acid, ulmic acid)의 추출 및 분리 방법 참조]. International Humic Substances Society (IHSS), an international society on natural humic substances, is a method of extracting physiologically active organic humic and fulvic acids to treat strong acids after treatment with strong bases (NaOH, pH 10). A method of precipitating a humic acid in (HCl, pH 1) to separate it from the dissolved fulvic acid (IHSS website: http://www.ihss.gatech.edu/). Since the purification method of humic acid and fulvic acid, adsorption chromatography (adsorption chromatography), cation-exchange chromatography, dialysis (dialysis), etc. are used. Extraction and separation of various organic acids (humic acid, fulvic acid, ulmic acid) from
본 발명이 해결하고자 하는 과제는 천연 휴믹 물질로부터 휴믹산과 풀빅산을 효율적으로 분리하는 추출법과 규격 설정 및 품질관리를 위한 분석법을 개발하는 것이다.The problem to be solved by the present invention is to develop an extraction method for efficient separation of humic acid and fulvic acid from natural humic materials and an analysis method for specification and quality control.
상기 기술적 과제를 달성하기 위하여 본 발명은According to an aspect of the present invention,
(a) 천연 휴믹 물질에 NaOH 수용액을 혼화하여 알칼리 추출 과정 가진 후 불용물을 제거하고 용액만을 취하는 단계;(a) mixing NaOH aqueous solution with a natural humic material to have an alkali extraction process, removing insoluble materials and taking only the solution;
(b) 상기 (a)단계를 거친 용액에 pH가 1.5가 될 때까지 염산을 부가하고 원심분리하여 침전물을 얻는 단계; 및(b) adding hydrochloric acid to the solution passed through step (a) until the pH is 1.5 and centrifuging to obtain a precipitate; And
(c) 상기 (b)단계의 침전물을 염산과 불산 혼합액으로 세척하는 단계를 포함하는 것을 특징으로 하는 휴믹산 추출방법을 제공한다.
(c) it provides a humic acid extraction method comprising the step of washing the precipitate of step (b) with a mixture of hydrochloric acid and hydrofluoric acid.
다른 기술적 과제를 달성하기 위하여 본 발명은The present invention to achieve another technical problem
(a) 천연 휴믹 물질에 NaOH 수용액을 혼화하여 알칼리 추출 과정 가진 후 침전물을 제거하고 상등액을 취하는 단계; 및 (a) mixing NaOH aqueous solution with a natural humic material to have an alkali extraction process, removing the precipitate and taking a supernatant; And
(b) 상기 (a)단계의 상등액에 duats을 가하여 pH가 1.8이 될 때까지 처리하고 원심분리하여 다시 침전물을 제거한 후, 공경 0.45 μm 멤브레인 필터로 여과하는 단계를 포함하는 것을 특징으로 하는 풀빅산 추출 방법을 제공한다.(b) adding biats to the supernatant of step (a) until the pH is 1.8, and centrifuging to remove the precipitate again, followed by filtration with a pore 0.45 μm membrane filter. Provide an extraction method.
본 발명에 따른 추출법은 종래의 방법에 비하여 고효율 및 고순도로 천연 휴믹 물질로부터 휴믹산과 풀빅산을 추출할 수 있는 장점을 가지고 있다. 본 발명에 대한 시험 결과 천연 휴믹 물질 원료 대비 휴믹산 17.0% (± 0.6%)와 풀빅산 3.1% (± 0.1%)를 얻어내었다. 각 유기산의 순도는 크로마토그래피 분석을 통해 95% 이상임을 확인하였다.Extraction method according to the present invention has the advantage of extracting humic acid and fulvic acid from natural humic materials with high efficiency and high purity compared to the conventional method. Test results of the present invention yielded 17.0% (± 0.6%) of humic acid and 3.1% (± 0.1%) of fulvic acid compared to natural humic materials. Purity of each organic acid was confirmed to be 95% or more through chromatographic analysis.
도 1: IHSS에서 추천하는 토양 원료로부터 다양한 유기산 (humic acid, fulvic acid, ulmic acid)의 추출 및 분리 방법
도 2: 천연 휴믹 물질로부터 생리활성 유기산인 휴믹산의 추출 및 분리 방법
도 3: 천연 휴믹 물질로부터 추출한 휴믹산 사진
도 4: Sigma사의 휴믹산과 천연 휴믹 물질로부터 추출한 휴믹산의 size-exclusion HPLC 크로마토그램.
