CN108152419A - A kind of two-dimensional liquid chromatography for measuring peptide molecular weight and application thereof - Google Patents
A kind of two-dimensional liquid chromatography for measuring peptide molecular weight and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of two-dimensional liquid chromatography for measuring peptide molecular weight and its application, which includes the first dimension liquid-phase chromatographic column, the second dimension liquid-phase chromatographic column, two-dimension switching device, the second dimension sampling device and two sample loops；First dimension liquid-phase chromatographic column therein and the second dimension liquid-phase chromatographic column are connect with two-dimension switching device；Second dimension liquid-phase chromatographic column is gel chromatographic columns；Two-dimension switching device and the second dimension sampling device are all multiple-way valves, and sample loop one is connected between two valve ports of two-dimension switching device, and sample loop two is connected between two valve ports of the second dimension sampling device.The two-dimensional liquid chromatography of the present invention is using gel chromatographic columns as two-dimensional liquid chromatography, efficiently separating for liquid chromatogram can be tieed up by means of first, pre-separation is carried out to target components or full constituent, again its peptide molecular weight situation is measured through the second dimension gel chromatography analysis, complexity of the analyzed component in gel column can be substantially reduced, so as to improve its separative efficiency and precision of analysis.
Description
Technical field
The invention belongs to Instrumental Analysis fields, and in particular to a kind of two-dimensional liquid chromatography and its use for measuring peptide molecular weight
On the way.
Background technology
Gel liquid chromatogram is widely used in the survey of albumen, the measure of peptide molecular weight or the distribution of enzymolysis product middle-molecular-weihydroxyethyl
It is fixed.However as gradually rising for sample complexity, the single column analysis of traditional one-dimensional gel liquid chromatogram because its resolution ratio is relatively low,
The phenomenon that co-elute is often presented in chromatographic peak, be overlapped to some extent with the chromatographic peak presence of other substances, can not meet sample
Analysis demand.
To improve chromatographic isolation effect, it is more that researcher reports a kind of homologous series different size gel liquid-phase chromatographic column
The method that column series connection is carried out at the same time chromatographic isolation, since this method uses the method directly connected to connect gel chromatographic columns, very greatly
Gel chromatographic columns are still limited by degree in itself, therefore its chromatographic resolution rate can only be improved to a certain extent, in addition, using not
The multicolumn series connection of same specification, there are string peak phenomenon of the different solutes in different columns, i.e., first go out in the first root chromatogram column originally
The solute at peak may due to non-sieve effect in second column after appearance, in addition, multicolumn connect when due to different chromatographic columns
Molecular weight efficiently separates that section is inconsistent, unknown solute molecule may be caused to retain, i.e., macromolecular substances may be subsequent
Be trapped in small molecule chromatographic column, develop thus a kind of novel peptide molecular weight assay method be instantly urgent need to resolve the problem of.
Two-dimensional liquid chromatography is far above traditional one-dimensional liquid phase color as a kind of emerging chromatographic separation technology, separative efficiency
Spectrum.But since the analyze speed of gel chromatography is limited by column material maximum pressure drop, it can not quickly be analyzed, and
Its resolution ratio is extremely low, therefore it is used frequently as the first dimension liquid chromatogram, and seldom as two-dimensional liquid chromatography, this
Outside, it when gel chromatography carries out peptide molecular weight measure, if assisting carrying out grain size and molecular weight determination without laser light scattering instrument, needs to create
The standard curve of molecular weight determination is built, and currently existing two-dimensional liquid chromatography scheme lacks to two-dimensional liquid chromatography sample introduction
It supports, therefore gel chromatography is seriously obstructed in the application and development of two-dimensional liquid chromatography.
Invention content
Existing resolution ratio is low during in order to which existing gel chromatography being overcome to measure peptide molecular weight, the second dimension gel chromatography can not be straight
The problem of sample creates standard curve is tapped into, primary and foremost purpose of the invention is to provide a kind of using gel chromatography as the second dimension liquid phase color
The two-dimensional liquid chromatography of spectrum.It, can be by means of the height of the first dimension liquid chromatogram using gel chromatographic columns as two-dimensional liquid chromatography
Effect separation carries out target components or full constituent pre-separation, then measures its peptide molecular weight through the second dimension gel chromatography analysis
Situation can substantially reduce complexity of the analyzed component in gel column, so as to improve its separative efficiency and precision of analysis.
