CN115925514A - Method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography - Google Patents
Method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography Download PDFInfo
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- CN115925514A CN115925514A CN202310010132.1A CN202310010132A CN115925514A CN 115925514 A CN115925514 A CN 115925514A CN 202310010132 A CN202310010132 A CN 202310010132A CN 115925514 A CN115925514 A CN 115925514A
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- 150000002009 diols Chemical class 0.000 title claims abstract description 44
- 241000208125 Nicotiana Species 0.000 title claims abstract description 40
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 48
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 238000002386 leaching Methods 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000004587 chromatography analysis Methods 0.000 claims description 20
- 238000000746 purification Methods 0.000 claims description 20
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical group O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- AINBZKYUNWUTRE-UHFFFAOYSA-N ethanol;propan-2-ol Chemical group CCO.CC(C)O AINBZKYUNWUTRE-UHFFFAOYSA-N 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical group CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 4
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- 238000013375 chromatographic separation Methods 0.000 abstract description 7
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- 238000000926 separation method Methods 0.000 description 14
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
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- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- LHORCXXUZJAMPU-UHFFFAOYSA-N 1,7,11-trimethyl-4-(propan-2-yl)cyclotetradecane Chemical compound CC(C)C1CCC(C)CCCC(C)CCCC(C)CC1 LHORCXXUZJAMPU-UHFFFAOYSA-N 0.000 description 1
- 241000222519 Agaricus bisporus Species 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
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Abstract
The invention relates to the field of chemical preparation, in particular to a method for multi-dimensionally preparing cembratriene diol in liquid chromatography purified tobacco leaves, which comprises the following steps: a1 Adding a solvent into a tobacco sample for leaching to obtain an extracting solution; a2 Purifying and eluting the extracting solution obtained in the step A1) by a first-dimension liquid chromatographic column, then purifying and eluting by a second-dimension liquid chromatographic column, and then carrying out fraction and elution by a third-dimension liquid chromatographic column after trapping and elution by a trapping column array so as to provide purified alpha-cematriene diol and beta-cematriene diol. The method applies multidimensional chromatography, and separates cembratriene diol from tobacco with multiple dimensions and high efficiency. The cembratriene diol is a compound with relatively poor thermal stability, and the multidimensional chromatographic separation process is not contacted with the external environment, so that the cembratriene diol is more suitable for separating substances with relatively poor thermal stability; and multiple detectors detect and eliminate interference, and the result is reliable.
Description
Technical Field
The invention relates to the field of chemical preparation, and in particular relates to a method for multi-dimensionally preparing cembratriene diol in liquid chromatography purified tobacco leaves.
Background
The cembratriene diol is an important tobacco flavor precursor, and not only is an important precursor of cigarette flavor components, but also has various biological activities such as tumor cell growth inhibition, prostaglandin synthesis inhibition, nicotine sensation inhibition, neuroprotective agents and the like.
The variety of compounds in tobacco is various, and the existing method for purifying and analyzing cembratriene diol has various characteristics, but has the defects of residual derivatization reagent, low solvent recovery rate, complicated pretreatment and the like. As a conventional separation method, an organic solvent extraction method is disclosed in the publication no: CN105001052A discloses a method for extracting cembrane agaricus bisporus from tobacco inflorescence, which comprises the steps of extracting and concentrating by using an organic solvent to obtain a crude extract, removing high-grade alkane from the crude extract by using a solvent precipitation method, removing pigment by using activated carbon to obtain a tobacco diterpene extract, and separating the obtained extract by using a silica gel column chromatography to obtain two diterpene compounds. Publication No.: CN102206138A discloses a method for separating and purifying two aroma precursors in tobacco, which adopts the steps of leaching, liquid-liquid extraction, normal phase silica gel column chromatographic separation, normal phase cyano column chromatographic separation, reverse phase C18 column chromatographic separation and the like to obtain two aroma precursors. The existing traditional purification and separation methods have stable separation effect, but the separation efficiency still needs to be improved, and the development of a new high-efficiency separation and purification method is the development direction of separation in the future.
Disclosure of Invention
The invention provides a method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography, aiming at the problems in the prior art.