도 5: Sigma사의 휴믹산 표준액의 size-exclusion HPLC 크로마토그램.
도 6: Sigma사의 휴믹산 표준액의 size-exclusion HPLC 분석 결과로 작성된 검량선.
도 7: 천연 휴믹 물질로부터 생리활성 풀빅산의 추출 및 분리 방법
도 8: 천연 휴믹 물질로부터 추출한 풀빅산 용액 및 풀빅산 분말 사진.
도 9: 천연 휴믹 물질로부터 추출한 풀빅산 분말 표준액의 흡광도를 이용한 검량선
도 10: 도 10: 천연 휴믹 물질로부터 분리한 풀빅산 분말 메탄올 용액 (5 mg/mL)의 역상 HPLC 크로마토그램.Figure 1: Extraction and separation of various organic acids (humic acid, fulvic acid, ulmic acid) from the soil raw materials recommended by IHSS
Figure 2: Extraction and separation of humic acid, a bioactive organic acid from natural humic materials
Figure 3: Humic acid picture extracted from natural humic materials
Figure 4: Size-exclusion HPLC chromatogram of humic acid extracted from humic acid from Sigma and natural humic materials.
Figure 5: Size-exclusion HPLC chromatogram of Sigma's humic acid standard solution.
Figure 6: Calibration curve prepared by the size-exclusion HPLC analysis of Sigma's humic acid standard solution.
7: Extraction and separation of bioactive fulvic acid from natural humic materials
8: Fulvic acid solution and fulvic acid powder photograph extracted from natural humic substance.
9: Calibration curve using absorbance of fulvic acid powder standard solution extracted from natural humic substance
Figure 10: Reverse phase HPLC chromatogram of fulvic acid powder methanol solution (5 mg / mL) isolated from natural humic material.
1. 천연 1. Natural 휴믹Humic 물질로 By substance 휴믹산Humic acid ( ( humichumic acidacid ) 추출법 개발 ) Extraction method development
천연 휴믹 물질에 함유된 휴믹산을 추출하는 방법으로 1차 알칼리 추출 후 2차 염산 처리하고 다시 알칼리로 용해하는 추출법을 이용하여 생리활성을 가진 휴믹산을 추출하는 방법을 확립하였다(도 2: 천연 휴믹 물질로부터 생리활성 유기산인 휴믹산의 추출 및 분리 방법 ). As a method of extracting humic acid contained in a natural humic substance, a method of extracting a humic acid having physiological activity by using an extraction method of primary hydrochloric acid extraction followed by secondary hydrochloric acid treatment and dissolving with alkali was established (FIG. 2: natural humic substance) Method of extracting and separating humic acid from bioactive organic acids).
본 방법에서는 천연 휴믹 물질에 0.1 M NaOH를 천연 휴믹 물질:0.1 M NaOH = 1:10 (g:mL)으로 처리하여 3시간 동안 혼화하면서 알칼리 추출 과정을 가진 후, 불용물을 제거하고 용액만을 취한 후 6 M HCl을 용액의 pH가 1.5가 될 때까지 처리하고 3000rpm에서 원심분리하여 침전물을 얻어내었다. In this method, 0.1 M NaOH is treated with natural humic substance: 0.1 M NaOH = 1:10 (g: mL) and mixed for 3 hours, followed by alkali extraction process, insolubles are removed, and only solution is taken. 6 M HCl was then treated until the pH of the solution was 1.5 and centrifuged at 3000 rpm to obtain a precipitate.
침전물을 0.1 M HCl과 0.3 M hydrofluoric acid (HF) 혼합액으로 3차례에 걸쳐 세척하고, cut-off 10,000 Da의 dialysis membrane으로 투석한 후, 동결건조하여 휴믹산 분말을 얻어내었다 (도 3: 천연 휴믹 물질로부터 추출한 휴믹산 사진).
The precipitate was washed three times with a 0.1 M HCl and 0.3 M hydrofluoric acid (HF) mixture, dialyzed with a cut-off 10,000 Da dialysis membrane, and lyophilized to obtain a humic acid powder (FIG. 3: natural humic material). Humic acid photo extracted from).
