CN101620182B - Multi-wavelength nucleic acid protein chromatographic separation detecting system - Google Patents
Multi-wavelength nucleic acid protein chromatographic separation detecting system Download PDFInfo
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- CN101620182B CN101620182B CN2009100328405A CN200910032840A CN101620182B CN 101620182 B CN101620182 B CN 101620182B CN 2009100328405 A CN2009100328405 A CN 2009100328405A CN 200910032840 A CN200910032840 A CN 200910032840A CN 101620182 B CN101620182 B CN 101620182B
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Abstract
The invention relates to a multi-wavelength nucleic acid protein chromatographic separating and detecting device, which is composed of a controller, an ultraviolet light source, an interference filter for splitting beam, a sample cell, a photoelectric cell, a signal amplifier, a logarithm converter and an A/D converter; the device adopts a two-channel A/D converter, the ultraviolet light source is formed into two beams of monochromatic light with different wavelengths after being light-split by two interference filters, two beams of the monochromatic light are respectively transmitted on the photoelectric cell after being reflected on the sample cell by a light path and a slit, the photoelectric cell obtains two paths of electrical signal which are proportional to transmission light intensity by a photoelectric converter, the electrical signal is connected with the signal amplifier to amplify the signal, is connected with the logarithm converter to carry out logarithmic conversion of the signal, the signal obtained by the logarithmic conversion is proportional to light adsorption in the sample cell and is connected with the two-channel A/D converter which outputs digital signal; a control terminal and an output terminal of the A/D converter are connected with the controller which is connected with an interface circuit for carrying out signal processing of a host computer server.
Description
One, technical field:
The present invention is the dedicated separation detection method and the equipment of biological chemistry and field of biological pharmacy, especially relates to multi-wavelength nucleic acid protein chromatographic method for separating and detecting and system.
Two, background technology:
In scientific research and production run, exist biomacromolecules such as amounts of protein, nucleic acid and polypeptide analysis, separate and purifying work, press for efficiently analyze fast, separation and preparation method.The biochemical isolation technics that extensively adopts mainly contains precipitate and separate, chromatography, electrophoretic separation, centrifuging, membrane separation technique at present.And the chromatography technology with its special advantages all the time in occupation of the leading position of biological macromolecule purifying technology.Tomographic system comprises two phases: stationary phase and mobile phase.When mobile phase flow through the stationary phase that is added with sample, because the allocation proportion difference of each component between two-phase, each component will move and disconnected from each other coming with friction speed.According to the form of stationary phase matrix, chromatography can be divided into that ply of paper is analysed, thin-layer chromatography and column chromatography.The gel chromatography of using always in the biochemical technology, ion-exchange chromatography etc. adopt column chromatography method usually.The column chromatography system mainly contains two parts and forms chromatography column system and detector system.According to research object design chromatography column system, comprise cylinder, stationary phase, moving phase (eluant, eluent), flow velocity etc., formed the field of specializing in.Detecting device is column chromatography systematic analysis, separation and purifying work " eyes ", mainly contains ultraviolet (UV) absorption, differential refraction, specific inductive capacity etc.Wherein ultraviolet absorption detector is the widest detecting device of column chromatography system applies, and it has 1. highly sensitive, and minimal detectable concentration can reach 10
-10It is little that g/mL is influenced by operating conditions and external environment 2., and being suitable for gradient elution does not 3. have the material composition of no uv absorption and respond 4. that non-destructive detects can connect with other detecting devices characteristics and advantages such as use.The isolation identification that both can be used for small amount of matter can be used for the separation and purification and the preparation of big quantity of material again.In occupation of the leading position of separation and purification of biological macromolecule technology, utilization rate accounts for about 70%.Though traditional core acid albumin chromatography device is furnished with 280nm, 254nm equiwavelength selects, but can only select single wavelength during actual the use detects, therefore, tradition chromatography tripping device can only utilize the known one-component of material property wavelength separated (albumen or nucleic acid etc.), can not separate unknown blending ingredients, more can not analyzing proteins or the purity of nucleic acid.The big molecule chromatography tripping device that uses is made of parts such as nucleic acid-protein detector, chromatographic column, constant flow pump, fraction collector and registering instruments at present.As a kind of easy analysis separation means and method, prolonged application is in education experiment, scientific research and commercial production.The operation of retouching links such as spectrum at adjustment at zero point, sensitivity selection, reading conversion, service recorder instrument is the same substantially, and only detects down at single wavelength (280nm or 254nm).Can only utilize the known one-component of material property wavelength separated (albumen or nucleic acid), can not separate unknown blending ingredients, more can not analyzing proteins or the purity of nucleic acid.Traditional core acid albumin detector is being operated and the following several respects deficiency of technical ubiquity: 1, error.First data of instrument detecting are the light transmission rate value.Since instrument the analog-to-digital conversion apparatus error is failed to obtain good rectification building-out, there is bigger error in actual converted result.Adjust though require the user must carry out zero point before sample introduction, method is: adjusting transmitance earlier is 100%, and then to transfer absorbance be 0, make this point (T=100%, A=0) both have met lambert--Beer law (A=lg (1/T)).But, can't guarantee that (generally getting 0-2A) in measurement range all meets lambert--Beer law.In fact difference both exist error, the error that has surpasses 50%.Therefore, the science of instrument detecting can not get guaranteeing.
