CN115097048A - Regeneration and activation method for normal phase silica gel column in high performance liquid chromatograph - Google Patents
Regeneration and activation method for normal phase silica gel column in high performance liquid chromatograph Download PDFInfo
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- CN115097048A CN115097048A CN202210834854.4A CN202210834854A CN115097048A CN 115097048 A CN115097048 A CN 115097048A CN 202210834854 A CN202210834854 A CN 202210834854A CN 115097048 A CN115097048 A CN 115097048A
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 239000000741 silica gel Substances 0.000 title claims abstract description 52
- 229910002027 silica gel Inorganic materials 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000007788 liquid Substances 0.000 title claims abstract description 29
- 238000011069 regeneration method Methods 0.000 title description 12
- 230000004913 activation Effects 0.000 title description 11
- 230000008929 regeneration Effects 0.000 title description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 65
- 238000005406 washing Methods 0.000 claims abstract description 36
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 28
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960000583 acetic acid Drugs 0.000 claims abstract description 10
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 10
- 230000003213 activating effect Effects 0.000 claims abstract description 9
- 239000002798 polar solvent Substances 0.000 claims abstract description 9
- 239000008213 purified water Substances 0.000 claims abstract description 9
- 230000001172 regenerating effect Effects 0.000 claims abstract description 9
- 150000007524 organic acids Chemical class 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 238000011010 flushing procedure Methods 0.000 claims description 9
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 230000000052 comparative effect Effects 0.000 description 27
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 26
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 26
- 235000005282 vitamin D3 Nutrition 0.000 description 26
- 239000011647 vitamin D3 Substances 0.000 description 26
- 229940021056 vitamin d3 Drugs 0.000 description 26
- 239000007910 chewable tablet Substances 0.000 description 13
- 229940068682 chewable tablet Drugs 0.000 description 13
- 230000000694 effects Effects 0.000 description 10
- 238000000926 separation method Methods 0.000 description 9
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 3
- 239000000945 filler Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical group [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 150000002009 diols Chemical group 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000003891 environmental analysis Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
Abstract
The invention discloses a method for regenerating and activating a normal phase silica gel column in a high performance liquid chromatograph, which comprises the following steps: (1) installing a normal phase silica gel column which has reduced performance and cannot meet the use requirement in a high performance liquid chromatograph, and setting the column temperature; (2) washing with isopropanol; (3) washing with a polar solvent; (4) washing with an aqueous solution of an organic acid with a volume percentage of 0-2%; (5) washing with purified water; (6) washing with isopropanol; (7) washing with 2.5 vol% glacial acetic acid and 2.5 vol% 2, 2-dimethoxypropane in n-hexane solution; (8) washing with isopropanol, and storing for later use. The method is simple and convenient to operate, can restore the performance of the normal phase silica gel column and effectively prolong the service life of the normal phase silica gel column.
Description
Technical Field
The invention relates to a normal phase filler chromatographic column in a high performance liquid chromatograph, in particular to a method for regenerating and activating a normal phase silica gel column in the high performance liquid chromatograph.
Background
High Performance Liquid Chromatography (HPLC) is a chromatographic method for separating and analyzing chemical mixtures, and compared with other separation methods, the method has the advantages that the equipment and consumables can be selected from a wide range of materials, the materials can be flexibly obtained according to the application target, and the method is almost a universal separation method. At the same time, it also has excellent analytical accuracy, in most cases ± 0.5% or even more accurate.
High Performance Liquid Chromatography (HPLC) is divided into normal phase chromatography and reverse phase chromatography. Reverse phase chromatography is generally suitable for separating non-polar or weakly polar compounds, the more polar components flow out first, and the method is most widely applied to modern liquid chromatography, and more than 70% of separation and analysis are performed in reverse phase chromatography. In contrast, in normal phase chromatography systems, polar compounds are more readily retained by the stationary phase and are typically used for separating polar compounds.
The normal phase chromatography adopts a separation method of a polar stationary phase (such as a stationary phase with diol group, amino group and cyano group, silica gel, aluminum oxide and the like) and a nonpolar mobile phase (such as n-hexane and the like), is mainly suitable for separating medium and small molecular compounds with nonpolar to medium polarity, is mainly applied to separation and analysis of fat-soluble vitamins, steroid hormones, medicines and the like in biochemical analysis, and has wide application in the aspects of pesticides, petrochemical industry, fine chemical industry, environmental analysis and the like.
