CN102580352B - Separation method of natural product - Google Patents
Separation method of natural product Download PDFInfo
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- CN102580352B CN102580352B CN201210022094.3A CN201210022094A CN102580352B CN 102580352 B CN102580352 B CN 102580352B CN 201210022094 A CN201210022094 A CN 201210022094A CN 102580352 B CN102580352 B CN 102580352B
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- 238000000926 separation method Methods 0.000 title claims abstract description 36
- 229930014626 natural product Natural products 0.000 title claims abstract description 25
- 239000003480 eluent Substances 0.000 claims abstract description 43
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000004780 2D liquid chromatography Methods 0.000 claims abstract description 18
- 239000007791 liquid phase Substances 0.000 claims description 81
- 238000010790 dilution Methods 0.000 claims description 44
- 239000012895 dilution Substances 0.000 claims description 44
- 238000001802 infusion Methods 0.000 claims description 38
- 239000012071 phase Substances 0.000 claims description 20
- 230000011218 segmentation Effects 0.000 claims description 19
- 239000000945 filler Substances 0.000 claims description 17
- 238000012546 transfer Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 11
- 239000000284 extract Substances 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 7
- 238000012856 packing Methods 0.000 claims description 7
- GPWDPLKISXZVIE-UHFFFAOYSA-N cyclo[18]carbon Chemical compound C1#CC#CC#CC#CC#CC#CC#CC#CC#C1 GPWDPLKISXZVIE-UHFFFAOYSA-N 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 17
- 230000008569 process Effects 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 2
- 238000004811 liquid chromatography Methods 0.000 abstract 2
- 230000007547 defect Effects 0.000 abstract 1
- 238000007865 diluting Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 239000002245 particle Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 240000004980 Rheum officinale Species 0.000 description 4
- 235000008081 Rheum officinale Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 241000269420 Bufonidae Species 0.000 description 2
- 239000004952 Polyamide Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000000401 methanolic extract Substances 0.000 description 2
- 229920002647 polyamide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002435 venom Substances 0.000 description 2
- 210000001048 venom Anatomy 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 238000005192 partition Methods 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
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Abstract
The invention discloses a separation method of a natural product and relates to a separation method of the natural product. The separation method comprises the following steps: carrying out loading working station, carrying out working section of first-dimensional liquid chromatography separation; diluting eluent flowing through a first-dimensional liquid chromatographic column; sectionally enriching the eluent; and carrying out working section of second-dimensional liquid chromatography separation. In the separating process, a comprehensive two-dimensional liquid chromatography method of medium-low pressure liquid and preparative high performance liquid is adopted, so that the defects in the prior art can be well overcome, not only is the large-capacity sample preparation capability of the medium-low pressure liquid kept, but also the high-efficiency separation capability of the high performance liquid chromatography is ensured and a great number of intermediate links are reduced.
Description
Technical field
The present invention relates to a kind of separation method of natural products.
Background technology
The separation and preparation of natural origin composition is the large bottleneck in Chinese medicine and researches on natural drugs process always.Because natural products is complex system, in a common sample, comprised tens hundreds of kind components even, and the difference of measuring between each component is conventionally very large.Therefore, in order to reach the object of component separation, often will be in conjunction with multiple chromatograph packing material, carry out column chromatography lock out operation repeatedly.This process is loaded down with trivial details, very long and uninteresting: first by column chromatography, natural extracts is separated and the stream part separating is carried out to Fractional Collections, detect stream part of determining required merging by TLC, after concentrating, according to circumstances carry out again further chromatographic column separation (sometimes may need repeatedly to repeat said process).In this course, relate to the operation that solvent is concentrated and sample shifts, the concentrated not only time consumption and energy consumption of solvent, and often change because heating makes unsettled structure; The transfer process of sample is also followed the loss of sample.Finally, the sample preparative high efficiency liquid phase that is difficult to separate separates, this technique first will be by solid sample with suitable dissolution with solvents, but has the solvent tool of better solubility often to have stronger solvent effect to solid sample, can greatly reduce the separating effect of preparative high efficiency liquid phase; And the weak solvent of solvent effect is often difficult to dissolved solid sample.To sum up, use conventional methods and be is prepared by Separation of Natural Products, natural products often separates in tediously long, various column chromatography, solvent is concentrated, sample shifts and sample dissolution operation in incur loss or structure changes, researcher has to strengthen inventory in the hope of obtaining desirable result.Therefore, how to reduce to greatest extent manual operation, realize automatic partition from, the bottleneck being formed to raise the efficiency, to break through conventional method, seems extremely urgent.