The invention provides a method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparation liquid chromatography, which comprises the following steps:
a1 Adding a solvent into a tobacco sample for leaching to obtain an extracting solution;
a2 Purifying and eluting the extracting solution obtained in the step A1) by a first dimension liquid chromatography column, then purifying and eluting by a second dimension liquid chromatography column, and then carrying out fraction and elution by a third dimension liquid chromatography column after trapping and elution by a trapping column array so as to provide purified alpha-cematriene diol and beta-cematriene diol.
Preferably, in the step A1), the tobacco leaf sample is crushed and sieved tobacco leaf powder; the aperture of the sieving screen of the tobacco sample is less than or equal to 300 meshes;
preferably, in the step A1), the ratio of the added mass g of the tobacco leaf sample to the added volume of the solvent is 100;
preferably, in step A1), the solvent is 85-95% aqueous ethanol;
preferably, in step A1), the number of times of leaching is 2 to 4 times, preferably 3 times;
preferably, in step A1), the leaching time is 9-11h, preferably 10h;
preferably, in the step A1), filtering and deslagging are carried out after leaching, leaching solutions are combined, and the solvent is removed through decompression and recovery; preferably, in step A2), the first-dimension liquid chromatography column purification conditions are: the chromatographic column is LSI-100 chromatographic column with diameter of 20-800mm;
the mobile phase is water;
the operating pressure of the chromatographic column is 1-10MPa;
preferably, in step A2), the second-dimensional liquid chromatography column purification conditions are: the chromatographic column is a C18 chromatographic column with the diameter of 20-800mm;
the operating pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-50mL/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-water, the mobile phase A is ethanol, and the mobile phase B is water;
the running time is 80min; gradient elution.
The specific procedure of the gradient elution is as follows: 0-60min, phase A: the volume ratio of the phase B is 10:90-80:20; 60-80min, phase A: the volume ratio of the phase B is 80:20-10:90.
preferably, in step A2), the trapping and eluting conditions are: the grain diameter of the filler of the trapping column array trapping column is 10-200 um; the trapping column array trapping column is a C8 chromatographic column; the diameter of the trapping column array is 20-800mm;
the operating pressure of the trapping column is 1-10MPa;
the number of the trapping columns is 9;
preferably, in the step A2), the third-dimensional liquid chromatography column purification conditions are: the chromatographic column is a cyano chromatographic column with the diameter of 20-800mm;
the operating pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-50mL/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-isopropanol, wherein the mobile phase A is ethanol, and the mobile phase B is isopropanol; the running time is 40min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-40min, phase A: the volume ratio of the phase B is 95:5-90:10.
preferably, in step A2), the conditions for trapping and eluting by the trapping column array are as follows: the particle size of the filler of the trapping column is 10-200 um; the trapping column is a C8 chromatographic column;
the diameter of the trapping column is 20-800mm;
the operating pressure of the trapping column is 1-10MPa;
the number of the trapping columns is 9;
preferably, the first chromatographic column is eluted using a gel permeation mode, and the elution volume of the target component is 5 to 20 times the volume of the first chromatographic column.
Preferably, the second chromatography column, using reversed phase mode elution, the elution volume of the target component is 1 to 15 times the volume of the second chromatography column.
Preferably, the volume of the target fraction captured by the chromatographic columns of the capture column array is 3 to 20 times of the volume of the capture column.
Preferably, the third chromatographic column, eluting using a non-aqueous reverse phase mode, elutes the target component in a volume of 1 to 10 times the column volume.
Preferably, the ethanol-water solution has a volume ratio of 10.
Preferably, the first trapping column captures an elution volume of the second chromatography column from 5.0BV to 6.1 BV;
the second trapping column captures the elution volume of the second chromatography column from 6.3BV to 6.8 BV;
the third trap column captures the elution volume of the second chromatography column from 6.9BV to 7.9 BV;
the fourth trapping column captures the second chromatography column at an elution volume of 7.9BV to 8.6 BV;
the fifth trap column captures the elution volume of the second chromatography column from 9.0BV to 10.2BV;
the sixth trapping column captures the elution volume of the second chromatography column from 10.2BV to 13.6 BV.