Another object of the present invention is to provide purposes of the above-mentioned two-dimensional liquid chromatography in peptide molecular weight is measured, analysis
Method is using reversed-phase liquid chromatography, ion chromatography or hydrophilic Interaction Chromatography as the first dimension liquid chromatogram, using gel chromatography as the second dimension
Liquid phase chromatography analytical method, then it is aided with the sample introduction support of two-dimensional liquid chromatography for creating gel chromatography molecular weight calibration song
Line.When carrying out sample analysis, this method is pre-separated sample to reduce inflow gel using the first dimension liquid chromatogram
The sample complexity of chromatography and analysis difficulty, so as to improve the molecular weight determination accuracy of target components or full constituent.
The purpose of the present invention is achieved through the following technical solutions：
A kind of two-dimensional liquid chromatography, including first dimension liquid-phase chromatographic column, second dimension liquid-phase chromatographic column, two-dimension switching device,
Second dimension sampling device and two sample loops；
The first dimension liquid-phase chromatographic column and the second dimension liquid-phase chromatographic column is connect with two-dimension switching device；
The second dimension liquid-phase chromatographic column is gel chromatographic columns；
The two-dimension switching device and the second dimension sampling device are all multiple-way valves, and sample loop one is connected to two dimension switching dress
Between two valve ports put, sample loop two is connected between two valve ports of the second dimension sampling device；
The sample loop one is for storing the component from the first dimension liquid chromatogram, and sample loop two is used for Two-dimensional Liquid
The sample feeding of phase chromatography, the independent use of two sample loops can reduce the first dimension outflow component and two-dimensional liquid chromatography sample introduction
Cross contamination between component；
The preferred ten-way valve of the two-dimension switching device, the described second dimension sampling device are at least six-way valve；
The two-dimensional liquid chromatography further includes chromatogram pump, sampling device and detector；
When two-dimensional liquid chromatography needs loading standard product, standard items are connected by the second dimension sampling device with sample loop two
In the valve port injection sample loop two connect；Then the second dimension of rotation sampling device switches a screens, the connected relation hair of each valve port
It is raw to change；The mobile phase of two-dimensional liquid chromatography ties up sampling device, sample loop two and two-dimension switching device followed by second, most
The standard items in sample loop two are sent into the second dimension liquid-phase chromatographic column and are analyzed at last, establish standard curve afterwards after testing；It should
Link is unrelated with the first dimension liquid-phase chromatographic column；
When carrying out the peptide molecular weight detection of unknown sample, first by unknown sample through the first dimension liquid-phase chromatographic column analysis, through inspection
After survey, the target components of subdivision are flowed to through two-dimension switching device in sample loop one, which is also referred to as two dimension sampling；Then two are rotated
It ties up switching device and switches a screens, the connected relation of each valve port changes；The mobile phase of two-dimensional liquid chromatography flows successively
Through the second dimension sampling device, two-dimension switching device and sample loop one, most the target components in sample loop one are sent into the second dimension at last
It is analyzed in liquid-phase chromatographic column, measures peptide molecular weight with calculating after testing；
Described first dimension liquid-phase chromatographic column be the reversed-phase liquid chromatography column that sample analysis can be carried out under the conditions of high water phase,
Ionic liquid phase chromatographic column or chromatographic column with hydrophilic function；
The chromatogram pump is more pumps or single pump, can be high pressure, middle pressure or low pressure liquid phase pump；
The detector is UV detector, Composition distribution or laser light scattering instrument.
Above-mentioned two-dimensional liquid chromatography can be used for the measure of peptide molecular weight, and assay method includes the following steps：
(1) peptide molecular weight standard items are analyzed to obtain its corresponding retention time by the one-dimensional of the second dimension liquid-phase chromatographic column,
And graph of relation between molecular weight logarithm-retention time is made with this, show that quantization therebetween is closed by linear fit
System's (being molecular weight standards calibration curve)；
(2) sample first passes around the pre-separation of the first dimension liquid-phase chromatographic column and obtains different outflow components；
(3) outflow component (eluting peak) is cut in two-dimension switching device and is transferred to the second dimension liquid-phase chromatographic column one by one；
(4) second dimension liquid-phase chromatographic columns carry out chromatographic isolation, detection sample outflow component (outflow to being transferred into the component come
Peak) retention time, with reference to the molecular weight standards calibration curve in step (1) can obtain its correspond to molecular size range.
The present invention is had the following advantages relative to the prior art and effect：
1st, peptide molecular weight two-dimensional liquid chromatography of the invention by the first dimension liquid-phase chromatographic column under high water phase to sample into
Row efficiently separates, and greatly reduces sample complexity of the target analysis component in gel chromatography, improves its separating effect, into
And improve the accuracy of molecular weight analysis.