Two-dimensional liquid chromatography is the technology growing up on high performance liquid chromatography basis, and it is combined different clastotypes and built two-dimentional system by the improvement technically and on equipment.Compared with one dimension liquid chromatographic system, the main advantage of two-dimensional liquid chromatography has been to provide larger peak capacity, thereby can more effectively solve the problem analysis (Wu Jianwei of complex system sample, Deng. multidimensional liquid chromatogram and the application in traditional Chinese medicine research thereof. Chinese herbal medicine, 2010,41 (6): 1020-1023).
Although existing two-dimensional liquid chromatography technology can meet the demand of analysis, cannot directly apply to the separation preparation of composition.This is to need more large sample amount to be processed owing to separating in preparation manipulation process, need to pass through the larger mobile phase of volume.When analysis operation, sample feeding amount is lower, is generally 20 μ l (μ g rank), therefore generally selects the analysis liquid-phase chromatographic column of small-scale; And in preparation manipulation, generally to select to process the more mesolow chromatographic column of large sample amount.In addition, in analytic process, the solvent effect of being brought by mobile phase, by reducing one dimension chromatogram flow velocity, reduce the processing of the sample size of two-dimentional chromatogram, can ignore substantially.But in preparative chromatography, solvent effect problem must solve, existing two-dimensional liquid chromatography technology cannot address the above problem.
Summary of the invention
The object of the present invention is to provide a kind of separation method of natural products, to solve the above-mentioned problems in the prior art.
The present invention includes following steps:
1) loading workshop section
By natural extracts solution direct injected to the first dimension liquid-phase chromatographic column to be separated, or natural extracts solution to be separated is added and mixed in sample filler, stir, after being dried, add to the top of described the first dimension liquid-phase chromatographic column;
2) the first dimension liquid chromatogram centrifugal station
With the first dimension eluent, the first dimension liquid-phase chromatographic column of loading is carried out to wash-out, described the first dimension eluent pumps in the first dimension liquid-phase chromatographic column by the first dimension infusion pump;
3) dilution of the first dimension liquid-phase chromatographic column eluent
The flow through eluent of the first dimension liquid-phase chromatographic column, in the blender of being located at after the first dimension liquid-phase chromatographic column, mix with dilution, described dilution is the solvent the first dimension liquid-phase chromatographic column to the de-ability of weak current, pumps in described blender by dilution dispenser pump;
4) segmentation enrichment of dilution
The eluent of the first dimension liquid-phase chromatographic column, through step 3) dilution after, under the control of transfer valve, segmentation is sent at least 1 enriching column and is carried out enrichment, and the volume that flows through each time the first dimension liquid-phase chromatographic column eluent of enriching column is 0.5～5 times of described enriching column column volume;
5) wash-out of enriched substance on enriching column
The enriched substance of segmentation enrichment in enriching column, under the control of described transfer valve, segmentation, by the second dimension eluent wash-out, is sent into the second dimension liquid-phase chromatographic column and is further separated;
6) two-dimensional liquid chromatography centrifugal station
With the second dimension eluent, sample to the second dimension liquid-phase chromatographic column of sending into carries out wash-out, described the second dimension eluent pumps in this second dimension liquid-phase chromatographic column by the second dimension infusion pump, and efflux carries out collection of illustrative plates detection by detector, collects stream part of corresponding chromatographic peak according to collection of illustrative plates.
In step 1) in, described the first dimension liquid-phase chromatographic column can adopt mesolow liquid-phase chromatographic column; Described the first dimension liquid-phase chromatographic column preferably uses the chromatograph packing material of Aquo System mobile phase, more preferably anti-phase carbon-18 filler (ODS).
In step 2) in, described the first dimension infusion pump can adopt mesolow pump.
In step 3) in, described the first dimension liquid-phase chromatographic column preferably uses the chromatograph packing material of Aquo System mobile phase, more preferably anti-phase carbon-18 filler (ODS); Described dilution can adopt water; The volume ratio of described eluent and dilution can be 1: (0.3～3).