Preferably, the volume ratio of the ethanol-isopropanol solution is 95.
The invention has the following beneficial effects:
the method applies multidimensional chromatography to separate cembratriene diol from tobacco in a multidimensional and high-efficiency manner. The cembratriene diol is a compound with relatively poor thermal stability, and the multidimensional chromatographic separation process is not contacted with the external environment, so that the cembratriene diol is more suitable for separating substances with relatively poor thermal stability; multiple detectors detect, interference is eliminated, and the result is reliable; complex pretreatment and intermediate treatment steps are not needed, the reagent dosage is reduced, and a new concept of safety and environmental protection is realized; the whole separation process is monitored by an upper computer, so that automatic production is realized, and manual intervention in the separation process is not needed; the purity of the cembratriene diol obtained by purification is more than 99 percent, the recovery rate is high, the process is simple, the large-scale production is easy, and the method has important significance for separation of the cembratriene diol.
Drawings
FIG. 1 is a schematic diagram of a multi-dimensional preparation liquid chromatography purification device for cembratriene diol in tobacco leaves according to the present invention;
FIG. 2 is a first dimension separation spectrum of cembratriene diol in the multi-dimensional preparative liquid chromatography purified tobacco leaves of the present invention;
FIG. 3 is a second dimension separation spectrogram in multi-dimensional preparative liquid chromatography purification of cembratriene diol in tobacco leaves according to the present invention;
FIG. 4 is a third dimension separation spectrum (peak 1: alpha-cembratriene diol; peak 2: beta-cembratriene diol) in multi-dimensional preparative liquid chromatography purified cembratriene diol in tobacco leaves according to the present invention.
Detailed Description
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.
As shown in fig. 1, the apparatus for multi-dimensionally preparing cembratriene diol in liquid chromatography purified tobacco leaves according to the present invention includes a first pump system, a first switching valve, a second switching valve, a third switching valve, a first detector, a fourth switching valve, a trapping column array, a fifth switching valve, a sixth switching valve, a second detector, and an automatic collector, which are connected in sequence; the first switching valve is connected with a sample feeding pump system, the second switching valve is connected with a first chromatographic column system, the sixth switching valve is connected with a third chromatographic column, and the automatic collector is externally connected with a waste liquid pool for collecting waste liquid;
the first switching valve, the second switching valve, the third switching valve and the sixth switching valve are six-way valves, have 6 connection sites of No. 1-6, and have two working states of A and B, when the switching valves are arranged at the position A, the No. 1 position of the switching valves is connected with the No. 2 position, the No. 3 position is connected with the No. 4 position, and the No. 5 position is connected with the No. 6 position; when the switching valve is arranged at the position B, the position 1 of the switching valve is connected with the position 6, the position 2 is connected with the position 3, and the position 4 is connected with the position 5;
the fourth switching valve and the fifth switching valve are ten-position selector valves which have 11 connection points of No. 1-11, and have ten working states of A2, A3, A4, A5, A6, A7, A8, A9, A10 and A11, when the switching valve is arranged at the position A2, the No. 1 position of the switching valve is connected with the No. 2 position, when the switching valve is arranged at the position A3, the No. 1 position of the switching valve is connected with the No. 3 position, when the switching valve is arranged at the position A4, the No. 1 position of the switching valve is connected with the No. 4 position, when the switching valve is arranged at the position A5, the No. 1 position of the switching valve is connected with the No. 5 position, when the switching valve is arranged at the position A6, the No. 1 position of the switching valve is connected with the No. 6 position, when the switching valve is arranged at the position A7, the No. 1 position of the switching valve is connected with the No. 7 position, when the switching valve is arranged at the position A8, the switching valve is arranged at the position A9, the No. 1 position of the switching valve is connected with the No. 9, when the switching valve is arranged at the position A10, the switching valve is connected with the No. 1 position, the switching valve is connected with the No. 11 position, and the switching valve is connected with the No. 11 position;
the trapping column array comprises a first trapping column, a second trapping column, a third trapping column, a fourth trapping column, a fifth trapping column, a sixth trapping column, a seventh trapping column, an eighth trapping column, a ninth trapping column and a bypass;