2. 천연 2. Natural 휴믹Humic 물질로부터 분리한 Separated from the substance 휴믹산Humic acid ( ( humichumic acidacid )의 분석 ) Analysis
(1) 천연 휴믹 물질 중 휴믹산의 중량 분석(1) Gravimetric analysis of humic acid in natural humic materials
천연 휴믹 물질 400 g을 가지고 분리한 휴믹산 분말의 무게는 68.1 g (± 2.3 g)으로 측정되었다. 따라서 천연 휴믹 물질중에 함유된 휴믹산의 함량은 17.0% (± 0.6%)로 계산되었다. The weight of the humic acid powder separated with 400 g of natural humic material was measured to be 68.1 g (± 2.3 g). Therefore, the content of humic acid in natural humic substances was calculated as 17.0% (± 0.6%).
(2) 휴믹산의 size-exclusion HPLC 분석(2) Size-exclusion HPLC analysis of humic acid
휴믹산의 size-exclusion HPLC는 Dionex HPLC system (Dionex P680A HPLC pump, Dionex ASI-100 automated sample injector, Dionex Thermostatted column compartment TCC-100, Dionex UVD 170U UV-Vis detector, Chromeleon CHM-1 software)을 이용하여 실행되었다. Humic acid size-exclusion HPLC is performed using the Dionex HPLC system (Dionex P680A HPLC pump, Dionex ASI-100 automated sample injector, Dionex Thermostatted column compartment TCC-100, Dionex UVD 170U UV-Vis detector, Chromeleon CHM-1 software) It became.
컬럼은 Shodex Protein KW-802.5 column (8 x 30 cm)를 사용하였고, 이동상은 deionized water를 사용하고, 유속은 1.0 mL/min이었으며, UV 260 nm에서 용출되는 물질을 검출하였다. 검액의 주입량은 20μL이었다. The column used Shodex Protein KW-802.5 column (8 x 30 cm), the mobile phase was deionized water, the flow rate was 1.0 mL / min, and the material eluted at
휴믹산 표준품을 Sigma사 (St. Louis, MO,USA)에서 구입한 humic acid를 사용하여 천연 휴믹 물질로부터 분리한 휴믹산과 비교 실험 하였다 (도 4: Sigma사의 휴믹산과 천연 휴믹 물질로부터 추출한 휴믹산의 size-exclusion HPLC 크로마토그램 참조). Humic acid standards were compared with humic acid isolated from natural humic materials using humic acid purchased from Sigma (St. Louis, MO, USA) (FIG. 4: Size- of humic acid extracted from humic acid from Sigma and natural humic materials. exclusion HPLC chromatogram).
실험 결과 Sigma사의 휴믹산은 5.0분에 검출되었고, 천연 휴믹 물질로부터 분리한 휴믹산은 5.1분에 검출되었다. 검출시간의 차이는 크지 않아 천연 휴믹 물질로부터 분리한 휴믹산이 Sigma사의 휴믹산과 비교하여 크기가 크게 다르지 않은 것으로 분석되었다. 천연 휴믹 물질로부터 휴믹산의 크로마토그램상의 순도는 100%에 가까운 것으로 측정되었다.As a result, Sigma's humic acid was detected at 5.0 minutes, and humic acid separated from natural humic material was detected at 5.1 minutes. The difference in the detection time was not so large that the humic acid separated from the natural humic material was not significantly different in size compared with the humic acid manufactured by Sigma. The purity on the chromatogram of humic acid from natural humic materials was determined to be close to 100%.
Size-exclusion HPLC를 이용하여 휴믹산을 정량분석하기 위하여 Sigma사의 휴믹산 표준액을 각 농도별 (10, 20, 50, 100 μg/mL)로 deionized water에 녹여 제조하고 위 분석조건에 따라 분석하였다 (도 5: Sigma사의 휴믹산 표준액의 size-exclusion HPLC 크로마토그램 참조). 검량선을 작성한 결과 y=0.8127x-2.1235의 식을 나타내었고, R 2=0.9999이상으로 좋은 직선성을 나타내었다 (도 6: Sigma사의 휴믹산 표준액의 size-exclusion HPLC 분석 결과로 작성된 검량선 참조). In order to quantify the humic acid using size-exclusion HPLC, Sigma's humic acid standard solution was prepared by dissolving in deionized water at each concentration (10, 20, 50, 100 μg / mL) and analyzed according to the above analysis conditions (Fig. 5). : Size-exclusion HPLC chromatogram of Sigma's humic acid standard solution. As a result of the calibration curve, the equation was y = 0.8127x-2.1235, and R 2 = 0.99999 or more. The linearity was good (see FIG. 6: calibration curve created by the size-exclusion HPLC analysis result of Sigma's humic acid standard solution).
