CN101703844A - Preparation-type two-dimensional column liquid chromatographic separation system and usage thereof - Google Patents
Preparation-type two-dimensional column liquid chromatographic separation system and usage thereof Download PDFInfo
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- CN101703844A CN101703844A CN200910237909A CN200910237909A CN101703844A CN 101703844 A CN101703844 A CN 101703844A CN 200910237909 A CN200910237909 A CN 200910237909A CN 200910237909 A CN200910237909 A CN 200910237909A CN 101703844 A CN101703844 A CN 101703844A
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Abstract
The invention discloses a preparation-type two-dimensional column liquid chromatographic separation system belonging to the technical field of chromatographic separation. The system comprises a one-dimensional chromatographic column (1), a two-dimensional chromatographic column (2), a first detector (6), a second detector (7), a first infusion pump (4) and a second infusion pump (5), and is characterized in that the outlet end of the first detector (6) connected with the one-dimensional chromatographic column (1) is provided with a concentrating column (3). The invention also discloses usage of the system. The system uses the concentrating column to replace the concentrating mode of performing offline reduced-pressure rotary evaporation on component groups flowing out from the one-dimensional chromatographic column or on-line heating-concentrating operation, needs no special instruments or no heating, cannot destroy the structure of samples, can be operated on line, and is beneficial to instrument automation, large in handling capacity and high in efficiency.
Description
Technical field
The invention belongs to the chromatographic separation technology field, be specifically related to a kind of preparation type two-dimensional column liquid chromatographic separation system and using method thereof.
Background technology
Along with the development of natural products research and the modernization of Chinese herbal medicine industry, it is a urgent task that various natural products are carried out separation and purification.Preparation type two-dimensional column liquid chromatographic realizes that full composition, highly purified natural goods separate, preparation type two dimension chromatogram comprises one dimension chromatographic column, two-dimentional chromatographic column and corresponding detector, the one dimension chromatographic column is the crude separation post, use eluant, eluent with the compound component group's wash-out in the one dimension chromatographic column, certain the compound component group who selects the drip washing of one dimension chromatographic column to go out is online to enter two-dimentional chromatographic column and carries out essence separation.If but the last sample volume of the compound component group who enters two-dimentional chromatographic column is excessive, can cause each component elution curve of two dimension wide, flat, overlapping between component, inferior separating effect is if the one dimension parting liquid that takes a morsel enters two-dimentional chromatographic column, good separating effect, but the product amount is few, and preparation efficiency is low.The method that overcomes above-mentioned defective at present is component group's off-line decompression rotary evaporation or the online heating evaporation solvent that the one dimension chromatographic column is flowed out, and all needs special equipment and complicated operation, and is wayward, and in addition, heating also might destroy compound structure.The present invention prepares the separation of type two-dimensional column liquid chromatographic and does not appear in the newspapers through document and patent retrieval.
Summary of the invention
The object of the present invention is to provide a kind of preparation type two-dimensional column liquid chromatographic separation system and using method thereof.
A kind of preparation type two-dimensional column liquid chromatographic separation system, comprise one dimension chromatographic column 1, two dimension chromatographic column 2, first detector 6, second detector 7, first infusion pump 4 and second infusion pump 5, it is improvement to existing preparation type two-dimensional column liquid chromatographic system, be characterized in evaporating column 3 being set at the port of export of first detector 6 that links to each other with one dimension chromatographic column 1, wherein, exit first flow divider 11 of first detector 6 is divided into two branch roads, a branch road is first outlet 8, another branch road links to each other with evaporating column 3 tops inlet, second infusion pump 5 converges with the branch road of porch, evaporating column 3 top, evaporating column 3 lower part outlets are divided into two branch roads through second flow divider 12, a branch road is second outlet 9, another branch road links to each other with the upper entrance of two-dimentional chromatographic column 2, the lower part outlet of two dimension chromatographic column 2 links to each other with second detector, 7 inlets, and 7 outlets of second detector are the 3rd outlet 10.
Described evaporating column 3 is the macroporous adsorption resin chromatography post.
The bed volume of described evaporating column 3 is the 1/4-1/6 of one dimension chromatographic column 1 bed volume.
Preparation type two-dimensional column liquid chromatographic separation system of the present invention is specially adapted to alkaloid and the flavones in the separation and purification kuh-seng.