In step 4) in, described transfer valve can adopt at least 1 two multiple-way valve or multiposition valve; Described enriching column preferably uses the chromatograph packing material of Aquo System mobile phase, more preferably anti-phase carbon-18 filler (ODS); The column volume of described enriching column can be 0.1～0.5 times of the first dimension liquid-phase chromatographic column column volume.
In step 5) in, described the second dimension liquid-phase chromatographic column can adopt preparative high-performance liquid chromatographic post.
In step 6) in, described the second dimension infusion pump can adopt high-pressure pump.
In the separation method of natural products, first sample separates through the first dimension liquid chromatogram (preferably mesolow chromatogram) unit, after the dilution that separated flow part is pumped with dilution dispenser pump mixes, the enriching column enrichment by transfer valve control is sent in segmentation, the sample retaining in enriching column, under the control of transfer valve, segmentation is pumped into preparative liquid chromatography post by two-dimensional liquid chromatography (preparation liquid phase) and further separates.By the separation method of this natural products, can realize the automation lock out operation of complex sample, there is the advantages such as separative efficiency is high, peak capacity is large, easy and simple to handle, can greatly save manpower and time.
Separation process of the present invention adopts the full two-dimensional liquid chromatography method of mesolow liquid phase × preparative high efficiency liquid phase, can solve well the existing shortcoming of prior art, both retained the large capacity sample preparation ability of mesolow liquid phase, guarantee again the efficient separating power of preparative high-performance liquid chromatographic, reduced a large amount of intermediate treatment links.
Accompanying drawing explanation
Fig. 1 is the separator structure composition schematic diagram that the embodiment of the present invention adopts.In Fig. 1, be respectively labeled as: 1, the first dimension infusion pump; 2, the first dimension liquid-phase chromatographic column; 3, dilution dispenser pump; 4, blender; 51, transfer valve; 6, enriching column; 7, the second dimension infusion pump; 8, the second dimension liquid-phase chromatographic column; 9, detector.
Fig. 2 is the separator structure composition schematic diagram that embodiment 1 adopts.In Fig. 2, be respectively labeled as: 1, the first dimension infusion pump (high pressure); 2, the first dimension liquid-phase chromatographic column (high pressure); 3, dilution dispenser pump (high pressure); 4, blender; 52, two six-way valves; 6, enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Fig. 3 is the chromatograms of embodiment 1.In Fig. 3, abscissa is time (min), and ordinate is spectral strength.
Fig. 4 is the separator structure composition schematic diagram that embodiment 2 adopts.In Fig. 4, be respectively labeled as: 1, the first dimension infusion pump (mesolow); 2, the first dimension liquid-phase chromatographic column (mesolow); 3, dilution dispenser pump (mesolow); 4, blender; 53, two ten-way valves; 61, the first enriching column; 62, the second enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Fig. 5 is the chromatograms of embodiment 2.In Fig. 5, abscissa is time (min), and ordinate is spectral strength.
Fig. 6 is the separator structure composition schematic diagram that embodiment 3 adopts.In Fig. 6, be respectively labeled as: 1, the first dimension infusion pump (mesolow); 2, the first dimension liquid-phase chromatographic column (mesolow); 3, dilution dispenser pump (mesolow); 4, blender; 54, multiposition valve; 6, enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Fig. 7 is the chromatograms of embodiment 3.In Fig. 7, abscissa is time (min), and ordinate is spectral strength.
The specific embodiment
Below in conjunction with embodiment, the present invention will be further described, but do not form any limitation of the invention.
Fig. 1 provides the separator structure composition schematic diagram that the embodiment of the present invention adopts.In Fig. 1, be respectively labeled as: 1, the first dimension infusion pump; 2, the first dimension liquid-phase chromatographic column; 3, dilution dispenser pump; 4, blender; 51, transfer valve; 6, enriching column; 7, the second dimension infusion pump; 8, the second dimension liquid-phase chromatographic column; 9, detector.
