CN103969375A - An elution method for liquid chromatography and inspection equipment thereof - Google Patents
An elution method for liquid chromatography and inspection equipment thereof Download PDFInfo
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- CN103969375A CN103969375A CN201310031435.8A CN201310031435A CN103969375A CN 103969375 A CN103969375 A CN 103969375A CN 201310031435 A CN201310031435 A CN 201310031435A CN 103969375 A CN103969375 A CN 103969375A
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Abstract
The invention provides an elution method for detection of glycosylated hemoglobin with liquid chramatography and inspection equipment thereof, the method including following steps: step (1): preparing eluents of three kinds of ion concentration; step (2): performing elution on ion exchange resin which contains a to-be-tested sample with the said eluents of three kinds of ion concentration; step (3): performing chromatographic analysis on the said liquid that undergoes elution, and determining the content of the elution component; step (4): determining the concentration of the glycosylated hemoglobin HbA1c. The method is capable of solving the problem of cross contamination caused in the detection of eluents of two kinds of ion concentration. Compared with the method using eluents of four kinds of ion concentration, the method simplifies the inspection procedure and the structure of the inspection equipment, improves the reliability of the operation of the equipment, reduces the cost of the equipment, and greatly improves the accuracy and stability of the inspection.
Description
Technical field
The present invention relates to liquid chromatography (LC) detection field, relate in particular to a kind of elution process and inspection machine thereof of low pressure ion exchange in solution chromatography detection glycosylated hemoglobin.
Background technology
The concentration of the topmost subfraction HbA1c of glycosylated hemoglobin in blood of human body directly reflects the trimestral average blood sugar level of diabetes patient, and this index is the important evidence of the clinical examination as diabetes patient, diagnosis, treatment.
Adopt clinically at present internationally recognized goldstandard liquid chromatography to detect Glycohemoglobin HbA1c, wherein high efficiency liquid phase chromatographic analysis method (HPLC) only adopts the elution process of two kinds of ion concentrations, uses a kind of high ion concentration eluent and a kind of low concentration eluent; By being set in machine, carrys out one mechanical six logical rotary distributor the eluent of distributively generated different ions concentration, so that the different glycosylated hemoglobin components in sample are carried out to wash-out; Due to proportioning variable concentrations eluent in employing machine, bring the complexity of instrument to increase, failure rate increases, and the cross pollution of eluent is serious, and the cost of the manufacture of instrument, maintenance, use is also high.And low pressure liquid phase chromatography analytic approach has the elution process of two kinds of ion concentrations of employing and the elution process of four kinds of ion concentrations; Adopt respectively the eluent of two kinds and four kinds different ions concentration; These two kinds of elution process are all, by the mode of the independent eluent of controlling a kind of ion concentration of an electrical isolation valve, the different glycosylated hemoglobin components of sample are carried out to wash-out; Two kinds of eluent wash-out separating effects are not thorough, directly affect the accuracy of testing result; And good compared with two kinds of eluents of four kinds of eluent separating effects, but the cost of the manufacture of the complexity of instrument, failure rate, instrument, maintenance and use all can increase thereupon.
Summary of the invention
In order to solve above technical matters, the invention provides elution process and inspection machine thereof that a kind of liquid chromatography (LC) detects glycosylated hemoglobin, can reduce the structure of eluent cross pollution, reduced inspection equipment and trace routine, raising equipment operational reliability, the equipment cost that reduces.
The invention provides a kind of elution process of liquid chromatography (LC), comprise following step:
Step (1): the eluent of three kinds of ion concentrations of configuration;
Step (2): have the ion exchange resin of sample to be tested to carry out wash-out to absorption the eluent of described three kinds of ion concentrations;
Step (3): carried out stratographic analysis to described by the liquid after wash-out, determine by the content of wash-out composition;
Step (4): the concentration of determining Glycohemoglobin HbA1c.
The present invention also provides a kind of elution process that detects glycosylated hemoglobin for liquid chromatography (LC), and described eluent comprises:
For the eluent A of resin cleaning, balance and wash-out glycosylated hemoglobin HbA1a and HbA1b, described eluent A by weight percentage, comprise: NaH2PO4 0.178-0.186%, Na2HPO40.056-0.076%, NaCl 0.116-0.120%, KCl 0.024-0.026%, NaN3 0.039-0.041%, surplus is deionized water;
For the eluent B of wash-out Glycohemoglobin HbA1c, described eluent B by weight percentage, comprising: NaH2PO4 0.148-0.168%, Na2HPO4 0.054-0.060%, NaCl 0.584-0.596%, KCl 0.040-0.044%, NaN3 0.039-0.041%, surplus are deionized water;
Be used for the eluent C of the regeneration of the non-glycosylated hemoglobin HbA0 of wash-out and resin, described eluent C by weight percentage, comprising: NaH2PO4 0.141-0.143%, Na2HPO4 0.082-0.088%, NaCl1.479-1.619%, NaN3 0.039-0.041%, surplus are deionized water.