Silica gel is a common normal phase chromatographic column filler, and the important parameter influencing the application effect is surface activity. The surface activity depends mainly on the type, distribution and reactivity of the surface hydroxyl groups, the most reactive surface is fully hydroxylated, and the surface activity is significantly reduced even in very small amounts by water or other polar compounds adsorbed on the surface. Thus, water should be avoided from entering the system during use of the normal phase chromatography.
With the use of the chromatographic column, impurities entering the chromatographic column are accumulated continuously, so that the separation effect of the normal-phase chromatographic column is reduced gradually, and the daily use requirement cannot be met finally. A common protocol for column maintenance is to wash the column with 100% isopropanol. Meanwhile, the surface activity of the chromatographic column is reduced due to the fact that a small amount of water is contained, and experts propose that the chromatographic column is washed and maintained by using a normal hexane solution containing 2.5% of glacial acetic acid and 2.5% of 2, 2-dimethoxypropane to remove residual water in the normal phase chromatographic column and recover the surface activity of the chromatographic column, and then the normal hexane solution is washed and removed by using 100% of isopropanol, so that the service performance of the chromatographic column can be recovered. After the treatment by the method, if the service performance of the normal phase chromatographic column still cannot be recovered, the manufacturer recommends replacing the chromatographic column for use. For some separation and analysis fields, the performance of the chromatographic column is reduced quickly, and if the chromatographic column is directly replaced, the operation cost of the chromatographic column is greatly increased.
Therefore, a method for activating and regenerating the normal phase silica gel column is established, so that the chromatographic column has extra service life, and the operation cost of related enterprises and institutions can be effectively reduced.
Disclosure of Invention
The invention aims to provide a method for regenerating and activating a normal phase silica gel column in a high performance liquid chromatograph, which is simple and convenient to operate, can restore the performance of the normal phase silica gel column and effectively prolong the service life of the normal phase silica gel column.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a method for regenerating and activating a normal phase silica gel column in a high performance liquid chromatograph, which comprises the following steps:
(1) a normal phase silica gel column which has reduced performance and cannot meet the use requirement is installed in a high performance liquid chromatograph, and the column temperature is set.
(2) Rinsing with isopropanol.
(3) Rinsing with a polar solvent.
(4) And (3) washing with an aqueous solution of 0-2% by volume of organic acid.
(5) Washed with purified water.
(6) Rinsing with isopropanol.
(7) Washed with 2.5% by volume glacial acetic acid and 2.5% by volume 2, 2-dimethoxypropane in n-hexane.
(8) Washing with isopropanol, and storing for later use.
Preferably, in the step (1), the column temperature is set to 28 to 80 ℃.
Preferably, in the step (2), the isopropanol washing flow rate is 0.1-2 ml/min, and the washing is 10-40 times of the column volume.
Preferably, in the step (3), the polar solvent is methanol or acetonitrile, the washing flow rate of the polar solvent is 0.1-2 ml/min, and the washing flow rate is 10-40 times of the column volume.
Preferably, in step (4); the organic acid is trifluoroacetic acid, the flushing flow rate of the aqueous solution of the organic acid with the volume percentage of 0-2% is 0.1-2 ml/min, and the flushing is 10-40 times of the column volume.
Preferably, in the step (5), the purified water is washed at a flow rate of 0.1 to 2ml/min by 10 to 40 times the column volume.
Preferably, in the step (6), the isopropanol washing flow rate is 0.1-2 ml/min, and the washing is 10-40 times of the column volume.
Preferably, in the step (7), the flushing flow rate of the glacial acetic acid with the volume percentage of 2.5% and the n-hexane solution of the 2, 2-dimethoxypropane with the volume percentage of 2.5% is 0.1-2 ml/min, and the flushing is 10-40 times of the column volume.
Preferably, in the step (8), the flow rate of washing with isopropanol is 0.1-2 ml/min, and the washing is 10-40 times of the column volume.
Therefore, the invention has the following beneficial effects: the method for regenerating and activating the normal phase silica gel column in the high performance liquid chromatograph is provided, the high performance liquid chromatograph is used for regenerating and activating the normal phase silica gel column through a specific solvent and a washing process, the operation is simple and convenient, the normal phase silica gel column with reduced performance and lost activity can be recovered to the service performance, and the service life of the normal phase silica gel column can be effectively prolonged (the service life of the column can be effectively prolonged by 2-8 times).
Drawings
FIG. 1 is a chromatogram of a normal phase silica gel column in comparative example 1.