The using method of above-mentioned preparation type two-dimensional column liquid chromatographic separation system, except that the use as follows of evaporating column 3, other parts use the method for preparing the type two-dimensional column liquid chromatographic as routine:
A, elder generation are with deionized water drip washing evaporating column, through 7 monitorings of second detector, until baseline stability;
B, will select pump into evaporating column 3 through one dimension look general post 1 isolated compound component group solution, treat in the solution that whole components are adsorbed on the filler of evaporating column solvent in the back emptying evaporating column;
C, in evaporating column 3, pump into eluent, flow velocity: 5-8mL/min, with wash-out of all components;
Behind D, the wash-out all components enter two-dimentional chromatographic column and carry out essence separation.
The long-pending one dimension chromatographic column that can reach of component colony after evaporating column 3 concentrates is separated 1/5th to 1/3rd of back volume, has promptly concentrated 3-5 doubly.
Preparation type two-dimensional column liquid chromatographic separation system of the present invention can be connected with computer realizes on-line operation.
Beneficial effect of the present invention: the evaporating column among the present invention has substituted from the concentrated mode of the component group's of one dimension chromatographic column outflow off-line decompression rotary evaporation or the operation that online heating concentrates, equipment is simple, instrument that need be special not, need not heat, normal-temperature operation can not destroyed compound structure, but on-line operation, help automation equipment, treating capacity is bigger, and efficient is higher.
Description of drawings
Fig. 1 is a preparation type two-dimensional column liquid chromatographic separation system of the present invention
Wherein, 1-one dimension chromatographic column; 2-two dimension chromatographic column; The 3-evaporating column; 4-first infusion pump; 5-second infusion pump; 6-first detector; 7-second detector; 8-first outlet; 9-second outlet; 10-the 3rd outlet; 11-first flow divider; 12-second flow divider
Fig. 2 utilizes chromatographic fractionation system of the present invention to alkaloidal separation and purification effect chromatogram in the kuh-seng;
Fig. 3 is that the chromatographic fractionation system of no evaporating column is to alkaloidal separation and purification effect chromatogram in the kuh-seng.
The specific embodiment
The extract of purifying substance to be separated is pumped into one dimension chromatographic column 1 by first infusion pump 4 carry out crude separation, use eluant, eluent with the compound component group in the one dimension chromatographic column 1 respectively wash-out come out, chosen compound component group can flow directly into evaporating column 3 after first detector detects, also can collect by first outlet, 8 places, the compound component group that first outlet, 8 places are collected pumps into evaporating column 3 by second infusion pump 5, use less eluent to come out through wash-out of compound component group that evaporating column 3 enrichments concentrate, eluent can flow directly into two-dimentional chromatographic column 2 and carry out the essence separation, also can collect by second outlet, 9 places, eluent after the collection pumps into two-dimentional chromatographic column 2 and carries out the essence separation, and smart component of separating goes out 10 places in the 3rd and collects behind second detector 7.
The alkaloid that embodiment 2 uses in the preparation type two-dimensional column liquid chromatographic separation system separation and purification kuh-seng of the present invention
Operating condition:
The one dimension chromatographic column: D101 macroporous absorbent resin post, filler are the opaque spherical particle of milky, low pole, particle diameter 0.3-1.25nm, specific surface 550 (m
2/ g), aperture 7-8nm; Column diameter 25mm, post height of bed 300mm
Two dimension chromatographic column: C
18Post, particle diameter 100 μ m; Column diameter 25mm, post height of bed 500mm
Evaporating column: adopt macroporous absorbent resin SP825 as filler, the concrete parameter of filler is as follows: light brown spherical particle, nonpolar, particle diameter 0.3-1.0mm, specific surface 1000 (m
2/ g), aperture 5.7nm; Column diameter 18mm, post height of bed 100mm
Detector: UV-detector, detect wavelength 280nm
The concrete operations step is as follows:
(1) pre-treatment of raw material: kuh-seng is pulverized with pulverizer, take by weighing the about 100g of powder, add 0.3% hydrochloric acid 1000mL, 24h vibrates in oscillator, filter. add 1000mL0.3% hydrochloric acid in the filter residue again, 24h vibrates in oscillator, filter. merge filtrate twice, regulate about pH to 9.0 with ammoniacal liquor, with chloroform extraction filtrate. behind the separatory, in the organic facies alkaloid. after rotary evaporation concentrates, dry. it is to make the about 5mL of saturated solution in 50% the ethanol water that the material after getting that 0.2g is above-mentioned and drying is dissolved in concentration of volume percent, is last all product;
(2) with deionized water drip washing evaporating column, through 7 monitorings of second detector, until baseline stability;
(3) the above-mentioned sample of 5mL is gone up sample with 5mL/min, use the gradient methanol with 5mL/min drip washing then, wherein, the volume ratio of methyl alcohol and water was followed successively by 1: 9,3: 7,5: 5,7: 3, obtain 4 component groups, component group's solution 600mL under methanol-water (volume ratio is 3: the 7) drip washing is pumped into evaporating column, after treating in the solution that whole components are adsorbed on the filler of evaporating column, solvent in the emptying post, with the 150mL absolute ethyl alcohol all components on the evaporating column is concentrated then and eluted, wherein, the absolute ethyl alcohol flow velocity is 6mL/min, this evaporating column with sample concentration 4 times;
(4) eluent that will collect from evaporating column pumps in the two-dimentional chromatographic column with the flow velocity of 9mL/min, is the speed drip washing with 9mL/min of 70% methanol aqueous solution with concentration of volume percent then, collects each component after testing behind the device.The chromatography result shows that the spectrum peak is better, and each component good separating effect is seen Fig. 2.