the outlet end of the first pump system is connected with the No. 1 position of the first switching valve through a pipeline, the No. 2 position of the first switching valve is connected with the No. 1 position of the second switching valve through a pipeline, and the No. 3 position of the first switching valve is connected with the outlet end of the sample injection pump system through a pipeline;
the No. 2 position of the second switching valve is connected to the inlet end of the first chromatographic column through a pipeline, the No. 3 position of the second switching valve is connected with the No. 5 position of the second switching valve through a pipeline, the No. 4 position of the second switching valve is connected with the outlet end of the first chromatographic column through a pipeline, and the No. 6 position of the second switching valve is connected to the No. 1 position of the third switching valve through a pipeline;
the No. 2 position of the third switching valve is connected to the inlet end of the second chromatographic column through a pipeline, the No. 3 position of the third switching valve is connected to the No. 5 position of the third switching valve through a pipeline, the No. 4 position of the third switching valve is connected to the outlet end of the second chromatographic column through a pipeline, and the No. 6 position of the third switching valve is connected to the inlet end of the first detector through a pipeline; the outlet end of the first detector is connected to the No. 1 position of the fourth switching valve through a pipeline.
No. 2 position of a fourth switching valve is connected with the inlet end of the first trapping column through a pipeline, no. 3 position of the fourth switching valve is connected with the inlet end of the second trapping column through a pipeline, no. 4 position of the fourth switching valve is connected with the inlet end of the third trapping column through a pipeline, no. 5 position of the fourth switching valve is connected with the inlet end of the fourth trapping column through a pipeline, no. 6 position of the fourth switching valve is connected with the inlet end of the fifth trapping column through a pipeline, no. 7 position of the fourth switching valve is connected with the inlet end of the sixth trapping column through a pipeline, no. 8 position of the fourth switching valve is connected with the inlet end of the seventh trapping column through a pipeline, no. 9 position of the fourth switching valve is connected with the inlet end of the eighth trapping column through a pipeline, no. 10 position of the fourth switching valve is connected with the inlet end of the ninth trapping column through a pipeline, and No. 11 position of the fourth switching valve is connected with No. 11 switching valve through a pipeline.
A position No. 1 of a fifth switching valve is connected with a position No. 1 of a sixth switching valve through a pipeline, a position No. 2 of the fifth switching valve is connected with the outlet end of the first trapping column through a pipeline, a position No. 3 of the fifth switching valve is connected with the outlet end of the second trapping column through a pipeline, a position No. 4 of the fifth switching valve is connected with the outlet end of the third trapping column through a pipeline, a position No. 5 of the fifth switching valve is connected with the outlet end of the fourth trapping column through a pipeline, a position No. 6 of the fifth switching valve is connected with the outlet end of the fifth trapping column through a pipeline, a position No. 7 of the fifth switching valve is connected with the outlet end of the sixth trapping column through a pipeline, a position No. 8 of the fifth switching valve is connected with the outlet end of the seventh trapping column through a pipeline, a position No. 9 of the fifth switching valve is connected with the outlet end of the eighth trapping column through a pipeline, and a position No. 10 of the fifth switching valve is connected with the outlet end of the ninth trapping column through a pipeline;
the No. 2 position of the sixth switching valve is connected with the inlet end of the third chromatographic column through a pipeline, the No. 3 position of the sixth switching valve is connected with the No. 5 position of the sixth switching valve through a pipeline, the No. 4 position of the sixth switching valve is connected with the outlet end of the third chromatographic column through a pipeline, the No. 5 position of the sixth switching valve is connected with the No. 3 position of the sixth switching valve through a pipeline, the No. 6 position of the sixth switching valve is connected with the inlet end of the second detector through a pipeline, the outlet end of the second detector is connected with the inlet end of the collector through a pipeline, and the outlet end of the collector is connected to a waste liquid pool through a pipeline,
the collector is used for collecting the target component;
in the above multidimensional liquid chromatography device for purifying cembratriene diol, the first chromatographic column is an LSI-100 chromatographic column, the second chromatographic column is a C18 chromatographic column, and the third chromatographic column is a cyano chromatographic column.