FIG. 2 is a chromatogram of a normal phase silica gel column with reduced performance and no satisfactory use requirement in example 1.
Fig. 3 is a chromatogram of the normal-phase silica gel column after regeneration and activation in example 1.
Fig. 4 is a chromatogram of the normal phase silica gel column after being subjected to regeneration activation in comparative example 1.
Fig. 5 is a chromatogram of the normal phase silica gel column after being subjected to regeneration activation in comparative example 2.
FIG. 6 is a chromatogram of a normal phase silica gel column after regeneration activation in comparative example 3.
FIG. 7 is a chromatogram of a normal phase silica gel column in comparative example 2.
FIG. 8 is a chromatogram of a normal phase silica gel column with reduced performance and no use requirement in example 2.
Fig. 9 is a chromatogram of the normal-phase silica gel column after regeneration activation in example 2.
FIG. 10 is a chromatogram of a normal phase silica gel column in comparative example 3.
Fig. 11 is a chromatogram of a normal phase silica gel column with decreased performance and failed to meet the use requirements in example 3.
FIG. 12 is a chromatogram of the normal phase silica gel column after regeneration activation in example 3.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
The following examples are given for systematic applicability of the vitamin D3 assay standard in vitamin D3 chewable tablet ii.
Comparative example 1
Normal phase silica gel column (SUPELCOSIL LC-SI, 4.6mm 250mm 5 μm) was loaded into high performance liquid chromatograph for vitamin D3 in vitamin D3 chewable tablet ii, under chromatographic conditions: n-hexane: n-pentanol = 997: 3, the flow rate is 1.0ml/min, the column temperature is 28 ℃, the detection wavelength is 254nm, and the obtained chromatogram is shown in figure 1.
Example 1
(1) A chromatogram for detecting vitamin D3 in vitamin D3 chewable tablet ii is shown in fig. 2 (the chromatographic conditions are the same as in comparative example 1) by using a normal phase silica gel column (the column specification is the same as in comparative example 1) which has a reduced performance and cannot meet the use requirement; the normal phase silica gel column, which has a decreased performance and cannot satisfy the use requirement, is installed in a high performance liquid chromatograph, and the column temperature is set at 50 ℃. .
(2) The column was rinsed 20 times its volume with isopropanol at a flow rate of 0.5 ml/min.
(3) The column volume was washed 30 times with a polar solvent (methanol) at a flow rate of 1 ml/min.
(4) The column volume was washed with purified water at a flow rate of 0.5ml/min for 20 times.
(5) The column volume was washed with purified water at a flow rate of 0.5ml/min for 20 times.
(6) The column volume was washed 40 times with isopropanol at a flow rate of 0.5 ml/min.
(7) A40 column volume wash was performed using 2.5% by volume glacial acetic acid and 2.5% by volume 2, 2-dimethoxypropane in n-hexane at a flow rate of 0.22 ml/min.
(8) The column volume was washed 40 times with isopropanol at a flow rate of 0.5ml/min and stored until use.
Loading the regenerated and activated forward silica gel column into a high performance liquid chromatograph, detecting vitamin D3 in vitamin D3 chewable tablet II, and obtaining a chromatogram as shown in figure 3 under the same chromatographic conditions as in comparative example 1.
Comparative example 1
Installing a normal phase silica gel column (the column specification is the same as that of the comparative example 1) with reduced performance and incapable of meeting the use requirement in a high performance liquid chromatograph, setting the column temperature at 50 ℃, and adopting a regeneration and activation method as follows: the column volume was washed with isopropanol at a flow rate of 0.5ml/min for 30 times and stored until use.
The regenerated and activated forward silica gel column was loaded into a high performance liquid chromatograph and used for detecting vitamin D3 in vitamin D3 chewable tablet II (the chromatographic conditions were the same as in comparative example 1), and the obtained chromatogram is shown in FIG. 4.
Comparative example 2
Installing a normal phase silica gel column (the column specification is the same as that of the comparative example 1) with reduced performance and incapable of meeting the use requirement in a high performance liquid chromatograph, setting the column temperature at 50 ℃, and adopting a regeneration and activation method as follows:
(1) washing 20 column volumes with isopropanol at a flow rate of 0.5 ml/min;
(2) washing 40 column volumes with methanol at a flow rate of 0.5 ml/min;
(3) the column volume was washed with isopropanol at a flow rate of 0.5ml/min for 30 times and stored until use.