Embodiment 1
A kind of method for separating and preparing of natural products, separation process adopts the full two-dimensional liquid chromatography method of high pressure liquid phase × preparative high efficiency liquid phase, transfer valve between described the first dimension liquid chromatogram and two-dimensional liquid chromatography is manual two six-way valves, its connected mode as shown in Figure 2, in Fig. 2, be respectively labeled as: 1, the first dimension infusion pump (high pressure); 2, the first dimension liquid-phase chromatographic column (high pressure); 3, dilution dispenser pump (high pressure); 4, blender; 52, two six-way valves; 6, enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Sample solution: rheum officinale water extract 500mg, is dissolved in 1ml water.
(1) loading workshop section
Get the aqueous solution of above-mentioned rheum officinale water extract, direct injected to the first dimension liquid-phase chromatographic column.
(2) first dimension liquid chromatogram centrifugal stations
■ first ties up infusion pump: high-pressure pump.
■ chromatographic column: preparative high-performance liquid chromatographic post, φ 2cm × 25cm (column volume: 78.5ml), filler is the ODS of particle diameter 5 μ m.
■ mobile phase: 30% → 60% methanol/water gradient elution, co-elute 800ml, flow velocity, 0.5ml/min.
The dilution of (3) first dimension liquid-phase chromatographic column eluents.
■ dilution: water.
■ mixed proportion: 1: 2 (dilution dispenser pump flow velocity: 1ml/min).
(4) segmentation enrichment of dilution
■ transfer valve: manual two six-way valves
■ enriching column
◆ number: 1
◆ column volume: the second dimension 0.14 times of column volume of liquid chromatogram (φ 3cm × 1.5cm, 10.6ml)
◆ filler: the ODS of 5 μ m
The ■ segmentation enrichment cycle: the every 30ml of eluent (about enriching column column volume 3 times) of the first dimension liquid-phase chromatographic column is by enriching column segmentation enrichment.
(5) wash-out of enriched substance on enriching column
Be enriched in the enriched substance in enriching column, by the path of two six-way valves of manual switchover, close the first dimension infusion pump, open the second dimension infusion pump, by the second dimension eluent wash-out, send into the second dimension liquid-phase chromatographic column and further separate.
(6) two-dimensional liquid chromatography centrifugal stations
■ second ties up infusion pump: high-pressure pump
■ chromatographic column: preparative high-performance liquid chromatographic post, φ 2cm × 15cm (column volume: 78.5ml), filler is the ODS of particle diameter 5 μ m
■ mobile phase: 20% → 40% acetonitrile/water gradient elution, 60min/ time, flow velocity, 8ml/min
■ detector: DAD detector
Complete after the separation of 1 enriched sample, the path of two six-way valves of manual switchover, opens the first dimension infusion pump, close the second dimension infusion pump, repeat the operation of workshop section 2～6, until sample is complete by wash-out, the chromatograms (separating for 1st～7 times) obtaining as shown in Figure 3.
A kind of method for separating and preparing of natural products, separation process adopts the full two-dimensional liquid chromatography method of mesolow liquid phase × preparative high efficiency liquid phase, transfer valve between described one dimension liquid chromatogram and two-dimensional liquid chromatography is automatically controlled two ten-way valves, its connected mode as shown in Figure 4, in Fig. 4, be respectively labeled as: 1, the first dimension infusion pump (mesolow); 2, the first dimension liquid-phase chromatographic column (mesolow); 3, dilution dispenser pump (mesolow); 4, blender; 53, two ten-way valves; 61, the first enriching column; 62, the second enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Sample solution: rheum officinale water extract 500mg, is dissolved in 1ml water.
(1) loading workshop section
Get the aqueous solution of above-mentioned rheum officinale water extract, mix in the polyamide filler of 1.0g, stir, solvent evaporated, the top of loading to the first dimension liquid-phase chromatographic column.
(2) first dimension liquid chromatogram centrifugal stations
■ first ties up infusion pump: mesolow infusion pump
■ chromatographic column: the mesolow post of φ 2.2cm × 20cm, fill with particle diameter 100 object polyamide
■ mobile phase: the methanol/water gradient elution of water % → 100%, co-elute 1000ml, flow velocity, 0.3ml/min
The dilution of (3) first dimension liquid-phase chromatographic column eluents
■ dilution: water
■ mixed proportion: 1: 0.3 (dilution dispenser pump flow velocity: 0.1ml/min).