2, reading.Reading was in the instrument effective range when traditional detection instrument was measured for assurance, on instrument panel, all be provided with sensitivity selecting arrangement (2.0A, 1.0A, 0.5A, 0.2A, 0.1A, 0.05A etc.), can bring thus: 1. when unknown sample concentration is had little understanding, do not know how to select sensitivity, need grope repeatedly; 2. the instrument displayed value is not the sample absorbance, and real absorbance is the product that instrument shows number and sensitivity; 3. as the sensitivity that when measuring, changes instrument will have serious consequences (baseline changes greatly).
3, loaded down with trivial details.The measurement result of traditional detection instrument under different sensitivities is the 0-10mv d. c. voltage signal mostly, and this signal is outputed on the registering instrument, selects suitable registering instrument chart drive speed, draws the absorbance spectrum on recording chart.Well imagine that the absorbance on the recording chart is not the sample absorbance, is subjected to the modulation of instrumental sensitivity yet.Wanting from the be absorbed parameter such as area, normalization at peak of recording chart will be consuming time, the very loaded down with trivial details thing of taking a lot of work.Moreover the information that the papery spectrogram provides is single and be difficult to preservation, more can not directly insert in article and use.
4, single.Though the traditional detection instrument may be furnished with 280nm, 254nm equiwavelength selects, can only select single wavelength when using and detect, can not carry out dual wavelength (280nm and 254nm) synchronous detection simultaneously.Therefore, traditional chromatography tripping device can only utilize material property wavelength separated known component.Need further to separate unknown materials, traditional chromatography tripping device is powerless.
Three, summary of the invention:
The present invention seeks to: the error that traditional chromatography tripping device exists is big, reading is directly perceived, registering instrument is retouched spectrum in order thoroughly to solve, can only single wavelength detection etc. problem.Proposing a kind of multi-wavelength nucleic acid protein chromatographic that relates to separates and detection system.
The technology of the present invention solution is: multi-wavelength nucleic acid protein chromatographic separates and detection method, become two monochromic beams of different wave length after the ultraviolet source beam split, two monochromic beams are by the sample cell of light path after slit is mapped to the nucleic acid protein chromatographic separation, be transmitted to respectively on two photoelectric tubes (R2868) through sample cell, photoelectric tube obtains and the directly proportional electric signal of transmitted intensity through opto-electronic conversion; Earlier signal is amplified, carry out signal then to number conversion, signal and the sample cell light absorption that obtain this moment are in direct ratio, form digital signal through double channel A/D conversion again.Become the two-beam of different wave length to inject wavelength (being respectively 280nm and 254nm) after ultraviolet source (comprising UA, UB, the UC wave band) beam split, monochromatic light is transmitted on the photoelectric tube respectively after slit is mapped to sample cell by light path (two group of 45 degree catoptron).
The step that the two monochromic beam light paths that light source produces detect is:
1), be provided with the light path selecting arrangement in the inner exit of ultraviolet source, this device is made up of stepper motor and anti-dazzling screen, has (two multiple is as 4 pairs) window on the anti-dazzling screen, and collector lens is housed on window, and the light after the convergence is injected and penetrated respective filter; 2), the two path signal of logarithmic amplifier output is input to A/D converter; 3), rotation and the A/D converter signal by the control step motor obtains to realize that synchronously assurance different wave length signal and A/D output are consistent; 4), then two ways of digital signals is carried out the shaping conversion, give interface circuit.
Multi-wavelength nucleic acid protein chromatographic separates and pick-up unit, by controller, ultraviolet source, the interference filter beam split, sample cell, photoelectric tube, signal amplifier, logarithmic converter, double channel A/D converter constitutes, ultraviolet source becomes the monochromatic light of different wave length after at least two kinds of interference filter beam split, monochromatic light is transmitted on the photoelectric tube respectively after being mapped to sample cell by light path and slit, photoelectric tube obtains two-way and the directly proportional electric signal of transmitted intensity through photoelectric commutator, electric signal connects signal amplifier and carries out the signal amplification, and be input to logarithmic amplifier (converter) and carry out signal number conversion, signal that logarithm is converted to and sample cell light absorption are in direct ratio, connect double channel A/D converter again, the digital signal of A/D converter output; The control end that is connected A/D converter by controller and output terminal also are connected the interface communication end of upper computer work; Computer work carries out the data transmission reception, screen is painted work such as spectrum, parameter analysis, spectrogram editor, data preservation.
