CN102323339A - Method for detecting stability of liquid phase chromatographic column - Google Patents
Method for detecting stability of liquid phase chromatographic column Download PDFInfo
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- CN102323339A CN102323339A CN201110129820A CN201110129820A CN102323339A CN 102323339 A CN102323339 A CN 102323339A CN 201110129820 A CN201110129820 A CN 201110129820A CN 201110129820 A CN201110129820 A CN 201110129820A CN 102323339 A CN102323339 A CN 102323339A
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Abstract
The invention relates to a method for effectively detecting the stability of a liquid phase chromatographic column, which mainly adopts a mode of two-time pulse sampling as follows: firstly, the liquid phase chromatographic column is connected according to the conventional connection direction, a first pulse sampling test is carried out, and the number of theoretical plates N1 is calculated; secondly, the chromatographic column is reconnected by turning upside down, i.e., the bottom of the original chromatographic column is used as the front end of the current chromatographic column, and the front end of the original chromatographic column is used as the bottom end of the current chromatographic column, a second pulse sampling test is carried out under the same conditions, and then the number of theoretical plates N2 is calculated. By analysis of degree of coincidence of chromatograms before and after the chromatographic column is turned upside down, the numbers of theoretical plates N1 and N2 are compared, accordingly, the stability or uniformity of the liquid phase chromatographic column can be effectively detected according to the degree of deviation. As the accompanying drawings of the abstract of the specification are shown, the chromatogram obtained from the chromatographic column with more stable filling has very high degree of coincidence, and accordingly, the stability of column efficiency is effectively confirmed.
Description
Technical field
The present invention relates to the chromatographic column technical field, be specifically related to a kind of method that detects the liquid chromatography column stability.
Background technology
Liquid chromatography technology is a kind of high efficiency separation purification technique, has obtained widespread use in a lot of fields, as: chemical industry, biology, food, medicine etc.Wherein, chromatographic column is the core of liquid chromatography, is the main place that realizes separating substances, can directly influence the performance of separation and purification, all is the core and the forward position of liquid chromatography technology research field all the time.
The chromatographic column post is imitated and stability is the important parameter in the chromatographic column quality investigation.Wherein, the chromatographic column post is imitated high more, shows that chromatographic column is strong more to the separating power of material; Chromatographic column stability is good more, shows that chromatographic column is good more for the analysis separation reappearance and the stability of material.Therefore, investigate significant to chromatographic column post effect and stability thereof.
In the chromatographic column research field, except buying commercial chromatographic column directly analyzes material or separate, liquid phase chromatography all can relate to the filling of chromatographic column, is about in stationary phase (the claiming filler again) gc column tube of packing into.Packing method is divided into dry method filling and wet method filling.The former is applicable to the filler that particle diameter is bigger, carries out can by means of apparatuses such as funnels; The latter is applicable to the filler of particle diameter little (like 20 μ m), uses related solvents to carry out homogenate, carries out can by means of high-pressure pump.In the chromatographic column filling process, usually based on the suitable packing method of used chromatographic column type selecting.The difference of treatment capacity per sample, liquid-phase chromatographic column can be divided into analytical column, semi-preparative column, preparative column, and in different chromatographic columns, packing material size is different.Generally speaking, the fill method that is applicable to the analytic type chromatographic column has equilibrium density method, equilibrium viscosity method, dynamic agitation method and homogenate method; Packing method research for semi-preparative column is less, and research shows that post effect and reappearance thereof that the homogenate filling is obtained are all effective than the dry method filling; Still there is dispute in the importance that the filling means are imitated for preparation property chromatographic column post, but in general, dress column method commonly used has dry packing method, homogenate dress post method, radially pressurization is technological, axial pressure is technological.
For the investigation of chromatographic column stability, adopt low flow velocity continuous sample introduction usually, investigate the chromatogram parameter between the different sample introductions, thereby confirm the stability of chromatographic column.But, because the filling process can have influence on the tight ness rating of filler, all once wait the filling quality, influence the chromatographic column post and imitates with stable, so the analysis that influences next step with separate.Therefore, the method for low flow velocity continuous sample introduction can not satisfy the needs of measuring filled-type liquid chromatography column stability, further sets up corresponding study on the stability method and investigates significant to the chromatogram column stability.
Summary of the invention
The objective of the invention is to set up a kind of method of measuring chromatographic column stability, the stability of effective evaluation chromatographic column.
