CN103869007A - Method for evaluating C18 reverse-phase filler preparative chromatographic column - Google Patents
Method for evaluating C18 reverse-phase filler preparative chromatographic column Download PDFInfo
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- CN103869007A CN103869007A CN201210552881.9A CN201210552881A CN103869007A CN 103869007 A CN103869007 A CN 103869007A CN 201210552881 A CN201210552881 A CN 201210552881A CN 103869007 A CN103869007 A CN 103869007A
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- phthalic acid
- dibutyl ester
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Abstract
The invention relates to a method for evaluating performance of a C18 preparative high-performance liquid column. The method is characterized in that with a 100% methanol as an elution solvent and with uracil and di-n-butyl phthalate as standard substances, performance evaluation of the C18 preparative high-performance liquid column is carried out. The method is used for evaluating the chromatographic column, can avoid a chromatographic column balancing process, directly performs column evaluation, has the characteristics of rapid and simple method and low cost, and is suitable for evaluating the performance of the C18 preparative high-performance liquid column.
Description
Technical field
The present invention relates to a kind of method of the C18 of evaluation preparative high efficiency liquid phase post.
Background technology
Preparative chromatography has powerful separating power, is separation and purification natural products, chiral drug, the main technique methods of albumen, polypeptide biomacromolecule.Chromatographic column is the core position of chromatographic resolution, affects separation efficiency, operating cost and product purity.The efficient preparative column of C18 filler filling has powerful separating power and the scope of application, is widely used in the separation and purification process of various complicated difficult sample separation.Post evaluation is the conventional step that uses of efficient preparative column, need regularly or irregularly to carry out, to understand column performance situation, the repeatability of detachment process between ensureing batch, high efficiency and validity, as load the optimization of process need post evaluation result guide parameters, and the undesirable post that needs of separating resulting is evaluated test column performance, and chromatographic column needs post evaluation to detect wash result after polluting the clear system of rear methyl alcohol.Conventional chromatogram post evaluation method adopts Methanol+Water as eluting solvent, the derivant of benzene and benzene is that standard substance is measured, before mensuration, need one section of chromatographic column equilibrium process, rinse chromatographic column with 10 to 20 times of column volume test solvent, by chromatographic column from filling or preserve to replace solvent to post and evaluate eluent (Liu Guoquan used, Yu Zhaolou, chromatographic column technology (second edition), 2005:95-100).Balance solvent load and column diameter are quadratic relationship.In the time adopting conventional post evaluation method to evaluate major diameter industry preparative column, the increase of column diameter causes solvent-oil ratio very big, and the equilibrium process complexity of chromatographic column simultaneously, increases time cost and labour intensity.
Methyl alcohol is efficient preparative chromatography use procedure common solvent, and 100% methyl alcohol can be applied to chromatographic column filling, cleans the aspects such as preservation.But taking 100% methyl alcohol as eluting solvent, a little less than the chromatogram of working standard retains, evaluation result is subject to the impact of the factors such as system bulk and inaccurate.
Summary of the invention
The present invention relates to a kind of method that C18 of evaluation prepares μ type high efficiency liquid phase post.After chromatographic column is connected in liquid chromatographic system, taking 100% methyl alcohol as eluting solvent, adopting uracil and the positive dibutyl ester of phthalic acid is that standard substance carries out C18 chromatographic column evaluation column performance.
Concrete steps are as follows:
1) preparation of standard solution
After the positive dibutyl ester of uracil and phthalic acid dissolves as standard solution.
2) post connects
C18 preparative high efficiency liquid phase post is connected in liquid chromatographic system.
3) reverse-phase chromatographic column evaluation
Standard solution is by six-way valve sample introduction, and 100% methyl alcohol is eluting solvent elution chromatography post, and ultraviolet detection record goes out peak situation.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.
Standard solution process for preparation: the volumetric concentration that 0.1-25g uracil and the positive dibutyl ester of 0.1-50g phthalic acid is dissolved in to 100mL is in 50-100% methyl alcohol or ethanol.
Adopt the present invention to have the following advantages compared with classic method:
1) to adopt 100% methyl alcohol be eluting solvent to this method.Can avoid chromatographic column equilibrium process, when chromatographic column filling, cleaning, preservation, can carry out immediately column performance evaluation, than traditional preparation method, this method does not need column equilibration process, saves solvent and time.