In the above multidimensional liquid chromatography apparatus for purifying cembratriene diol, the trapping column array comprises at least two trapping columns and a bypass connected in parallel; wherein the trapping column is a C8 chromatographic column.
The first chromatographic column, the second chromatographic column, the third chromatographic column and the trapping column can be glass columns or stainless steel columns, the diameter of the glass columns or stainless steel columns is 20-800mm, the particle size of the packing is 10-200 mu m, and the operating pressure is 1-10MPa.
The number of the above columns, the number of the trapping columns, and the number of bits of the switching valve can be increased or decreased according to the actual situation.
Based on any one of the above multidimensional liquid chromatography devices, the invention also provides a method for purifying cembratriene diol, which comprises the following steps:
1) First dimension preparative liquid chromatography purification
Loading the tobacco leaf extracting solution to a first chromatographic column, and eluting relative to the first chromatographic column by taking water as a flow; when the first detector detects the target fraction eluted from the first chromatographic column, the second chromatographic column is used for capturing all the target fraction by switching the chromatographic column switching device; enriching the target fraction into a second chromatographic column for second-dimension chromatographic separation; the first chromatographic column is an LSI-100 chromatographic column with the specification of 30 x 20mm; the volume ratio of the water is 100%, the elution volume of the target fraction is 5-20 times of the volume of the first chromatographic column, and the first detector is an ultraviolet-visible light detector.
2) Second dimension preparative liquid chromatography purification
Eluting with ethanol-water as flow phase to the second chromatographic column; when the second detector detects the target fraction eluted from the second chromatographic column, the trapping column switching device is switched, the trapping columns are sequentially used for trapping different target fractions, and the target fractions are enriched into the trapping column array for third-dimensional chromatographic separation; the second chromatographic column is a C18-10um-B chromatographic column with specification of 30 x 250mm, the volume ratio of ethanol to water is 10-95. The trapping column array comprises 9 trapping columns, the first to ninth trapping columns are C8-10um chromatographic columns with the specification of 20 x 20mm, the volume of target fraction trapped by the trapping columns is 3-20 times of the volume of the trapping columns, and the second detector is a differential detector.
3) Third dimension preparative liquid chromatography purification
Sequentially loading target fractions in the elution trapping column array to a third chromatographic column for three-dimensional purification by taking ethanol-isopropanol as a mobile phase, and collecting a third target fraction eluent by using a fraction collector when a second detector detects the corresponding target fraction in the elution state of the third chromatographic column; the third chromatographic column is a cyano CN-10um chromatographic column, the specification is 30 × 250mm, the volume ratio of ethanol to isopropanol is 95-50, and the elution volume of the target fraction is 1-10 times of the volume of the second chromatographic column.
4) And drying the third-dimensional target fraction eluent to obtain the product.
The specific purification steps of the invention are as follows:
Step 3, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position A; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the second column was in the pre-elution state. The first pump system transmits an ethanol-water mobile phase, enters a No. 1 position of a second switching valve from a No. 2 position of the first switching valve, enters a No. 1 position of the second switching valve from the No. 1 position of the first switching valve, enters a No. 1 position of a third switching valve from a No. 1 position of the second switching valve to a No. 6 position of the second switching valve, enters a second chromatographic column from a No. 1 position of the third switching valve to a No. 2 position of the third switching valve, enters a No. 4 position of the third switching valve through the second chromatographic column, enters a first detector from a No. 4 position of the third switching valve to a No. 6 position of the third switching valve, enters a No. 1 position of the fourth switching valve through the first detector, enters a No. 11 position of the fifth switching valve from the No. 1 position of the fourth switching valve to a No. 11 position of the fourth switching valve, enters a No. 1 position of the sixth switching valve from the No. 11 position of the fifth switching valve to the No. 1 position of the sixth switching valve, enters a No. 1 position of the sixth switching valve from the No. 1 position of the sixth switching valve, enters a second detector, and enters a waste liquid pool through the second detector.