This method mainly is the mode through two subpulse sample introductions, at first liquid-phase chromatographic column is connected according to conventional closure, carries out the test of pulse first time sample introduction, and theory of computation plate is counted N
1Then, chromatographic column turned upside down to be connected again, under the same conditions, carries out pulse second time sample introduction, and theory of computation plate is counted N
2,, and compare number of theoretical plate N through chromatographic behavior before and after analyzing
1And N
2, estimate liquid-phase chromatographic column stability or inhomogeneity situation according to its extent of deviation: general deviation is imitated stable 2% with the interior chromatographic column post that is regarded as; Deviation shows that chromatographic column stability is general when 2～5% scope; Deviation showed the chromatographic column less stable greater than 5% o'clock.Simultaneously, the repeatability of twice Pulse Chromatographic figure is one of foundation of estimating liquid-phase chromatographic column stability, and repeatability is good, shows the liquid-phase chromatographic column filling evenly, and is stable, otherwise, then load inhomogeneous, poor stability.
The present invention is the core means with the chromatographic column inversion, is inverted the post effect of front and back through measuring chromatographic column, compares the difference of chromatographic behavior, thereby confirms the stability of chromatographic column itself.This method is operated easily, and stability that can the effective evaluation liquid-phase chromatographic column has very high use value.
Description of drawings
Fig. 1: the present invention is applied to load stability chromatographic column gained chromatogram comparison preferably: the chromatogram that solid line records when being the forward connection, dotted line are the chromatogram that records after chromatographic column is inverted.
Fig. 2: the chromatographic column gained chromatogram that the present invention is applied to less stable compares: the chromatogram that solid line records when being the forward connection, dotted line are the chromatogram that records after chromatographic column is inverted.
Embodiment
The present invention has designed a kind of method that is used to detect the liquid chromatography column stability, imitates the stability of effective evaluation chromatographic column through the chromatographic column post before and after relatively chromatographic column is inverted.
Below just ring-like sieve plate of the present invention and the effect that produced, cooperate instantiation to be described in detail as follows:
Instance 1
1, be 25mm with internal diameter, length is that the upper end column cap of traditional chromatographic column of 150mm is turned on, and with washing with alcohol 300 order nonporous glass pearl powder, stirs into homogenate with ultrapure water again, adopts slurry packing to be filled in the nonporous glass pearl in the gc column tube equably; In order to obtain filling effect preferably, in the filling process chromatographic column is carried out ultrasonic, can be so that filling settlement be more even; Tighten the chromatogram column cap after filling up.Afterwards, connect chromatographic analysis device, the chromatographic column front end is connected with chromatogram feed liquor pipeline, this test is used to be Tianjin, island LC-20AT system.The analyzing and testing condition: moving phase is ultrapure water, two flow velocitys is set is respectively 5mmmin
-1, 10mmmin
-1Sample is 0.002molmL
-1Aqueous acetone solution, sample size are 20 μ L; UV-detector, the detection wavelength is 250nm.Difference sample introduction under different in flow rate, record chromatogram and theory of computation plate are counted N
1Then, connect the chromatographic column pipeline again: chromatographic column is inverted, and promptly the bottom of former chromatographic column links to each other with chromatogram sample introduction pipe as present front end; Former chromatographic column front end becomes present bottom, and goes out a kind pipeline and links to each other, and under the same conditions, carries out pulse second time sample introduction, the record chromatogram, and theory of computation plate is counted N
2Experimental result is as shown in table 1.
Table 1 filling effect chromatographic column is preferably put upside down front and back theoretical cam curve variation and deviation
Table 1 shows that under different in flow rate, the theoretical cam curve deviation was less before and after chromatographic column was put upside down, and relative standard deviation is less than 2%.Fig. 1 is that chromatogram relatively can find out that the chromatogram registration that records before and after being inverted is very high before and after chromatographic column was inverted.Can confirm that thus the chromatographic column filling effect is better, chromatographic column is comparatively stable.