The present invention also provides a kind of inspection machine that detects glycosylated hemoglobin for liquid chromatography (LC), comprising:
Control device, for sending instruction control sampling system;
Sampling apparatus, starts to carry out specimen sample, and sample is placed into mixed educating in cup, carries out haemolysis;
Peristaltic pump, for providing pipeline liquid flow required drive;
Electrical isolation valve, for switching sample to be tested, eluent A, eluent B and eluent C;
Pick-up unit, comprises photometer, chromatographic column and colorimetric pool, and when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample.
The elution process for liquid chromatography (LC) detection glycosylated hemoglobin that adopts this equipment, comprises following step:
Step (1): control device sends instruction control sampling apparatus to start to sample, is placed into mixed educating and in cup, carries out haemolysis;
Step (2): control device sends instruction and makes peristaltic pump running, and electrical isolation valve is sent to instruction, switches respectively sample, eluent A, eluent B and eluent C;
Step (3): control device monitors control to the temperature of chromatographic column in real time, when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample and returns to control device.
Preferably, also comprise: all parameters are showing screen display, and when completing after test, control device finally draws measurement result by the calculating of magnitude of voltage.
Preferably, peristaltic pump comprises the first peristaltic pump and the second peristaltic pump, and the rotating speed of described the first peristaltic pump is greater than the rotating speed of described the second peristaltic pump.
The invention has the beneficial effects as follows: the present invention has not only solved the problem of the eluent easy cross pollution of when inspection of two kinds of ion concentrations, and for the eluent of four kinds of ion concentrations, simplified inspection machine structure and trace routine, improved equipment operational reliability, reduced equipment cost, greatly improved the Stability and veracity detecting.
Brief description of the drawings
Fig. 1 is the structural representation that a kind of liquid chromatography (LC) provided by the invention detects the inspection machine of glycosylated hemoglobin.
The schematic diagram of the inspection machine that Fig. 2 adopts for the inventive method embodiment.
Embodiment
Below in conjunction with accompanying drawing, preferably embodiment of the present invention is described in further detail:
Embodiment 1
In the structural representation shown in Fig. 1, the eluent of three kinds of different ions concentration is set, that is: A eluent 1, B eluent 2, C eluent 3, the export pipeline of three kinds of eluent containers is controlled by electrical isolation valve 14 separately respectively, and can be respectively under the effect of the first peristaltic pump 4, the second peristaltic pump 7, need this resin 5 of sample through absorption, after wash-out, enter colorimetric pool 7 and carry out stratographic analysis, finally draw the concentration of glycosylated hemoglobin.
In the inspection machine schematic diagram shown in Fig. 2, power supply 11 is powered to whole system, control device 8 detects after the beginning test instruction that operator sends, sampling device 10 starts to sample, be placed into the mixed cup 17 of educating and carry out haemolysis, then control device 8 sends instruction the first peristaltic pump 4 and the second peristaltic pump 7 is turned round according to same direction, wherein the rotating speed of the first peristaltic pump 4 is greater than the rotating speed of the second peristaltic pump 7, then control device 8 sends instruction to electrical isolation valve 14, switch respectively sample to be tested, A eluent 1, B eluent 2, C eluent 3, control device 8 monitors control to the temperature of chromatographic column 5 in real time, when sample enters after colorimetric pool 6 by chromatographic column 5, photometer 12 carries out voltage sample and returns to control device 8, all parameters show on display screen 13.When completing after test, control device 8 finally draws measurement result by the calculating of magnitude of voltage.
Embodiment 2
1. the corresponding eluent of configuration is as shown in table 1:
Table 1
2. control device sends instruction control sampling apparatus and starts to sample, be placed into mixed educating and in cup, carry out haemolysis, then control device sends instruction and makes peristaltic pump running, then control device sends instruction to electrical isolation valve, switch respectively sample, eluent A, eluent B and eluent C, the control device in real time temperature to chromatographic column (25 DEG C) monitors control, and when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample and returns to control device.