Step 4, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position A; the fourth switching valve and the fifth switching valve are positioned at the A2 position; the sixth switching valve is at the position B; the second column was in the eluted state. The first pump system conveys the ethanol-water mobile phase, the second chromatographic column is eluted, the target object passes through the first detector, the target object is enriched by the first trapping column, and the waste liquid enters the waste liquid pool.
Step 5, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position A; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position B; the second column was in a post-elution impurity state. The first pump system conveys the ethanol-water mobile phase, the second chromatographic column is eluted, the waste liquid passes through the first detector, and the waste liquid enters the waste liquid pool.
And 6, repeating the steps 3-5, switching the fourth switching valve and the fifth switching valve to different states A2-A10, and trapping different target components by the first trapping column to the ninth trapping column respectively. Repeating the steps 1-5, loading and eluting for multiple times, and enriching trace components on the trapping column, and performing third-dimensional separation after a certain amount of target components are enriched.
Step 7, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position B; the fourth switching valve and the fifth switching valve are positioned at the A2 position; the sixth switching valve is at the position A; the third chromatographic column is in a sample loading state. The first pump system transmits an ethanol-water mobile phase, enters a No. 1 position of a second switching valve from a No. 2 position of the first switching valve, enters a No. 1 position of the second switching valve from the No. 1 position of the first switching valve, enters a No. 1 position of a third switching valve from a No. 1 position of the second switching valve to a No. 6 position of the second switching valve, enters a first detector from a No. 1 position of the third switching valve to a No. 6 position of the third switching valve, enters a No. 1 position of the fourth switching valve from the first detector, enters a first trapping column from the No. 1 position of the fourth switching valve to the No. 2 position of the fourth switching valve, enters a No. 2 position of a fifth switching valve from the No. 2 position of the fifth switching valve to the No. 1 position of the fifth switching valve, enters a third chromatographic column from the No. 1 position of the sixth switching valve to the No. 2 position of the sixth switching valve, enters a No. 4 position of the sixth switching valve from the third chromatographic column, enters a second detector from the No. 4 position of the sixth switching valve to the No. 6 position of the sixth switching valve, enters a waste liquid pool through the second detector.
Step 8, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position B; the fourth switching valve and the fifth switching valve are positioned at the A2 position; the sixth switching valve is at the position A; the third chromatographic column is in a pre-elution impurity state. The first pump system conveys the ethanol-water mobile phase, elutes the third chromatographic column, and the waste liquid enters the waste liquid pool after passing through the second detector.
9, the first switching valve is in the position A; the second switching valve is at the position B; the third switching valve is at the position B; the fourth switching valve and the fifth switching valve are in the A11 position; the sixth switching valve is at the position A; the third chromatography column is in an eluted state. The first pump system conveys the ethanol-water mobile phase, elutes the third chromatographic column, the target object is collected by the collector after passing through the second detector, and the waste liquid enters the waste liquid pool.
And 11, repeating the steps 7-10, switching the fourth switching valve and the fifth switching valve to different states A2-A10, and loading different target substances trapped by the first trapping column to the ninth trapping column to the third chromatographic column for third-dimensional purification.
And 12, repeating the steps 2 to 11. Thus, a complete process of sample introduction, enrichment, elution and regeneration is completed.
Example 1
Weighing 100g of crushed tobacco powder below 300 meshes, adding 1000mL of 90% ethanol solution, leaching for 3 times, 10 hours each time, filtering to remove residues, combining extracting solutions, and recovering under reduced pressure to remove part of ethanol solvent to obtain a crude extracting solution;
loading the crude extractive solution onto a first chromatographic column, wherein the first chromatographic column is an LSI-100 chromatographic column with a height of 30 × 20mm, and eluting the 15 th column volume by using water as a mobile phase; switching the valve to flow 5-15 column volumes of eluent into a second chromatographic column, applying the target component to the second chromatographic column, wherein the second chromatographic column is a 30 x 250mm c18-10um-B chromatographic column with ethanol-water as the mobile phase, the initial ethanol-water ratio is 10, and the 60min ratio is 80:20 The ratio at 80 minutes is 90:10, the flow rate is 40mL/min, and the detection wavelength of a first detector is 210nm; based on the first detector signal, the valve is switched and the trap column traps the target.