(4) segmentation cutting and the enrichment of dilution
■ transfer valve: automatically controlled two ten-way valves
■ enriching column
◆ number: 2
◆ column volume: 0.5 times (φ 3cm × 5cm, 35.3ml) of the approximately second dimension liquid chromatogram column volume
◆ filler: the ODS of 5 μ M
The ■ segmentation enrichment cycle: the every 18ml of eluent (about enriching column column volume 0.5 times) of the first dimension liquid-phase chromatographic column is by enriching column segmentation enrichment.
(5) wash-out of enriched substance on enriching column
Be enriched in the enriched substance in enriching column, by switching the path of two ten-way valves, by the second dimension eluent wash-out, send into the second dimension liquid-phase chromatographic column and further separate.
(6) two-dimensional liquid chromatography centrifugal stations
■ second ties up infusion pump: high-pressure pump.
■ chromatographic column: preparative high-performance liquid chromatographic post, φ 2cm × 25cm (column volume: 78.5ml), filler is the ODS of particle diameter 5 μ m.
■ mobile phase: 30% → 70% methanol/water gradient elution, 60min/ time, flow velocity, 8ml/min.
■ detector: DAD detector.
When this workshop section's section is carried out, another enriching column carries out enrichment to the sample being eluted by the first dimension liquid chromatogram simultaneously.
Complete after the separation of 1 enriched sample, switch the path of two ten-way valves, repeat the operation of workshop section 2～6, until sample is complete by wash-out, the chromatograms (separating for 1st～7 times) obtaining as shown in Figure 5.
Embodiment 3
A kind of method for separating and preparing of natural products, separation process adopts the full two-dimensional liquid chromatography method of mesolow liquid phase × preparative high efficiency liquid phase, six valves that transfer valve between described one dimension liquid chromatogram and two-dimensional liquid chromatography is a pair of synchronous switching, its connected mode as shown in Figure 6, in Fig. 6, be respectively labeled as: 1, the first dimension infusion pump (mesolow); 2, the first dimension liquid-phase chromatographic column (mesolow); 3, dilution dispenser pump (mesolow); 4, blender; 54, multiposition valve; 6, enriching column; 7, the second dimension infusion pump (high pressure); 8, the first dimension liquid-phase chromatographic column (preparative high-performance liquid chromatographic post); 9, detector; 11, the first dimension eluent; 12, dilution; 13, the second dimension eluent.
Sample solution: dried venom of toads methanolic extract 200mg, is dissolved in 1ml methyl alcohol.
(1) loading workshop section
Get the methanol solution of above-mentioned dried venom of toads methanolic extract, mix in the ODS of 1.0g filler, stir, solvent evaporated, the top of loading to the first dimension liquid-phase chromatographic column.
(2) first dimension liquid chromatogram centrifugal stations
■ first ties up infusion pump: mesolow infusion pump.
■ chromatographic column: the mesolow post of φ 1cm × 20cm, fill with the ODS of particle diameter 75 μ M.
■ mobile phase: 50% → 100% methanol/water gradient elution, co-elute 400ml, flow velocity, 0.1ml/min.
The dilution of (3) first dimension liquid-phase chromatographic column eluents
■ dilution: water.
■ mixed proportion: 1: 3 (dilution dispenser pump flow velocity: 0.3 ml/min).
(4) segmentation cutting and the enrichment of dilution
■ transfer valve: a pair of automatically controlled six valves, synchronously switch.
■ enriching column.
◆ number: 6.
◆ column volume: the second dimension 0.1 times of column volume of liquid chromatogram (φ 1cm × 1.5cm, 1.18ml).
◆ filler: the ODS of 5 μ m.
The ■ segmentation enrichment cycle: the every 6ml of eluent (about enriching column column volume 5 times) of the first dimension liquid-phase chromatographic column is by enriching column segmentation enrichment.After completing an enrichment, switch stream one time, continue enrichment next time until complete 6 enrichments, or carry out wash-out.
(5) wash-out of enriched substance on enriching column
Complete after 6 enrichments, close the first dimension infusion pump, open the second dimension infusion pump, with the second dimension eluent wash-out, gradation is sent into the second dimension liquid-phase chromatographic column and is further separated.
(6) two-dimensional liquid chromatography centrifugal stations
■ second ties up infusion pump: high-pressure pump.