2nd, peptide molecular weight two-dimensional liquid chromatography of the invention realizes the direct injected of two-dimensional liquid chromatography, divides establishing
Son amount standard items calibration curve when can directly to two-dimensional liquid chromatography carry out individually sample introduction is analyzed, the analysis of standard items at this time is not
Need the sample introduction defect problem for by the first dimension liquid chromatogram, solving two-dimensional liquid chromatography.In addition, the liquid phase of two dimensions
Chromatography contains injector, and the independent operating of two dimension liquid chromatograies can be easily realized by the switching of two-dimentional switching part,
It realizes dual-use, substantially increases the utilization rate of instrument.
3rd, peptide molecular weight two-dimensional liquid chromatography of the invention can be both analyzed by heartcut to one-dimensional liquid chromatogram target
Molecular weight, molecular weight distribution, grafting situation for flowing out component etc. is analyzed, also can be by full two-dimension analysis to one-dimensional liquid phase color
Whole outflow components of spectrum carry out the analyses such as molecular weight, molecular weight distribution, branch distribution, composition distribution situation, in addition, full two dimension
Analysis collection of illustrative plates may further provide the distribution situation of peptide mixing sample on two dimensions, can be used as its separation and analytical plan
Reference.
4th, peptide molecular weight two-dimensional liquid chromatography of the invention uses gel chromatography as two-dimensional liquid chromatography, can be fine
The high water phase mobile phase of compatibility, be conducive to the on-line joining process that two-dimensional liquid chromatography is reacted with biological enzyme after column, be conducive to promote
The development with detecting is detached into online bioactive substance.
5th, in the present invention, two-dimentional switching valve used sample ring (sample loop one) and sample used in two-dimensional liquid chromatography sampling valve
Product ring (sample loop two), which is independently operated, can be reduced between the first dimension sample component and two-dimensional liquid chromatography sample introduction component
Cross contamination.
Description of the drawings
Fig. 1 is that the two-dimensional liquid chromatography of embodiment is in connection diagram when standard items load and analyze state；
Fig. 2 is the connection diagram when two-dimensional liquid chromatography of embodiment is in two dimension sampling and analyzes state；
Wherein, a- chromatogram pumps；B- sampling devices；C- first ties up liquid-phase chromatographic column；D- two-dimension switching devices；E- chromatogram pumps；
F- sampling devices；G- sample loops two；H- second ties up liquid-phase chromatographic column；I- detectors, j- sample loops one.
Fig. 3 is the standard items calibration curve of embodiment.
Fig. 4 is the sample C18 one-dimensional elution curves of embodiment.
Fig. 5 is the comprehensive two dimensional gas chromatography figure of embodiment sample.
Fig. 6 is the sample chromatogram separation figure of comparative example.
Specific embodiment
With reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Embodiment
A kind of two-dimensional liquid chromatography, as depicted in figs. 1 and 2, including first dimension liquid-phase chromatographic column c (4.6 × 250mm C18
Analyze chromatographic column (Dalian Yi Lite SinoChrom ODS-AP)), second dimension liquid-phase chromatographic column h (7.8 × 300mm gel chromatographies
Analytical column (Tosoh G2000SWXL)), two-dimension switching device d (electronic ten-way valve (DIONEX PD105-702)), second tie up into
Sampling device f (six-way valve) and two sample loops g and j；
The first dimension liquid-phase chromatographic column c and the second dimension liquid-phase chromatographic column h is connect with two-dimension switching device d；Sample loop
One j is connected between the valve port 4 and 7 of two-dimension switching device d, and two g of sample loop is connected to the valve port 1 and 4 of the second dimension sampling device f
Between；
The two-dimensional liquid chromatography further includes chromatogram pump a and e, and (high pressure liquid chromatography pumps (DIONEX ultimate DGP
3000SD)), sampling device b (DIONEX ultimate WPS 3000) and detector i (UV detector (DIONEX
ultimate DAD 3000))；
When two-dimensional liquid chromatography needs loading standard product as shown in Figure 1, by standard items by the second dimension sampling device f with
The valve port 4 of two g connections of sample loop is injected in two g of sample loop；Then the second dimension sampling device of rotation f switches a screens, each valve
The connected relation of mouth changes；The valve port 3,4 that the mobile phase of two-dimensional liquid chromatography ties up sampling device f followed by second,
1,2, the valve port 5 and 6 of two-dimension switching device d, the standard items in most at last two g of sample loop are sent into the second dimension liquid-phase chromatographic column h
It is analyzed, establishes standard curve afterwards after testing；
As shown in Fig. 2, unknown sample first is tieed up liquid-phase chromatographic column c through first when carrying out the peptide molecular weight detection of unknown sample
Analysis, after testing after, the valve port 3 and 4 of the target components of subdivision through two-dimension switching device d is flowed into one j of sample loop, the link
Claim two dimension sampling；Then rotation two-dimension switching device d switches a screens, and the connected relation of each valve port changes；Second dimension
The mobile phase of liquid chromatogram is followed by the valve port 3 and 2 of the second dimension sampling device f, the valve port 5 of two-dimension switching device d, and 4,7,6,
Target components in most at last one j of sample loop are sent into the second dimension liquid-phase chromatographic column h and are analyzed, and measure peptide with calculating after testing
Molecular weight.