2) this method employing uracil and the positive dibutyl ester of phthalic acid are that standard substance carries out the evaluation of chromatographic column evaluation column performance, and sample composition is simple, and standard substance, for conventional chemical reagent, easily obtains.
3) the positive dibutyl ester of phthalic acid is that rich shade spectrum retains material, and under 100% methanol-eluted fractions, retention factors can reach more than 1, can effectively avoid the impact of system bulk on evaluation result.
Brief description of the drawings
Fig. 1 is embodiment 1 Plays separating substances result.1: uracil, 2: the positive dibutyl ester of phthalic acid.
Embodiment
Now with accompanying drawing, the present invention is described in further details, embodiment only limits to illustrate the present invention in conjunction with the embodiments, but not limitation of the invention.
Embodiment 1
Evaluate Shiseido company and produce the efficient preparative column of commercialization, fixing is Superiorex UG120 type C18 filler mutually.Take 25g uracil and the positive dibutyl ester of 10g phthalic acid is dissolved in 100mL methyl alcohol, as standard solution.Chromatographic column (5 μ m, 20 × 250mm) is connected in liquid chromatographic system.Chromatographic column (5 μ m, 20 × 250mm) is connected in liquid chromatographic system.Standard solution passes through six-way valve sample introduction, sampling volume 10 μ l, and 100% methyl alcohol is eluent system elution chromatography post, ultraviolet detection record goes out peak situation, detects wavelength 300nm.Standard substance separate colors spectrogram is shown in accompanying drawing 1.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.The chromatographic column dead time is 2.56min, and the positive dibutyl ester retention factors of phthalic acid is 1.34, and chromatographic column post effect is 8464.
Evaluate Waters company and produce the efficient preparative column of commercialization, fixing is Nova-pak ODS type C18 filler mutually.Take 10g uracil and the positive dibutyl ester of 50g phthalic acid is dissolved in 100mL ethanol, as standard solution.Chromatographic column (4 μ m, 40 × 250mm) is connected in liquid chromatographic system.Standard solution passes through six-way valve sample introduction, sampling volume 10 μ l, and 100% methyl alcohol is eluent system elution chromatography post, ultraviolet detection record goes out peak situation, detects wavelength 220nm.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.The chromatographic column dead time is 2.65min, and the positive dibutyl ester retention factors of phthalic acid is 1.02, and chromatographic column post effect is 8964.
Embodiment 3
Evaluate Agilent company and produce the efficient preparative column of commercialization, fixing is XDB type C18 filler mutually.Take 0.1g uracil and the positive dibutyl ester of 0.5g phthalic acid is dissolved in 100mL 50% ethanol, as standard solution.Chromatographic column (5 μ m, 20 × 250mm) is connected in liquid chromatographic system.Standard solution passes through six-way valve sample introduction, sampling volume 10 μ l, and 100% methyl alcohol is eluent system elution chromatography post, ultraviolet detection record goes out peak situation, detects wavelength 300nm.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.The chromatographic column dead time is 2.66min, and the positive dibutyl ester retention factors of phthalic acid is 2.01, and chromatographic column post effect is 3864.
Embodiment 4
Evaluate the preparative high efficiency liquid phase post oneself loading, fixing is that Agilent company produces Microsorb type C18 filler mutually.Take 0.5g uracil and the positive dibutyl ester of 0.1g phthalic acid is dissolved in 100mL 50% methyl alcohol, as standard solution.Chromatographic column (5 μ m, 50 × 200mm) is connected in liquid chromatographic system.Standard solution passes through six-way valve sample introduction, sampling volume 100 μ l, and 100% methyl alcohol is eluent system elution chromatography post, ultraviolet detection record goes out peak situation, detects wavelength 203nm.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.The chromatographic column dead time is 2.76min, and the positive dibutyl ester retention factors of phthalic acid is 1.31, and chromatographic column post effect is 5864.