■ chromatographic column: preparative high-performance liquid chromatographic post, φ 1cm × 15cm (column volume: 11.8ml), filler is the ODS of particle diameter 5 μ m.
■ mobile phase: 0 → 30min 28% → 54% acetonitrile, 30min → 60min 54% acetonitrile wash-out, flow velocity, 2ml/min.
■ detector: DAD detector.
Complete after the separation of 6 enriched sample, switch the path of multiposition valve, repeat the operation of workshop section 6, complete after the separation of 6 enriched sample, repeat the operation of workshop section 2～6, until sample is complete by wash-out, the chromatograms (separating for 1st～7 times) obtaining as shown in Figure 7.
Embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (11)
1. a separation method for natural products, is characterized in that comprising the following steps:
1) loading workshop section
By natural extracts solution direct injected to the first dimension liquid-phase chromatographic column to be separated, or natural extracts solution to be separated is added and mixed in sample filler, stir, after being dried, add to the top of described the first dimension liquid-phase chromatographic column;
2) the first dimension liquid chromatogram centrifugal station
With the first dimension eluent, the first dimension liquid-phase chromatographic column of loading is carried out to wash-out, described the first dimension eluent pumps in the first dimension liquid-phase chromatographic column by the first dimension infusion pump;
3) dilution of the first dimension liquid-phase chromatographic column eluent
The flow through eluent of the first dimension liquid-phase chromatographic column, in the blender of being located at after the first dimension liquid-phase chromatographic column, mix with dilution, described dilution is the solvent the first dimension liquid-phase chromatographic column to the de-ability of weak current, pumps in described blender by dilution dispenser pump; Described dilution adopts water; The volume ratio of described eluent and dilution is 1: 0.3～3;
4) segmentation enrichment of dilution
The eluent of the first dimension liquid-phase chromatographic column, after step 3) dilution, under the control of transfer valve, segmentation is sent at least 1 enriching column and is carried out enrichment, and the volume that flows through each time the first dimension liquid-phase chromatographic column eluent of enriching column is 0.5～5 times of described enriching column column volume; The column volume of described enriching column is 0.1～0.5 times of the first dimension liquid-phase chromatographic column column volume;
5) wash-out of enriched substance on enriching column
The enriched substance of segmentation enrichment in enriching column, under the control of described transfer valve, segmentation, by the second dimension eluent wash-out, is sent into the second dimension liquid-phase chromatographic column and is further separated;
6) two-dimensional liquid chromatography centrifugal station
With the second dimension eluent, sample to the second dimension liquid-phase chromatographic column of sending into carries out wash-out, described the second dimension eluent pumps in this second dimension liquid-phase chromatographic column by the second dimension infusion pump, and efflux carries out collection of illustrative plates detection by detector, collects stream part of corresponding chromatographic peak according to collection of illustrative plates.
2. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 1), and described the first dimension liquid-phase chromatographic column adopts mesolow liquid-phase chromatographic column; Described the first dimension liquid-phase chromatographic column uses the chromatograph packing material of Aquo System mobile phase.
3. the separation method of a kind of natural products as claimed in claim 2, is characterized in that described the first dimension liquid-phase chromatographic column uses anti-phase carbon-18 filler.
4. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 2) in, described the first dimension infusion pump adopts mesolow pump.
5. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 3), and described the first dimension liquid-phase chromatographic column uses the chromatograph packing material of Aquo System mobile phase.
6. the separation method of a kind of natural products as claimed in claim 5, is characterized in that described the first dimension liquid-phase chromatographic column uses anti-phase carbon-18 filler.
7. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 4), and described transfer valve adopts at least 1 two multiple-way valve or multiposition valve.
8. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 4), and described enriching column uses the chromatograph packing material of Aquo System mobile phase.
9. the separation method of a kind of natural products as claimed in claim 8, is characterized in that described enriching column uses anti-phase carbon-18 filler.
10. the separation method of a kind of natural products as claimed in claim 1, is characterized in that in step 5), and described the second dimension liquid-phase chromatographic column adopts preparative high-performance liquid chromatographic post.
The separation method of 11. a kind of natural products as claimed in claim 1, is characterized in that in step 6), and described the second dimension infusion pump adopts high-pressure pump.
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