CN102323339B - 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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- CN102323339B CN102323339B CN201110129820.7A CN201110129820A CN102323339B CN 102323339 B CN102323339 B CN 102323339B CN 201110129820 A CN201110129820 A CN 201110129820A CN 102323339 B CN102323339 B CN 102323339B
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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 chromatographic column technical field, be specifically related to a kind of method that detects stability of liquid phase chromatographic column.
Background technology
Liquid chromatography technology is a kind of efficient separating and purifying technology, is widely applied 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 affect the performance of separation and purification, is all core and the forward position of liquid chromatography technology research field all the time.
Chromatographic column post effect is the important parameter in chromatographic column quality investigation with stability.Wherein, chromatographic column post effect is higher, shows that chromatographic column is stronger to the separating power of material; Column stability is better, shows that chromatographic column is better for Analyze & separate reappearance and the stability of material.Therefore, to chromatographic column post effect and stability thereof, investigate significant.
In chromatographic column research field, except buy that business chromatographic column is directly analyzed material or separation, liquid phase chromatography all can relate to the filling of chromatographic column, is about to fixedly phase (claiming again filler) and packs in gc column tube.Packing method is divided into dry method filling and wet method filling.The former is applicable to the filler that particle diameter is larger, by means of apparatuses such as funnels, carries out filling; The latter is applicable to the filler of particle diameter little (as 20 μ m), uses related solvents to carry out homogenate, by means of high-pressure pump, carries out filling.In chromatographic column filling process, conventionally according to the suitable packing method of chromatographic column type selecting used.The difference for the treatment of 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 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 homogenate filling obtains are all effective than dry method filling; Still there is dispute for the importance of preparation property chromatographic column post effect in filling means, but in general, and conventional dress column method has dry packing method, homogenate dress post method, radially pressurize technology, axial pressure technology.
For the investigation of Column stability, conventionally adopt low flow velocity continuous sample introduction, investigate the chromatogram parameter between different sample introductions, thereby determine the stability of chromatographic column.But, because filling process can have influence on the tight ness rating of filler, equal filling quality such as once, affect chromatographic column post and imitate and stability, and then the analysis that affects next step is with separated.Therefore, the method for low flow velocity continuous sample introduction can not meet the needs of measuring filled-type stability of liquid phase chromatographic column, further sets up corresponding study on the stability method and investigates significant to Column stability.
Summary of the invention
The object of the invention is to set up a kind of method of measuring Column stability, the stability of effective evaluation chromatographic column.
The method is mainly the mode by twice Pulsed Sampling, first liquid-phase chromatographic column is connected according to conventional closure, carries out Pulsed Sampling test for the first time, calculates theoretical plate and counts N
1; Then, chromatographic column is turned upside down and reconnected, under the same conditions, carry out Pulsed Sampling for the second time, calculate theoretical plate and count N
2, by chromatographic behavior before and after analyzing, and compare number of theoretical plate N
1and N
2, according to its extent of deviation, evaluate stability of liquid phase chromatographic column or inhomogeneity situation: general deviation is stablized with the interior chromatographic column post effect that is considered as 2%; Deviation, when 2～5% scope, shows that Column stability is general; When deviation is greater than 5%, show that Column stability is poor.Meanwhile, the repeatability of twice Pulse Chromatographic figure be evaluate stability of liquid phase chromatographic column according to one of, repeatability is good, shows liquid-phase chromatographic column filling evenly, stable, otherwise, load inhomogeneous, poor stability.
The present invention be take chromatographic column inversion as core means, and the post of being inverted front and back by measuring chromatographic column is imitated, and compares the difference of chromatographic behavior, thereby determines the stability of chromatographic column itself.This method easily operates, and stability that can effective evaluation liquid-phase chromatographic column, has very high use value.
Accompanying drawing explanation
Fig. 1: the present invention is applied to load the comparison of the good chromatographic column gained of stability chromatogram: the chromatogram that solid line records while being forward connection, dotted line is the chromatogram recording after chromatographic column is inverted.
Fig. 2: the present invention is applied to the chromatographic column gained chromatogram comparison of less stable: the chromatogram that solid line records while being forward connection, dotted line is the chromatogram recording after chromatographic column is inverted.
Embodiment
The present invention has designed a kind of method for detection of stability of liquid phase chromatographic column, and the chromatographic column post of being inverted front and back by comparing chromatographic column is imitated, the stability of effective evaluation chromatographic column.