Embodiment 5
Evaluate the preparative high efficiency liquid phase post oneself loading, fixing is that Fuji company produces Chromatorex type C18 filler mutually.Take 0.5g uracil and the positive dibutyl ester of 0.5g phthalic acid is dissolved in 100mL 50% methyl alcohol, as standard solution.Chromatographic column (5 μ m, 100 × 210mm) is connected in liquid chromatographic system.Standard solution passes through six-way valve sample introduction, sampling volume 100 μ l, and 100% methyl alcohol is eluent system elution chromatography post, ultraviolet detection record goes out peak situation, detects wavelength 254nm.With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.The chromatographic column dead time is 2.58min, and the positive dibutyl ester retention factors of phthalic acid is 1.21, and chromatographic column post effect is 5664.
Claims (6)
1. a method of evaluating C18 reverse phase filler preparative chromatography post, is characterized in that: taking 100% methyl alcohol as eluting solvent, the positive dibutyl ester of uracil and phthalic acid is standard substance, carries out the evaluation of C18 preparative high efficiency liquid phase column performance; Concrete steps are as follows:
1) preparation of standard solution
After the positive dibutyl ester of uracil and phthalic acid dissolves as standard solution;
2) post connects
C18 preparative high efficiency liquid phase post is connected in liquid chromatographic system;
3) reverse-phase chromatographic column evaluation
Standard solution is by six-way valve sample introduction, and 100% methyl alcohol is eluting solvent elution chromatography post, and ultraviolet detection record goes out peak situation; With chromatographic retention examination post dead time and the retention factors of uracil and the positive dibutyl ester of phthalic acid, with the positive dibutyl ester chromatographic retention of phthalic acid and peak width computer chromatography post post effect.
2. method according to claim 1, is characterized in that: it is as standard solution in 50-100% methyl alcohol or ethanol that the positive dibutyl ester of uracil and phthalic acid is dissolved in volumetric concentration.
3. method according to claim 1, is characterized in that: in standard solution, uracil concentration is 0.1-25%.
4. method according to claim 1, is characterized in that:
In standard solution, the positive dibutyl ester concentration of phthalic acid is 0.1-50%.
5. method according to claim 1, is characterized in that: preparative high efficiency liquid phase post can be selected the preparative high efficiency liquid phase post of commercialization or oneself filling.
6. method according to claim 1, is characterized in that: it is arbitrary wavelength between 203nm to 300nm that ultraviolet detects wavelength.
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Citations (3)
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| CN101776668A (en) * | 2010-03-13 | 2010-07-14 | 兰州中科安泰分析科技有限责任公司 | Filling method of C18 HPLC (High Performance Liquid Chromatography) column |
| CN102323339A (en) * | 2011-05-19 | 2012-01-18 | 周鑫 | Method for detecting stability of liquid phase chromatographic column |
| CN102460147A (en) * | 2009-06-24 | 2012-05-16 | 弗·哈夫曼-拉罗切有限公司 | Characterization of re-useable chromatography equipment |
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Patent Citations (3)
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|---|---|---|---|---|
| CN102460147A (en) * | 2009-06-24 | 2012-05-16 | 弗·哈夫曼-拉罗切有限公司 | Characterization of re-useable chromatography equipment |
| CN101776668A (en) * | 2010-03-13 | 2010-07-14 | 兰州中科安泰分析科技有限责任公司 | Filling method of C18 HPLC (High Performance Liquid Chromatography) column |
| CN102323339A (en) * | 2011-05-19 | 2012-01-18 | 周鑫 | Method for detecting stability of liquid phase chromatographic column |
Non-Patent Citations (8)
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| MCCALLEY, DV: "Comparison of the performance of conventional C-18 phases with others of alternative functionality for the analysis of basic compounds by reversed-phase high-performance liquid chromatography", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 844, no. 1, 4 June 1999 (1999-06-04), pages 23 - 38 * |
| ZHANG, MENGYAO ET.AL: "Preparation and Chromatographic Evaluation of Columns for Reversed-Phase Liquid Chromatography", 《ACTA CHIMICA SLOVENICA》, vol. 58, no. 1, 31 January 2011 (2011-01-31), pages 60 - 68 * |
| 刘国诠 等: "《色谱柱技术》", 31 January 2009, article "液相色谱柱的填装、高效液相色谱柱的清洗与再生", pages: 94 - 284 * |
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Application publication date: 20140618 |