천연 휴믹 물질로부터 분리한 휴믹산의 중량당 size-exclusion HPLC 피크 면적 (peak area/weight)은 Sigma사의 휴믹산의 2배 (동일 중량 샘플에서 천연 휴믹 물질 휴믹산 피크면적/Sigma사 휴믹산 피크면적 x 100 = 197%)에 달하는 것으로 나타났다. 이는 천연 휴믹 물질로부터 분리한 휴믹산이 Sigma사의 휴믹산보다 순도가 더 높을 것이라는 예측을 가능하게 한다.
The size-exclusion HPLC peak area (weight area / weight) per weight of humic acid isolated from the natural humic material was twice that of Sigma's humic acid (the peak area of the natural humic material Humic acid / Sigma humic acid in the same weight sample x 100 = 197). %). This makes it possible to predict that humic acids isolated from natural humic materials will be of higher purity than Sigma's humic acids.
3. 천연 3. Natural 휴믹Humic 물질로부터 From matter 풀빅산Fulvic Mountain ( ( fulvicfulvic acidacid ) 추출법 개발 ) Extraction method development
천연 휴믹 물질에 함유된 풀빅산을 추출하는 방법으로 1차 알칼리 추출 후 2차 염산 추출법을 이용하여 pH를 조절하여 생리활성을 가진 풀빅산을 추출하는 공정을 확립하였다 (도 7: 천연 휴믹 물질로부터 생리활성 풀빅산의 추출 및 분리 방법 참조). As a method of extracting fulvic acid contained in the natural humic substance, a process of extracting fulvic acid having physiological activity by adjusting the pH by using a second hydrochloric acid extraction method after the first alkali extraction was established (FIG. 7: from the natural humic substance). Extraction and isolation of bioactive fulvic acid).
본 방법에서는 천연 휴믹 물질에 0.1 M NaOH를 처리하여 3시간 동안 혼화하면서 알칼리 추출 과정을 가진 후, 침전물을 제거하고 상등액에 6 M HCl을 용액의 pH가 1.8이 될 때까지 처리하고 3000 rpm에서 원심분리하여 다시 침전물을 제거한 후, 공경 0.45 μm 멤브레인 필터로 여과하여 풀빅산이 함유된 노란색의 여액을 얻었다 (도 8:천연 휴믹 물질로부터 추출한 풀빅산 용액 및 풀빅산 분말 사진 참조 ). In this method, the natural humic material was treated with 0.1 M NaOH and mixed for 3 hours, followed by an alkali extraction process. Then, the precipitate was removed, and the supernatant was treated with 6 M HCl until the pH of the solution reached 1.8 and centrifuged at 3000 rpm. After separation and removal of the precipitate, it was filtered through a pore 0.45 μm membrane filter to obtain a yellow filtrate containing fulvic acid (see Fig. 8: Fulvic acid solution and fulvic acid powder photograph extracted from natural humic material).
이를 가지고 0.1 M NaOH로 pH를 조정하여 적절한 pH를 가진 풀빅산 용액을 얻어내었다. 필요에 따라 풀빅산 용액을 동결건조하여 노란색 분말을 얻어내었다 (도 8: 천연 휴믹 물질로부터 추출한 풀빅산 용액 및 풀빅산 분말 사진 참조).
The pH was adjusted with 0.1 M NaOH to obtain a fulvic acid solution with an appropriate pH. If necessary, the fulvic acid solution was lyophilized to obtain a yellow powder (see FIG. 8: Fulvic acid solution and fulvic acid powder photograph extracted from natural humic material).
5. 천연 5. Natural 휴믹Humic 물질로부터 분리한 Separated from the substance 풀빅산Fulvic Mountain ( ( fulvicfulvic acidacid )의 분석 ) Analysis
(1) 천연 휴믹 물질중 풀빅산의 중량 분석(1) Gravimetric analysis of fulvic acid in natural humic materials
천연 휴믹 물질 400 g을 가지고 분리한 풀빅산 분말의 무게는 12.6 g (± 0.6 g)으로 측정되었다. 따라서 천연 휴믹 물질중에 함유된 풀빅산의 함량은 3.1% (± 0.1%)로 계산되었다.