Instance 2
With internal diameter is 25mm, and length is that the upper end column cap of traditional chromatographic column of 150mm is turned on, and with washing with alcohol 300 order nonporous glass pearl powder, stirs into homogenate with ultrapure water again, adopts slurry packing to be filled in the nonporous glass pearl in the gc column tube equably; Do not carry out ultrasonicly in the filling process, filler relies on gravity to carry out sedimentation, may cause filling settlement inhomogeneous, high density granular and the first sedimentation of bulky grain; Tighten the chromatogram column cap after filling up.Afterwards, connect chromatographic analysis device, the chromatographic column front end is connected with chromatogram feed liquor pipeline, this test is used to be Tianjin, island LC-20AT system.Test according to condition shown in the instance 1 and step, that is: two flow velocitys are set are respectively 5mmmin
-1, 10mmmin
-1, 20mmmin
-1Sample is 0.002molmL
-1Aqueous acetone solution, sample size are 20 μ L; UV-detector, the detection wavelength is 250nm.Difference sample introduction under different in flow rate, record chromatogram and theory of computation plate are counted N
1Then, connect the chromatographic column pipeline again: chromatographic column is inverted, and promptly the bottom of former chromatographic column links to each other with chromatogram sample introduction pipe as present front end; Former chromatographic column front end becomes present bottom, and goes out a kind pipeline and links to each other, and under the same conditions, carries out pulse second time sample introduction, the record chromatogram, and theory of computation plate is counted N
2Experimental result is as shown in table 2.
Theoretical cam curve changed and deviation before and after the relatively poor chromatographic column of table 2 filling effect was put upside down
Table 2 shows that under the different in flow rate, the theoretical cam curve deviation was all bigger before and after chromatographic column was put upside down, and relative standard deviation is about 10%, greater than 5%.Fig. 2 is that chromatogram relatively can find out that the chromatogram registration that records before and after being inverted is very low before and after chromatographic column was inverted.Can confirm that thus the chromatographic column filling effect is relatively poor, chromatographic column is unstable, has also confirmed the influence of filling process for chromatographic column simultaneously.
Claims (4)
1. method that can effectively detect the liquid chromatography column stability.This method mainly is the mode through two subpulse sample introductions, at first liquid-phase chromatographic column is connected according to conventional closure, carries out the test of pulse first time sample introduction, and theory of computation plate is counted N
1Then, chromatographic column turned upside down to be connected again, under the same conditions, carries out pulse second time sample introduction, and theory of computation plate is counted N
2,, and compare number of theoretical plate N through chromatographic behavior before and after analyzing
1And N
2, just can estimate liquid-phase chromatographic column stability or inhomogeneity situation effectively according to its extent of deviation.
2. the method for detection liquid chromatography column stability according to claim 1 is characterized in that, chromatographic column turns upside down, and to connect again be the characteristics of this method.
3. the method for detection liquid chromatography column stability according to claim 1 is characterized in that, the repeatability of twice Pulse Chromatographic figure is one of foundation of estimating liquid-phase chromatographic column stability; Repeatability is good; The liquid-phase chromatographic column filling is all even stable, otherwise, then load inhomogeneous and poor stability.
4. the method for detection liquid chromatography column stability according to claim 1 is characterized in that, number of theoretical plate N
1And N
2Be to estimate two of the stable foundation of liquid-phase chromatographic column, deviation is greater than 5%, and the chromatographic column poor stability loads bad; Deviation is between 2～5%, and stability is all general with the filling effect; Deviation is less than between 2%, good stability, and filling is evenly.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103869007A (en) * | 2012-12-18 | 2014-06-18 | 中国科学院大连化学物理研究所 | Method for evaluating C18 reverse-phase filler preparative chromatographic column |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1148336A1 (en) * | 1998-11-18 | 2001-10-24 | Eisai Co., Ltd. | Diffusion promoting apparatus for low flow velocity gradient high-speed liquid chromatography |
| US6344172B1 (en) * | 1991-09-30 | 2002-02-05 | Perseptive Biosystems, Inc. | Protein chromatography system |
| 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 |
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- 2011-05-19 CN CN201110129820.7A patent/CN102323339B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6344172B1 (en) * | 1991-09-30 | 2002-02-05 | Perseptive Biosystems, Inc. | Protein chromatography system |
| EP1148336A1 (en) * | 1998-11-18 | 2001-10-24 | Eisai Co., Ltd. | Diffusion promoting apparatus for low flow velocity gradient high-speed liquid chromatography |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 姜静等: "实验室HPLC柱的填装", 《仪器仪表与分析监测》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103869007A (en) * | 2012-12-18 | 2014-06-18 | 中国科学院大连化学物理研究所 | Method for evaluating C18 reverse-phase filler preparative chromatographic column |
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