Number position of the multiple-way valve is only to show system connection relation and use, and specifically used position can supply with manufacturer
Original position difference, the name of number position and sequence can be since the arbitrary interfaces of multiple-way valve according to counterclockwise or clockwise since 1
Sequence name.
Above-mentioned two-dimensional liquid chromatography can be used for the measure of peptide molecular weight, and assay method includes the following steps：
(1) respectively using sampling device difference sample introduction bovine serum albumin (BSA), cromoci (CTC), Aprotinin
(APR)、Trp-Ser-Arg-Glu-Glu-Gln-Glu-Arg-Glu-Glu(WSREEQEREE)、Gly-Gly-Tyr-Arg
(GGYR) and Gly-Gly-Gly (GGG) and second dimension liquid-phase chromatographic column in analyzed (sampling volume is respectively 5 μ L),
Its retention time is obtained, molecular weight logarithm and retention time relation curve are made with this and makees linear fit acquisition standard items correction is bent
Line such as Fig. 3；
(2) analysis condition that Elastin peptide (30 μ L of sampling volume) is tieed up using first, first in 18 chromatographic column of the first Victoria C
Its isolated the first dimension elution curve of middle progress is as shown in Figure 4；
(3) two-dimension switching device chooses its one-dimensional outflow component (27.15-28.20min) and is transferred to the second dimension progress gel
Chromatography, transfer interval 0.35min, analysis result are as shown in Figure 5.
First dimension analysis part mobile phase is made of the acetonitrile (A) respectively containing 0.1% trifluoroacetic acid and water (B), gradient elution
Condition：0-8.00min (88%A), 8.00-20.00min (88-65%A), 20.00-24.00min (65-88%A) and
24.00-45.00min (88%A), flow velocity 0.3mL/min.
Second dimension analysis part mobile phase is made of acetonitrile (40%), water (60%), trifluoroacetic acid (0.1%), using etc.
Degree elution, flow velocity 1mL/min, Detection wavelength 214nm, frequency acquisition 10HZ.
Comparative example
Gel chromatography analysis is carried out to above-mentioned Elastin peptide sample using the one-dimensional liquid-phase chromatography method of the prior art.Point
Analyzer device：3000 high performance liquid chromatography of DIONEX ultimate (is equipped with quaternary Gradient chromatography pump, autosampler, DAD detections
Device)；Chromatographic column is 7.8 × 300mm gel chromatographies analytical column (TosohG2000SWXL)；Flowing phase composition is：Acetonitrile (40%),
Water (60%) and trifluoroacetic acid (0.1%)；Elution requirement is isocratic elution, and elution curve is as shown in Figure 6.
From the figure 3, it may be seen that molecular weight standards calibration curve is created in the second dimension gel chromatography using the second dimension sampling device
Good correlation, coefficient R are obtained under this condition2=0.959.
Fig. 4 is Elastin peptide in the one-dimensional analysis result of reversed-phase liquid chromatography, and as seen from the figure, Elastin peptide sample can
Preferable separating effect is obtained on reversed-phase liquid chromatography, can be divided into more chromatographic peak, on this basis using full two dimension point
Each peak is transferred to the analysis of the second dimension gel chromatography by analysis method one by one, full two dimension RPLC-SEC separation figure (Fig. 5) is obtained, with reference to Fig. 3
Standard curve, by can intuitively find out in figure the first dimension chromatography outflow full constituent molecular weight distribution situation.