Embodiment 3
1. the corresponding eluent of configuration is as shown in table 2:
Table 2
2. control device sends instruction control sampling apparatus and starts to sample, be placed into mixed educating and in cup, carry out haemolysis, then control device sends instruction and makes peristaltic pump running, then control device sends instruction to electrical isolation valve, switch respectively sample, eluent A, eluent B and eluent C, the control device in real time temperature to chromatographic column (25 DEG C) monitors control, and when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample and returns to control device.
Embodiment 4
1. the corresponding eluent of configuration is as shown in table 3:
Table 3
While coordinating inspection machine to detect for glycosylated hemoglobin the eluent of above-described embodiment, its preci-sion and accuracy is tested, concrete test method is as follows:
Precision test: get concentration value and be third party's glycosylated hemoglobin standard items of 9.45%, the eluent of use embodiment and inspection machine revision test thereof 10 times, calculate the mean value X of 10 tests, and calculate standard deviation S according to formula (1), calculate coefficient of variation CV according to formula (2).
Standard deviation
---------(1)
Wherein: N is test number (TN), T is each test value.
Coefficient of variation CV=(S/X) × 100%-----------------------------------------------(2)
Accuracy test: get concentration value and be third party's glycosylated hemoglobin standard items of 5.0%, use the eluent of embodiment and inspection machine duplicate detection thereof 3 times, calculates the mean value X of 3 tests, and according to formula (3) calculating relative deviation B.
Relative deviation B=[(X-5.0)/5.0] × 100%----------------------------------------(3)
Experimental test result is as shown in table 5, table 6:
Table 5 precision test
| Sequence number | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| 1 | 9.45 | 9.59 | 9.51 |
| 2 | 9.53 | 9.46 | 9.45 |
| 3 | 9.38 | 9.49 | 9.58 |
| 4 | 9.48 | 9.55 | 9.42 |
| 5 | 9.52 | 9.47 | 9.57 |
| 6 | 9.41 | 9.52 | 9.36 |
| 7 | 9.50 | 9.41 | 9.42 |
| 8 | 9.43 | 9.48 | 9.38 |
| 9 | 9.49 | 9.45 | 9.54 |
| 10 | 9.44 | 9.48 | 9.46 |
| Average | 9.46 | 9.49 | 9.47 |
| Standard deviation | 0.05 | 0.06 | 0.05 |
| The coefficient of variation (%) | 0.52 | 0.60 | 0.52 |
Table 6 accuracy test
| Sequence number | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| 1 | 5.02 | 5.10 | 5.07 |
| 2 | 5.11 | 5.03 | 5.09 |
| 3 | 5.08 | 5.04 | 5.03 |
| Average | 5.07 | 5.06 | 5.06 |
| Standard items target value | 5.0 | 5.0 | 5.0 |
| Relative deviation (%) | 1.4 | 1.2 | 1.2 |
Test findings shows, the testing result that the inventive method and inspection machine draw meets clinical detection requirement completely.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (6)
1. an elution process for liquid chromatography (LC), mainly for detection of the concentration of Glycohemoglobin HbA1c, is characterized in that, comprises following step:
Step (1): the eluent of three kinds of ion concentrations of configuration;
Step (2): have the ion exchange resin of sample to be tested to carry out wash-out to absorption the eluent of described three kinds of ion concentrations;
Step (3): carried out stratographic analysis to described by the liquid after wash-out, determine by the content of wash-out composition;
Step (4): the concentration of determining Glycohemoglobin HbA1c.
2. the elution process for liquid chromatography (LC) as claimed in claim 1, is characterized in that, described eluent comprises:
For the eluent A of resin cleaning, balance and wash-out glycosylated hemoglobin HbA1a and HbA1b, described eluent A by weight percentage, comprise: NaH2PO4 0.178-0.186%, Na2HPO40.056-0.076%, NaCl 0.116-0.120%, KCl 0.024-0.026%, NaN3 0.039-0.041%, surplus is deionized water;
For the eluent B of wash-out Glycohemoglobin HbA1c, described eluent B by weight percentage, comprising: NaH2PO4 0.148-0.168%, Na2HPO4 0.054-0.060%, NaCl 0.584-0.596%, KCl 0.040-0.044%, NaN3 0.039-0.041%, surplus are deionized water;
Be used for the eluent C of the regeneration of the non-glycosylated hemoglobin HbA0 of wash-out and resin, described eluent C by weight percentage, comprising: NaH2PO4 0.141-0.143%, Na2HPO4 0.082-0.088%, NaCl1.479-1.619%, NaN3 0.039-0.041%, surplus are deionized water.