Just annular sieve plate of the present invention and the effect that produces below, coordinate instantiation to be described in detail as follows:
Example 1
1, by internal diameter, be 25mm, the upper end column cap of traditional chromatographic column that length is 150mm is turned on, and washs 300 order nonporous glass pearl powder, then stir into homogenate with ultrapure water with ethanol, adopts slurry packing that nonporous glass pearl is filled in gc column tube equably; In order to obtain good filling effect, in filling process, chromatographic column is carried out ultrasonic, can be so that filling settlement be more even; After filling up, tighten chromatogram column cap.Afterwards, connect chromatographic analysis device, chromatographic column front end is connected with chromatogram feed liquor pipeline, this test is used is Shimadzu LC-20AT system.Analyzing and testing condition: mobile phase is ultrapure water, arranges two flow velocitys and is respectively 5mmmin
-1, 10mmmin
-1; Sample is 0.002molmL
-1aqueous acetone solution, sample size is 20 μ L; UV-detector, detection wavelength is 250nm.Under different in flow rate, difference sample introduction, records chromatogram and calculates theoretical plate and count N
1; Then, reconnect chromatographic column pipeline: chromatographic column is inverted, and the bottom of former chromatographic column, as present front end, is connected with chromatogram sample feeding pipe; Former chromatographic column front end becomes present bottom, and goes out sample pipeline and is connected, and under the same conditions, carries out Pulsed Sampling for the second time, records chromatogram, calculates theoretical plate and counts N
2.Experimental result is as shown in table 1.
The good chromatographic column of table 1 filling effect is put upside down front and back theoretical cam curve and is changed and deviation
Table 1 shows, under different in flow rate, chromatographic column put upside down before and after theoretical cam curve deviation less, relative standard deviation is less than 2%.Fig. 1 is chromatogram comparison before and after chromatographic column is inverted, and can find out, the chromatogram registration recording before and after being inverted is very high.It can be confirmed that chromatographic column filling effect is better, chromatographic column is comparatively stable.
Example 2
By internal diameter, be 25mm, the upper end column cap of traditional chromatographic column that length is 150mm is turned on, and washs 300 order nonporous glass pearl powder, then stir into homogenate with ultrapure water with ethanol, adopts slurry packing that nonporous glass pearl is filled in gc column tube equably; In filling process, do not carry out ultrasonicly, filler relies on gravity to carry out sedimentation, may cause filling settlement inhomogeneous, high density granular and the sedimentation of bulky grain elder generation; After filling up, tighten chromatogram column cap.Afterwards, connect chromatographic analysis device, chromatographic column front end is connected with chromatogram feed liquor pipeline, this test is used is Shimadzu LC-20AT system.According to condition shown in example 1 and step, test, that is: two flow velocitys are set and are respectively 5mmmin
-1, 10mmmin
-1, 20mmmin
-1; Sample is 0.002molmL
-1aqueous acetone solution, sample size is 20 μ L; UV-detector, detection wavelength is 250nm.Under different in flow rate, difference sample introduction, records chromatogram and calculates theoretical plate and count N
1; Then, reconnect chromatographic column pipeline: chromatographic column is inverted, and the bottom of former chromatographic column, as present front end, is connected with chromatogram sample feeding pipe; Former chromatographic column front end becomes present bottom, and goes out sample pipeline and is connected, and under the same conditions, carries out Pulsed Sampling for the second time, records chromatogram, calculates theoretical plate and counts N
2.Experimental result is as shown in table 2.
The poor chromatographic column of table 2 filling effect is put upside down front and back theoretical cam curve and is changed and deviation
Table 2 shows, under different in flow rate, chromatographic column put upside down before and after theoretical cam curve deviation all larger, relative standard deviation, in 10% left and right, is greater than 5%.Fig. 2 is chromatogram comparison before and after chromatographic column is inverted, and can find out, the chromatogram registration recording before and after being inverted is very low.It can be confirmed that chromatographic column filling effect is poor, chromatographic column is unstable, has also confirmed the impact of filling process for chromatographic column simultaneously.
Claims (1)
1. can effectively detect a method for stability of liquid phase chromatographic column, the method is the mode by twice Pulsed Sampling, first liquid-phase chromatographic column is connected according to conventional closure, carries out Pulsed Sampling test for the first time, calculates theoretical plate and counts N1; Then, chromatographic column is turned upside down and reconnected, under the same conditions, carry out Pulsed Sampling for the second time, calculate theoretical plate and count N2, by chromatographic behavior before and after analyzing, and comparison number of theoretical plate N1 and N2, according to its extent of deviation, just can effectively evaluate stability of liquid phase chromatographic column or inhomogeneity situation, it is characterized in that: the repeatability of twice Pulse Chromatographic figure be evaluate stability of liquid phase chromatographic column according to one of, repeatability is good, liquid-phase chromatographic column filling is all even stable, otherwise, load inhomogeneous and poor stability; Number of theoretical plate N1 and N2 be evaluate stability of liquid phase chromatographic column according to two, deviation is greater than 5%, Column stability is poor and filling is bad, deviation is between 2-5%, stability and filling are general, deviation is less than 2%, good stability and filling are evenly.
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