The regenerated and activated forward silica gel column was loaded into a high performance liquid chromatograph and used for detecting vitamin D3 in vitamin D3 chewable tablet II (the chromatographic conditions were the same as in comparative example 1), and the obtained chromatogram is shown in FIG. 5.
Comparative example 3
Installing a normal phase silica gel column (the column specification is the same as that of the comparative example 1) with reduced performance and incapable of meeting the use requirement in a high performance liquid chromatograph, setting the column temperature at 50 ℃, and adopting a regeneration and activation method as follows:
(1) washing 20 column volumes with isopropanol at a flow rate of 0.5 ml/min;
(2) washing 40 times of column volume with 2.5 vol% glacial acetic acid and 2.5 vol% 2, 2-dimethoxypropane in n-hexane at flow rate of 0.22 ml/min;
(3) the column volume was washed 30 times with isopropanol at a flow rate of 0.5ml/min and stored for further use.
The regenerated and activated forward silica gel column was loaded into a high performance liquid chromatograph and used for detecting vitamin D3 in vitamin D3 chewable tablet ii (the chromatographic conditions were the same as in comparative example 1), and the obtained chromatogram is shown in fig. 6.
As can be seen by comparing fig. 1 to 6: when the chromatographic column fails and the separation degree is obviously reduced, the conventional regeneration method cannot repair the chromatographic column, but the method can obviously repair the separation degree of the chromatographic column so as to meet the original application requirement.
Comparative example 2
Normal phase Silica gel column (Nano Chrom Core Silica, 4.6mm 250mm 5 μm) was loaded into high performance liquid chromatograph for measuring vitamin D3 in vitamin D3 chewable tablet ii under chromatographic conditions: n-hexane: n-pentanol = 997: 3, the flow rate is 1.0ml/min, the column temperature is 28 ℃, the detection wavelength is 254nm, and the obtained chromatogram is shown in FIG. 7.
Example 2
(1) A chromatogram for detecting vitamin D3 in vitamin D3 chewable tablet ii is shown in fig. 8 (the chromatographic conditions are the same as in comparative example 2) by using a normal phase silica gel column (the column specification is the same as in comparative example 2) which has a reduced performance and cannot meet the use requirement; the normal phase silica gel column, which had decreased performance and failed to satisfy the use requirements, was installed in a high performance liquid chromatograph, and the column temperature was set at 28 ℃.
(2) The column volume was washed 30 times with isopropanol at a flow rate of 0.5 ml/min.
(3) The column volume was washed 30 times with methanol at a flow rate of 0.5 ml/min.
(4) A30 column volume wash was performed with 1% trifluoroacetic acid in water at a flow rate of 0.5 ml/min.
(5) The column volume was washed with purified water at a flow rate of 0.5ml/min for 30 times.
(6) The column volume was washed 30 times with isopropanol at a flow rate of 0.5 ml/min.
(7) A30 column volume wash was performed using 2.5% by volume glacial acetic acid and 2.5% by volume 2, 2-dimethoxypropane in n-hexane at a flow rate of 0.5 ml/min.
(8) The column volume was washed 30 times with isopropanol at a flow rate of 0.5ml/min and stored for further use.
The regenerated and activated forward silica gel column was loaded into a high performance liquid chromatograph and used for detecting vitamin D3 in vitamin D3 chewable tablet II (the chromatographic conditions were the same as in comparative example 2), and the obtained chromatogram is shown in FIG. 9.
As can be seen from comparison of fig. 3, fig. 7, fig. 8 and fig. 9, although the chromatographic columns of different specifications have slight differences in the retention time and the resolution, the results of the column effect restoration of the chromatographic columns by the method of the present invention remain unchanged, and the method has universal applicability.
Comparative example 3
Mixing normal phase silica gel column (Welch Ultimate SiO) 2 4.6mm 250mm 5 μm) was loaded into a high performance liquid chromatograph and used for measuring and detecting vitamin D3 in vitamin D3 chewable tablet ii, the chromatographic conditions were as follows:n-hexane: n-pentanol = 997: 3, the flow rate is 1.0ml/min, the column temperature is 28 ℃, the detection wavelength is 254nm, and the obtained chromatogram is shown in FIG. 10.
Example 3
(1) A chromatogram for detecting vitamin D3 in vitamin D3 chewable tablet ii is shown in fig. 11 (the chromatographic conditions are the same as in comparative example 3) by using a normal phase silica gel column (the column specification is the same as in comparative example 3) which has a reduced performance and cannot meet the use requirement; the normal phase silica gel column, which has a decreased performance and cannot satisfy the use requirement, is installed in a high performance liquid chromatograph, and the column temperature is set at 30 ℃.