The first trap column captures a second fraction that elutes in the second chromatography column at a volume of 5.0BV to 6.1 BV; capturing a third fraction at an elution volume of 6.3BV to 6.8BV of the second chromatography column by a second trap column; capturing a fourth fraction on the third trap column at an elution volume of 6.9BV to 7.9BV on the second column; the fourth trap column captures a fifth fraction, which is an elution volume at the second chromatography column from 7.9BV to 8.6 BV; the fifth trapping column captures a sixth fraction which elutes at a volume of 9.0BV to 10.2BV in the second chromatography column; the sixth trap column captures a seventh fraction (containing cembratriene diol) at an elution volume of 10.2BV to 13.6BV of the second chromatography column.
And (3) switching a valve, connecting the trapping column with a third chromatographic column, eluting the trapping column, and loading the sample to the third chromatographic column, wherein the third chromatographic column is a 30 x 250mm CN-10um chromatographic column. And (3) loading the fraction collected by the sixth collecting column to a third chromatographic column for separation, wherein ethanol-isopropanol is used as a mobile phase, and the initial ethanol-isopropanol ratio is 95: ratio 90 at 5,40 min: 10, the flow rate is 40mL/min, and the detection wavelength is 210nm; according to the second detector signal, the valve is switched, the fraction collector collects the target substances, the elution volume of the first target substance (alpha-cembratriene diol) is 2.25BV-2.49BV, the elution volume of the second target substance (beta-cembratriene diol) is 4.73BV-4.97BV, the target fraction is subjected to freeze-drying treatment, and the detection results are shown in Table 1:
table 1 table of product name, purity and yield in example 1
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (9)
1. A method for purifying cembratriene diol in tobacco leaves by multi-dimensional preparative liquid chromatography is characterized by comprising the following steps:
a1 Adding a solvent into a tobacco sample for leaching to obtain an extracting solution;
a2 Purifying and eluting the extracting solution obtained in the step A1) by a first-dimension liquid chromatographic column, then purifying and eluting by a second-dimension liquid chromatographic column, and then carrying out fraction and elution by a third-dimension liquid chromatographic column after trapping and elution by a trapping column array so as to provide purified alpha-cematriene diol and beta-cematriene diol.
2. The process for multi-dimensional preparative liquid chromatography purification of cembratriene diol in tobacco leaves according to claim 1, comprising at least one of the following technical features:
b1 In the step A1), the tobacco leaf sample is crushed and sieved tobacco leaf powder; the aperture of the sieving screen of the tobacco sample is less than or equal to 300 meshes;
b2 In the step A1), the ratio of the added mass g of the tobacco leaf sample to the added volume of the solvent is 100;
b3 In step A1), the solvent is 85-95% ethanol aqueous solution;
b4 In step A1), the leaching is carried out 2 to 4 times, preferably 3 times;
b5 In step A1), the leaching time is 9-11h, preferably 10h;
b6 In the step A1), filtering and deslagging are carried out after leaching, the leaching liquor is combined, and the solvent is removed by decompression and recovery;
b7 In step A2), the first-dimension liquid chromatography column purification conditions are: the chromatographic column is LSI-100 chromatographic column with diameter of 20-800mm;
the mobile phase is water;
the operating pressure of the chromatographic column is 1-10MPa;
b8 In step A2), the second-dimensional liquid chromatography column purification conditions are: the chromatographic column is a C18 chromatographic column with the diameter of 20-800mm; preferably 30mm;
the operating pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-3000L/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-water, the mobile phase A is ethanol, and the mobile phase B is water;
the running time is 80min; gradient elution.
The specific procedure of the gradient elution is as follows: 0-60min, phase A: the volume ratio of the phase B is 10:90-80:20;
60-80min, phase A: the volume ratio of the phase B is 80:20-10:90.
b9 In step A2), the trapping and eluting conditions are as follows: the grain diameter of the filler of the trapping column array trapping column is 10-200 um; the trapping column array trapping column is a C8 chromatographic column; the diameter of the trapping column array is 20-800mm;
the operating pressure of the trapping column is 1-10MPa;
the number of the trapping columns is 9;
b10 In step A2), the third-dimensional liquid chromatography column purification conditions are: the chromatographic column is a cyano chromatographic column with the diameter of 20-800mm; preferably 30mm.