Control experiment is: under the same terms, directly pump into two-dimentional chromatographic column through the above-claimed cpd component group of one dimension chromatographic column crude separation without evaporating column and carry out essence and separate, the chromatography result shows, spectrum peak width, flat, and overlapping between component, inferior separating effect is seen Fig. 3.
Claims (4)
1. one kind prepares the type two-dimensional column liquid chromatographic separation system, comprise one dimension chromatographic column (1), two dimension chromatographic column (2), first detector (6), second detector (7), first infusion pump (4) and second infusion pump (5), it is characterized in that, the port of export at first detector (6) that links to each other with one dimension chromatographic column (1) is provided with evaporating column (3), wherein, exit first flow divider (11) of first detector (6) is divided into two branch roads, a branch road is first outlet (8), another branch road links to each other with evaporating column (3) top inlet, second infusion pump (5) converges with the branch road of evaporating column (3) porch, top, evaporating column (3) lower part outlet is divided into two branch roads through second flow divider (12), a branch road is second outlet (9), another branch road links to each other with the upper entrance of two-dimentional chromatographic column (2), and the lower part outlet of two-dimentional chromatographic column (2) links to each other with second detector (7) inlet.
2. preparation type two-dimensional column liquid chromatographic separation system according to claim 1 is characterized in that described evaporating column (3) is the macroporous adsorption resin chromatography post.
3. preparation type two-dimensional column liquid chromatographic separation system according to claim 1 and 2 is characterized in that, the column volume of described evaporating column (3) is the 1/4-1/6 of one dimension chromatographic column (1) volume.
4. the using method of the described preparation type of claim 1 two-dimensional column liquid chromatographic separation system is characterized in that, comprises following operating procedure:
A, elder generation are with deionized water drip washing evaporating column, through second detector (7) monitoring, until baseline stability;
B, will select pump into evaporating column (3) through one dimension chromatographic column (1) isolated compound component group solution, treat in the solution that whole components are adsorbed on the filler of evaporating column solvent in the back emptying evaporating column;
C, pump into eluent in evaporating column (3), flow velocity is 5-8mL/min, with wash-out of all components;
Behind D, the wash-out all components enter two-dimentional chromatographic column and carry out essence separation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569223A (en) * | 2015-01-31 | 2015-04-29 | 苏州汇通色谱分离纯化有限公司 | Method for preparing impurity standard substance of pesticide through two-dimensional multi-time central cutting preparative chromatography |
| CN106924995A (en) * | 2015-12-30 | 2017-07-07 | 中美华世通生物医药科技（武汉）有限公司 | Continuous chromatography system and chromatography method |
| CN109557199A (en) * | 2018-11-20 | 2019-04-02 | 西安交通大学 | A kind of two-dimensional liquid chromatography full-automatic valve control system and method |
| CN112098571A (en) * | 2020-09-10 | 2020-12-18 | 江南大学 | Method for quickly separating polar components in frying oil on line |
-
2009
- 2009-11-25 CN CN2009102379098A patent/CN101703844B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569223A (en) * | 2015-01-31 | 2015-04-29 | 苏州汇通色谱分离纯化有限公司 | Method for preparing impurity standard substance of pesticide through two-dimensional multi-time central cutting preparative chromatography |
| CN106924995A (en) * | 2015-12-30 | 2017-07-07 | 中美华世通生物医药科技（武汉）有限公司 | Continuous chromatography system and chromatography method |
| CN109557199A (en) * | 2018-11-20 | 2019-04-02 | 西安交通大学 | A kind of two-dimensional liquid chromatography full-automatic valve control system and method |
| CN112098571A (en) * | 2020-09-10 | 2020-12-18 | 江南大学 | Method for quickly separating polar components in frying oil on line |
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