(2) The column volume was washed 30 times with isopropanol at a flow rate of 1 ml/min.
(3) The column volume was washed 30 times with methanol at a flow rate of 1 ml/min.
(4) The column volume was washed 30 times with 1ml/min flow rate by washing with 1% by volume aqueous trifluoroacetic acid.
(5) The column volume was washed 30 times with purified water at a flow rate of 1 ml/min.
(6) The column volume was washed 30 times with isopropanol at a flow rate of 1 ml/min.
(7) The column volume was washed 30 times with 2.5% by volume glacial acetic acid and 2.5% by volume 2, 2-dimethoxypropane in n-hexane at a flow rate of 1 ml/min.
(8) The column volume was washed 30 times with isopropanol at a flow rate of 1ml/min and stored for further use.
The regenerated and activated forward silica gel column was loaded into a high performance liquid chromatograph and used for detecting vitamin D3 in vitamin D3 chewable tablet ii (the chromatographic conditions were the same as in comparative example 3), and the obtained chromatogram is shown in fig. 12.
As can be seen from comparison of fig. 3, 10, 11 and 12, although the chromatographic columns of different specifications have slight differences in the retention time and the resolution, the results of the column effect restoration by the method of the present invention remain unchanged, and the method has universal applicability.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (9)
1. A method for regenerating and activating a normal phase silica gel column in a high performance liquid chromatograph is characterized by comprising the following steps:
(1) installing a normal phase silica gel column which has reduced performance and cannot meet the use requirement in a high performance liquid chromatograph, and setting the column temperature;
(2) washing with isopropanol;
(3) washing with a polar solvent;
(4) washing with an aqueous solution of an organic acid with a volume percentage of 0-2%;
(5) washing with purified water;
(6) washing with isopropanol;
(7) flushing with glacial acetic acid with the volume percentage of 2.5 percent and n-hexane solution of 2, 2-dimethoxypropane with the volume percentage of 2.5 percent;
(8) washing with isopropanol, and storing for later use.
2. The method as set forth in claim 1, wherein in the step (1), the column temperature is set to 28 to 80 ℃.
3. The method as claimed in claim 1, wherein in step (2), the flow rate of washing with isopropyl alcohol is 0.1-2 ml/min, and the column volume is 10-40 times the washing flow rate.
4. The method as claimed in claim 1, wherein in step (3), the polar solvent is methanol or acetonitrile, the polar solvent washing flow rate is 0.1-2 ml/min, and the polar solvent is washed 10-40 times the column volume.
5. The method of regenerating and activating a normal phase silica gel column in a high performance liquid chromatograph according to claim 1, wherein in the step (4); the organic acid is trifluoroacetic acid, the flushing flow rate of the aqueous solution of the organic acid with the volume percentage of 0-2% is 0.1-2 ml/min, and the flushing is 10-40 times of the column volume.
6. The method as claimed in claim 1, wherein in the step (5), the purified water is washed at a flow rate of 0.1 to 2ml/min at a rate of 10 to 40 column volumes.
7. The method as claimed in claim 1, wherein in step (6), the flow rate of washing with isopropyl alcohol is 0.1-2 ml/min, and the washing is 10-40 times the column volume.
8. The method as claimed in claim 1, wherein in step (7), the flow rate of flushing the 2.5% by volume glacial acetic acid and the 2.5% by volume n-hexane solution of 2, 2-dimethoxypropane is 0.1-2 ml/min, and the flushing is 10-40 times the column volume.
9. The method as claimed in claim 1, wherein in step (8), the flow rate of washing with isopropyl alcohol is 0.1-2 ml/min, and the column volume is 10-40 times the washing flow rate.
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| WO2004089504A1 (en) * | 2003-04-08 | 2004-10-21 | Novo Nordisk A/S | Regeneration of chromatographic stationary phases |
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| WO2004089504A1 (en) * | 2003-04-08 | 2004-10-21 | Novo Nordisk A/S | Regeneration of chromatographic stationary phases |
| CN105561631A (en) * | 2015-12-15 | 2016-05-11 | 袁泳 | Regenerating method of normal phase padding column in high performance liquid chromatograph |
| CN214150580U (en) * | 2020-12-31 | 2021-09-07 | 林蕾 | Liquid chromatographic column maintenance device |
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