The two monochromic beam structures that light source produces are: be provided with the light path selecting arrangement in the inner exit of ultraviolet source, this device is made up of stepper motor and anti-dazzling screen, have alternately corresponding (as 4 pairs) window of output of two wavelength light on the anti-dazzling screen, corresponding respectively the monochromatic light output of two kinds of wavelength.Collector lens is housed on window, and the light after the convergence is injected optical filter; The two path signal of logarithmic amplifier output is input to A/D converter; When driving the anti-dazzling screen rotation, in light path, exports by stepper motor the monochromatic light of two kinds of wavelength respectively; The present invention can also adopt the light of the different wavelength of three beams as required.Corresponding three photoelectric tubes of employing (R2868) have (three multiple is as 6 pairs) window on the anti-dazzling screen, collector lens is housed on window, and the light after the convergence is injected corresponding optical filter.
Beneficial effect of the present invention is: the present invention is foundation with the Lambert-Beer's law, designs in detection method, light source design, light path conversion.Comprise at testing circuit that simulation is amplified, logarithm amplifies, aspects such as A/D conversion, digital control, embedded technology and computer work are as the same.On probation through overtesting and user, finished the development of " dual wavelength nucleic acid-protein detector " and computer software workstation, mix parts such as chromatographic column, constant flow pump, fraction collector, constituted complete " dual wavelength nucleic acid protein chromatographic separation detecting system ".This system has realized the good detection effect: the present invention carries out real-time synchronous detection and designs chromatography computer software workstation the absorbance of sample cell liquid with two kinds of wavelength (as 280nm and 254nm) in the chromatography process.Draw out the dual wavelength chromatogram of more abundant information, in the dual wavelength collection of illustrative plates, carry out ratio computing (as using the 280nm absorbance), obtain the purity collection of illustrative plates of albumen and nucleic acid divided by the 254nm absorbance.
1), absorbance A and transmitance T be in strict conformity with Lambert-Beer's law (A=Lg (1/T)), both (A and T) corresponding errors are less than 1% arbitrarily; 2), system adjusts absorbance to 0.000, transmittance to 100% automatically; 3), dual wavelength (as 280nm and 254nm) synchronous detection; 4), data acquisition, computer interface (com port and USB interface) and software workstation, detection, collection, software workstation integral system are integrated; 5), chromatographic analysis software has data acquisition, retouches spectrum, analytical parameters, preservation, printing, edition function in real time.6), analytical parameters has peak height, peak width, peak area, normalization, retention time, area content, purity, chromatographic column resolution etc.
Four, description of drawings
Fig. 1 is that hardware of the present invention constitutes synoptic diagram
Fig. 2 is a detection system block diagram of the present invention
Fig. 3,4 is two kinds and detects collection of illustrative plates
Five, embodiment
As shown in drawings, according to hardware block diagram with, multi-wavelength nucleic acid protein chromatographic of the present invention separates and pick-up unit, by controller, ultraviolet source, the interference filter beam split, sample cell, photoelectric tube, signal amplifier, logarithmic converter, double channel A/D converter constitutes, ultraviolet source becomes the monochromatic light of different wave length after at least two kinds of interference filter beam split beam split, monochromatic light is transmitted on the photoelectric tube respectively after being mapped to sample cell by light path and slit, photoelectric tube obtains two-way and the directly proportional electric signal of transmitted intensity through photoelectric commutator, electric signal connects signal amplifier to carry out signal and amplifies, and connects logarithmic amplifier (converter) and carry out signal to number conversion, and signal that logarithm is converted to and sample cell light absorption are in direct ratio, connect double channel A/D converter again, the digital signal of A/D converter output; The control end that is connected A/D converter by controller and output terminal also are connected the interface communication end of upper computer work; Computer work carries out the data transmission reception, screen is painted work such as spectrum, parameter analysis, spectrogram editor, data preservation.