3. an inspection machine that adopts method described in claim 1, comprising:
Sampling apparatus, starts to sample, and is placed into mixed educating and in cup, carries out haemolysis;
Control device, for sending sampling apparatus described in instruction control;
Peristaltic pump, for providing pipeline liquid flow required drive;
Electrical isolation valve, for switching sample to be tested, eluent A, eluent B and eluent C;
Pick-up unit, comprises photometer, chromatographic column and colorimetric pool, and when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample.
4. adopt an elution process for equipment as claimed in claim 3, it is characterized in that, comprise following step:
Step (1): control device sends instruction control sampling apparatus and starts to carry out specimen sample, and sample is placed into mixed educating in cup, carries out haemolysis;
Step (2): control device sends instruction and makes peristaltic pump running, and electrical isolation valve is sent to instruction, switches respectively sample to be tested, eluent A, eluent B and eluent C;
Step (3): control device monitors control to the temperature of chromatographic column in real time, when sample enters after colorimetric pool by chromatographic column, photometer carries out voltage sample and returns to control device.
5. method as claimed in claim 4, is characterized in that, also comprises: all parameters are showing screen display; When completing after test, control device finally draws measurement result by the calculating of magnitude of voltage.
6. method as claimed in claim 4, is characterized in that, peristaltic pump comprises the first peristaltic pump and the second peristaltic pump, and the rotating speed of described the first peristaltic pump is greater than the rotating speed of the second peristaltic pump.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865326A (en) * | 2015-05-28 | 2015-08-26 | 江苏奥迪康医学科技有限公司 | Three-gradient elution analysis method of glycosylated hemoglobin analyzer |
| CN105771316A (en) * | 2016-05-03 | 2016-07-20 | 哈尔滨珍宝制药有限公司 | Automatic control system and method for panax notoginseng saponins column chromatography |
| CN105974141A (en) * | 2016-05-09 | 2016-09-28 | 温冬梅 | Novel multi-mode automatic analysis system of glycated hemoglobin HbA1c |
| CN110441463A (en) * | 2019-07-04 | 2019-11-12 | 江苏奥迪康医学科技股份有限公司 | A kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c |
| CN111562339A (en) * | 2020-06-17 | 2020-08-21 | 深圳普门科技股份有限公司 | Method and equipment for detecting hemoglobin substances |
| CN115445245A (en) * | 2022-07-29 | 2022-12-09 | 广州双桥（重庆）有限公司 | Sequential simulated moving bed chromatographic separation system and cleaning method thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865326A (en) * | 2015-05-28 | 2015-08-26 | 江苏奥迪康医学科技有限公司 | Three-gradient elution analysis method of glycosylated hemoglobin analyzer |
| CN105771316A (en) * | 2016-05-03 | 2016-07-20 | 哈尔滨珍宝制药有限公司 | Automatic control system and method for panax notoginseng saponins column chromatography |
| CN105771316B (en) * | 2016-05-03 | 2017-12-22 | 哈尔滨珍宝制药有限公司 | Arasaponin column chromatography procedure automatic control system and method |
| CN105771316B8 (en) * | 2016-05-03 | 2020-08-04 | 哈尔滨珍宝制药有限公司 | Automatic control system and method for panax notoginseng saponins column chromatography process |
| CN105974141A (en) * | 2016-05-09 | 2016-09-28 | 温冬梅 | Novel multi-mode automatic analysis system of glycated hemoglobin HbA1c |
| CN110441463A (en) * | 2019-07-04 | 2019-11-12 | 江苏奥迪康医学科技股份有限公司 | A kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c |
| CN111562339A (en) * | 2020-06-17 | 2020-08-21 | 深圳普门科技股份有限公司 | Method and equipment for detecting hemoglobin substances |
| CN111562339B (en) * | 2020-06-17 | 2022-11-15 | 深圳普门科技股份有限公司 | Method and equipment for detecting hemoglobin substances |
| CN115445245A (en) * | 2022-07-29 | 2022-12-09 | 广州双桥（重庆）有限公司 | Sequential simulated moving bed chromatographic separation system and cleaning method thereof |
| CN115445245B (en) * | 2022-07-29 | 2024-06-11 | 广州双桥（重庆）有限公司 | Sequential simulated moving bed chromatographic separation system and cleaning method thereof |
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Inventor after: Zeng Ying Inventor after: Xu Yan Inventor after: Liu Xiancheng Inventor after: Peng Guoqing Inventor after: Hu Wenyong Inventor after: Mei Linxiang Inventor before: Xu Yan Inventor before: Liu Xiancheng Inventor before: Peng Guoqing Inventor before: Hu Wenyong Inventor before: Mei Linxiang |
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Application publication date: 20140806 |