The weight of fulvic acid powder separated with 400 g of natural humic substance was measured as 12.6 g (± 0.6 g). Therefore, the content of fulvic acid in natural humic substances was calculated as 3.1% (± 0.1%).
(2) 풀빅산의 흡광도 분석(2) Absorbance Analysis of Fulvic Acid
건조한 풀빅산 분말을 물에 녹여 10 mg/mL 농도로 만든 후, 물로 희석하여 0.10, 0.25, 0.50, 1.0, 2.0, 5.0 mg/mL 농도의 표준액을 제조하였다. 각 표준액을 공경 0.45 μm 멤브레인 필터로 여과한 후 Shimazhu사의 UV-MINI-1240 모델 UV/Vis spectrophotometer로 UV 280 nm에서 흡광도를 측정하였다. 각 표준액의 흡광도를 가지고 검량선을 작성한 결과 y=0.2746x-0.0031의 식의 직선을 얻어졌고, R2=0.9998로 좋은 직선성을 나타냄을 확인하였다 (도 9: 천연 휴믹 물질로부터 추출한 풀빅산 분말 표준액의 흡광도를 이용한 검량선 참조). 검량선을 이용하여 1% 풀빅산 용액의 비흡광도 (E1 %)를 측정한 결과 2.743의 값을 얻었다.
The dry fulvic acid powder was dissolved in water to make a concentration of 10 mg / mL, and then diluted with water to prepare a standard solution of 0.10, 0.25, 0.50, 1.0, 2.0, 5.0 mg / mL. Each standard solution was filtered with a pore 0.45 μm membrane filter and absorbance was measured at 280 nm with a UV-MINI-1240 model UV / Vis spectrophotometer manufactured by Shimazhu. As a result of preparing a calibration curve with absorbance of each standard solution, a straight line of y = 0.2746x-0.0031 was obtained, and it was confirmed that R2 = 0.9998 showed good linearity (FIG. 9: Fulvic acid powder standard solution extracted from natural humic material). See calibration curve using absorbance). The specific absorbance (E 1 % ) of the 1% fulvic acid solution was measured using a calibration curve to obtain a value of 2.743.
(3) 풀빅산의 역상분배 HPLC 분석(3) Reverse Phase Distribution HPLC Analysis of Fulvic Acid
천연 휴믹 물질로부터 분리한 풀빅산을 정량할 수 있는 역상분배 HPLC (reversed-phase HPLC) 분석법을 개발하였다. HPLC는 Dionex HPLC system (Dionex P680A HPLC pump, Dionex ASI-100 automated sample injector, Dionex Thermostatted column compartment TCC-100, Dionex UVD 170U UV-Vis detector, Chromeleon CHM-1 software)을 이용하여 실행되었다. 컬럼은 Alltech사의 Prosphere 300 C4 column (4.6 x 250 mm, 5 micron particle size)을 사용하였고, 유속은 1.0 mL/min이었으며, UV 215 nm에서 용출되는 물질을 검출하고 검액의 주입량은 20 μL이었다. 이동상은 deionized water (이동상 A)와 methanol (이동상 B)을 기울기 용리 (gradient elution)하는 조건을 설정하였으며, 기울기용리 조건은 검체 주입부터 4분 동안은 이동상 B 10%를 유지하다가 8분까지 이동상 B를 80%까지 점차적으로 높인 후, 1분 후 다시 이동상 B 10%로 낮춘 후 20분까지 유지하였다. A reversed-phase HPLC (reversed-phase HPLC) assay was developed to quantify fulvic acid isolated from natural humic materials. HPLC was performed using a Dionex HPLC system (Dionex P680A HPLC pump, Dionex ASI-100 automated sample injector, Dionex Thermostatted column compartment TCC-100, Dionex UVD 170U UV-Vis detector, Chromeleon CHM-1 software). Alltech's Prosphere 300 C4 column (4.6 x 250 mm, 5 micron particle size) was used. The flow rate was 1.0 mL / min. The material eluted at
천연 휴믹 물질로부터 분리한 풀빅산 분말을 5 mg/mL로 메탄올에 녹인 샘플을 설정된 조건에서 분석한 결과 retention time 3.2분에서 풀빅산 피크가 검출되는 것을 확인하였다 (도 10: 천연 휴믹 물질로부터 분리한 풀빅산 분말 메탄올 용액 (5 mg/mL)의 역상 HPLC 크로마토그램 참조).