The two monochromic beam structures that produced by ultraviolet source are: be provided with the light path selecting arrangement in the inner exit of ultraviolet source, this device is made up of stepper motor and anti-dazzling screen, have (4 pairs) window on the anti-dazzling screen, collector lens is housed on window, the light after the convergence is injected optical filter; The two path signal of logarithmic amplifier output is input to A/D converter;
The present invention is foundation with the Lambert-Beer's law, amplifies in light source design, light path design, simulation amplification, logarithm, aspects such as A/D conversion, digital control, embedded technology and computer work use the brand-new design theory.Use through long-time research, test and user, finished the development of " dual wavelength nucleic acid-protein detector " and computer software workstation, mix parts such as chromatographic column, constant flow pump, fraction collector, constituted complete " dual wavelength nucleic acid protein chromatographic piece-rate system ".This system has realized:
1, absorbance A and transmitance T are in strict conformity with Lambert-Beer's law (A=Lg (1/T)), and both (A and T) corresponding errors are less than 1% arbitrarily;
2, system adjusts absorbance to 0.000, transmittance to 100% automatically;
3, dual wavelength (as 280nm and 254nm) synchronous detection;
4, data acquisition, computer interface (com port and USB interface) and software workstation, detection, collection, software workstation integral system are integrated;
5, chromatographic analysis software has data acquisition, retouches spectrum, analytical parameters, preservation, printing, editting function in real time;
6, analytical parameters has peak height, peak width, peak area, normalization, retention time, area content, purity, chromatographic column resolution etc.;
(2) by lambert--Beer law as can be known, the absorbance A (λ) of component under different wave length, only with this component under extinction coefficient K (λ) is relevant, that is: A (λ 1)=K (λ 1) Cb A (λ 2)=K (λ 2) Cb
A(λ1)/A(λ2)＝K(λ1)/K(λ2)
This ratio is a constant.The absorbance of sample cell liquid is carried out synchronous detection and draw with two kinds (as 280nm, 254nm) or two or more wavelength, obtain the dual wavelength or the multi-wavelength chromatogram of more abundant information.The present invention carries out detecting in real time synchronously with dual wavelength (as 280nm and 254nm), carries out computing (using the 280nm absorbance divided by the 254nm absorbance) in the dual wavelength collection of illustrative plates that obtains, and just obtains albumen, nucleic acid purity collection of illustrative plates.Can know where be pure protein (ratio is greater than 1.7), where be pure nucleic acid (ratio is less than 0.5).
Fig. 3 is haemoglobin and lactochrome biased sample solution 2mg, chromatography column length 20cm, and internal diameter 1.0cm, stationary phase are sephadex G 25, the chromatography collection of illustrative plates that obtains with 0.05mol phosphate buffer wash-out.Because haemoglobin different with the lactochrome molecular weight (the haemoglobin molecule amount is greater than the lactochrome molecular weight), residence time of chromatographic column with difference.First peak is the haemoglobin component, and second peak is the lactochrome component, can see that two components separate fully.
Fig. 4 is that the purity of carrying out obtaining after the ratio computing (using 280nm (light color) absorbance divided by 254nm (dark color) absorbance (black) curve, that is: A280/A254) on Fig. 3 basis is composed.Document is pointed out: ratio is greater than 1.7 part pure proteins in the purity spectrum, and ratio is pure nucleic acid less than 0.5 part.Therefore, haemoglobin component purity is higher in the sample, and lactochrome component protein purity is lower.
Claims (2)
1. multi-wavelength nucleic acid protein chromatographic separates and pick-up unit, by controller, ultraviolet source, interference filter, sample cell, photoelectric tube, signal amplifier, logarithmic converter, double channel A/D converter constitutes, it is characterized in that described ultraviolet source is two monochromic beams that become different wave length after two interference filter beam split, two monochromic beams are transmitted on the photoelectric tube respectively after being mapped to sample cell by light path and slit, photoelectric tube obtains two-way and the directly proportional electric signal of transmitted intensity through photoelectric commutator, described electric signal connects signal amplifier to carry out after signal amplifies, connect logarithmic converter and carry out signal number conversion, signal that logarithm is converted to and sample cell light absorption are in direct ratio, connect double channel A/D converter again, double channel A/D converter output digital signal; The control end that is connected double channel A/D converter by controller and output terminal also are connected the interface circuit of the signal Processing of upper computer work.
2. multi-wavelength nucleic acid protein chromatographic according to claim 1 separates and pick-up unit, it is characterized in that ultraviolet source produces the output of two monochromic beams, be provided with the light path selecting arrangement in the inner exit of ultraviolet source, the light path selecting arrangement is made up of stepper motor and anti-dazzling screen, stepper motor drives the anti-dazzling screen rotation, has 4 pairs of windows on anti-dazzling screen, corresponding respectively the monochromatic light output of two kinds of wavelength, collector lens is housed on window, and the light after the convergence is injected interference filter; The two path signal of logarithmic converter output is input to double channel A/D converter.
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| CN106018309A (en) * | 2016-05-05 | 2016-10-12 | 武汉光昱科技有限公司 | Multifunctional nucleic acid protein detection system |
| CN109781672A (en) * | 2019-03-18 | 2019-05-21 | 广东工业大学 | A kind of strength of fluid recognition methods and equipment |
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