(the figure compared with traditional one-dimensional gel liquid chromatogram in comparative example 1 is there are the analysis result at many unknown co-elute peaks
6), two-dimensional liquid chromatography of the present invention can obviously improve separating effect and precision of analysis of the solute in the second dimension gel chromatography, this
The advance and novelty of two-dimensional liquid chromatography of the present invention are absolutely proved.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of two-dimensional liquid chromatography, it is characterised in that cut including the first dimension liquid-phase chromatographic column, the second dimension liquid-phase chromatographic column, two dimension
Changing device, the second dimension sampling device and two sample loops；
The first dimension liquid-phase chromatographic column and the second dimension liquid-phase chromatographic column is connect with two-dimension switching device；
The second dimension liquid-phase chromatographic column is gel chromatographic columns；
The two-dimension switching device and the second dimension sampling device are all multiple-way valves, and sample loop one is connected to two-dimension switching device
Between two valve ports, sample loop two is connected between two valve ports of the second dimension sampling device；
When two-dimensional liquid chromatography needs loading standard product, standard items are connect by the second dimension sampling device with sample loop two
In valve port injection sample loop two；Then the second dimension of rotation sampling device switches a screens, and the connected relation of each valve port changes
Become；The mobile phase of two-dimensional liquid chromatography is followed by the second dimension sampling device, sample loop and two-dimension switching device, most sample at last
Standard items in product ring two are sent into the second dimension liquid-phase chromatographic column and are analyzed；
Carry out unknown sample peptide molecular weight detection when, first by unknown sample through first dimension liquid-phase chromatographic column analysis, after testing after,
The target components of subdivision are flowed into through two-dimension switching device in sample loop one, which is also referred to as two dimension sampling；Then rotation two dimension is cut
Changing device switches a screens, and the connected relation of each valve port changes；The mobile phase of two-dimensional liquid chromatography is followed by
Two-dimentional sampling device, two-dimension switching device and sample loop one, most the target components in sample loop one are sent into the second dimension liquid phase at last
It is analyzed in chromatographic column.
2. two-dimensional liquid chromatography according to claim 1, it is characterised in that：The two-dimension switching device is ten-way valve.
3. two-dimensional liquid chromatography according to claim 1, it is characterised in that：The second dimension sampling device is at least six
Port valve.
4. two-dimensional liquid chromatography according to claim 1, it is characterised in that：The two-dimensional liquid chromatography includes chromatography
Pump, sampling device and detector.
5. two-dimensional liquid chromatography according to claim 1, it is characterised in that：The first dimension liquid-phase chromatographic column is reverse phase
Liquid-phase chromatographic column, ionic liquid phase chromatographic column or chromatographic column with hydrophilic function.
6. two-dimensional liquid chromatography according to claim 4, it is characterised in that：The chromatogram pump is more pumps or single pump.
7. two-dimensional liquid chromatography according to claim 4, it is characterised in that：The chromatogram pump is high pressure, middle pressure or low
Press liquid phase pump.
8. two-dimensional liquid chromatography according to claim 4, it is characterised in that：The detector is UV detector, shows
Difference detector or laser light scattering instrument.
9. application of the claim 1-8 any one of them two-dimensional liquid chromatography in peptide molecular weight is measured.
10. application according to claim 9 is characterized in that its assay method includes the following steps：
(1) peptide molecular weight standard items analyze to obtain its corresponding retention time by the one-dimensional of the second dimension liquid-phase chromatographic column, and with
This makees the graph of relation between molecular weight logarithm-retention time, and quantitative relationship therebetween is obtained by linear fit；
(2) sample first passes around the pre-separation of the first dimension liquid-phase chromatographic column and obtains different outflow components；
(3) outflow component is cut in two-dimension switching device and is transferred to the second dimension liquid-phase chromatographic column one by one；
(4) second dimension liquid-phase chromatographic columns carry out chromatographic isolation to being transferred into the component come, when detection sample flows out the reservation of component
Between, show that it corresponds to molecular size range with reference to the molecular weight standards calibration curve in step (1).
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Effective date of registration: 20211117 Address after: 510075 No. n2248, floor 3, Xingguang Yingjing, No. 117, Shuiyin Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee after: GUANGZHOU ZHAOJI BIOTECHNOLOGY CO.,LTD. Address before: 510640 No. five, 381 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: SOUTH CHINA University OF TECHNOLOGY Effective date of registration: 20211117 Address after: 528400 No. 8, Perfect Road, South China Modern Chinese Medicine City, Nanlang Town, Zhongshan City, Guangdong Province Patentee after: PERFECT (GUANGDONG) COMMODITY Co.,Ltd. Address before: 510075 No. n2248, floor 3, Xingguang Yingjing, No. 117, Shuiyin Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGZHOU ZHAOJI BIOTECHNOLOGY CO.,LTD. |