As a result of analyzing the sample dissolved in methanol at 5 mg / mL in the fulvic acid powder separated from the natural humic substance under the set conditions, it was confirmed that the fulvic acid peak was detected at the retention time of 3.2 minutes (FIG. 10: separated from the natural humic substance). Reverse-phase HPLC chromatogram of fulvic acid powder methanol solution (5 mg / mL)).
Claims (2)
(b) 상기 (a)단계를 거친 용액에 pH가 1.5가 될 때까지 염산을 부가하고 원심분리하여 침전물을 얻는 단계; 및
(c) 상기 (b)단계의 침전물을 염산과 불산 혼합액으로 세척하는 단계를 포함하는 것을 특징으로 하는 휴믹산 추출방법.(a) mixing NaOH aqueous solution with a natural humic material to have an alkali extraction process, removing insoluble materials and taking only the solution;
(b) adding hydrochloric acid to the solution passed through step (a) until the pH is 1.5 and centrifuging to obtain a precipitate; And
(c) Humic acid extraction method comprising the step of washing the precipitate of step (b) with a mixture of hydrochloric acid and hydrofluoric acid.
(b) 상기 (a)단계의 상등액에 duats을 가하여 pH가 1.8이 될 때까지 처리하고 원심분리하여 다시 침전물을 제거한 후, 공경 0.45 μm 멤브레인 필터로 여과하는 단계를 포함하는 것을 특징으로 하는 풀빅산 추출 방법.
(a) mixing NaOH aqueous solution with a natural humic material to have an alkali extraction process, removing the precipitate and taking a supernatant; And
(b) adding biats to the supernatant of step (a) until the pH is 1.8, and centrifuging to remove the precipitate again, followed by filtration with a pore 0.45 μm membrane filter. Extraction method.
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| CN104826380A (en) * | 2015-04-21 | 2015-08-12 | 李计双 | Complete solid-liquid separation technology in production of humic acid product |
| CN106478961A (en) * | 2016-10-12 | 2017-03-08 | 西安兰心有机肥有限公司 | Plant humic acid and its manufacture craft |
| CN106701130A (en) * | 2017-01-16 | 2017-05-24 | 张辉 | Method for extracting fulvic acid through low-temperature carbonization |
| WO2018145459A1 (en) * | 2017-02-08 | 2018-08-16 | 中国环境科学研究院 | Equipment for use in extracting hydrophilic fulvic acid from soil |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104826380A (en) * | 2015-04-21 | 2015-08-12 | 李计双 | Complete solid-liquid separation technology in production of humic acid product |
| CN106478961A (en) * | 2016-10-12 | 2017-03-08 | 西安兰心有机肥有限公司 | Plant humic acid and its manufacture craft |
| CN106478961B (en) * | 2016-10-12 | 2018-09-25 | 赵月平 | Plant humic acid and its manufacture craft |
| CN106701130A (en) * | 2017-01-16 | 2017-05-24 | 张辉 | Method for extracting fulvic acid through low-temperature carbonization |
| CN106701130B (en) * | 2017-01-16 | 2019-07-12 | 张辉 | A kind of method that fulvic acid is extracted in low temperature distillation |
| WO2018145459A1 (en) * | 2017-02-08 | 2018-08-16 | 中国环境科学研究院 | Equipment for use in extracting hydrophilic fulvic acid from soil |
| KR20190032748A (en) * | 2017-09-20 | 2019-03-28 | 김영식 | Fulvic acid extracting method using electro-analysised water |
| CN110372881A (en) * | 2019-06-14 | 2019-10-25 | 天津科技大学 | A kind of wet solution processing waste dish prepares the methods and applications of humic acid |
| KR102083798B1 (en) | 2019-10-21 | 2020-05-26 | 변형완 | Humic acid paste production method and its paste |
| KR102134314B1 (en) | 2019-10-21 | 2020-07-16 | 변형완 | Manufacturing apparatus for humic acid paste |
| CN113959812A (en) * | 2021-11-09 | 2022-01-21 | 西南林业大学 | Method for extracting fulvic acid and humic acid from soil |
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