The operating pressure of the chromatographic column is 1-10MPa;
the flow rate of the mobile phase is 30-3000L/min, preferably 40mL/min;
the detection wavelength is 210nm; the mobile phase is ethanol-isopropanol, wherein the mobile phase A is ethanol, and the mobile phase B is isopropanol;
the running time is 40min; gradient elution;
the specific procedure of the gradient elution is as follows: 0-40min, phase A: the volume ratio of the phase B is 95:5-90:10.
b11 In step A2), the conditions for trapping and eluting by the trapping column array are as follows: the grain diameter of the filler of the trapping column is 10um-200um; the trapping column is a C8 chromatographic column;
the diameter of the trapping column is 20-800mm;
the operating pressure of the trapping column is 1-10MPa;
the number of trapping columns was 9.
3. The method for multidimensional preparation and liquid chromatography purification of cembratriene diol in tobacco leaves according to claim 2, wherein in B7), the first chromatographic column is eluted by using a gel permeation mode, and the elution volume of the target component is 5-20 times of the column volume of the first chromatographic column.
4. The method for purifying cembratriene diol in tobacco leaves through multidimensional preparation liquid chromatography according to claim 2, wherein in B8), the second chromatographic column is eluted by using a reversed-phase mode, and the elution volume of the target component is 1-15 times of that of the second chromatographic column.
5. The method for purifying cembratriene diol in tobacco leaves through multidimensional preparation and liquid chromatography according to claim 2, wherein in B9), the volume of the target fraction captured by the chromatographic columns of the capture column array is 3-20 times that of the capture column.
6. The method for multi-dimensionally preparing liquid chromatography purified cembratriene diol in tobacco leaves according to claim 2, wherein in B10), the third chromatographic column is eluted using a non-aqueous reverse phase mode, and the elution volume of the target component is 1 to 10 times of the column volume.
7. The method for purifying cembratriene diol in tobacco leaves through multidimensional preparative liquid chromatography according to claim 2, wherein in B8), the volume ratio of the ethanol-water solution is 10-95.
8. The method for multidimensional preparative liquid chromatography purification of cembratriene diol in tobacco leaves according to claim 2, wherein in B9), the first trapping column captures an elution volume of the second chromatography column from 5.0BV to 6.1 BV;
the second trapping column captures the elution volume of the second chromatography column from 6.3BV to 6.8 BV;
the third trapping column captures an elution volume of the second chromatography column from 6.9BV to 7.9 BV;
the fourth trapping column captures an elution volume of the second chromatography column from 7.9BV to 8.6 BV;
the fifth trap column captures the elution volume of the second chromatography column from 9.0BV to 10.2BV;
the sixth trapping column captures the elution volume of the second chromatography column from 10.2BV to 13.6 BV.
9. The method for purifying cembratriene diol in tobacco leaves through multidimensional preparative liquid chromatography according to claim 2, wherein in B10), the volume ratio of the ethanol-isopropanol solution is 95-50.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102206138A (en) * | 2010-12-30 | 2011-10-05 | 上海烟草集团有限责任公司 | Method for separating and purifying two fragrance precursors from tobacco |
| CN103561595A (en) * | 2011-04-27 | 2014-02-05 | R·J·雷诺兹烟草公司 | Tobacco-derived components and materials |
| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206138A (en) * | 2010-12-30 | 2011-10-05 | 上海烟草集团有限责任公司 | Method for separating and purifying two fragrance precursors from tobacco |
| CN103561595A (en) * | 2011-04-27 | 2014-02-05 | R·J·雷诺兹烟草公司 | Tobacco-derived components and materials |
| CN110140993A (en) * | 2011-04-27 | 2019-08-20 | R·J·雷诺兹烟草公司 | Component and material from tobacco |
| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
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