CN104034833B - A kind of Nano filling capillary chromatography microtrabeculae preparation facilities and preparation method thereof - Google Patents
A kind of Nano filling capillary chromatography microtrabeculae preparation facilities and preparation method thereof Download PDFInfo
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Abstract
A kind of Nano filling capillary chromatography microtrabeculae preparation facilities and preparation method thereof, relates to Nano filling capillary chromatography microtrabeculae and prepares.Described Nano filling capillary chromatography microtrabeculae preparation facilities is provided with centrifuge and centrifugal filling device, and centrifugal filling device is provided with detachable PEEK joint, PEEK interface, PTFE sleeve pipe, capillary tube and centrifuge tube.Taking nanoparticle and be scattered in acetone, it is 1～50 μ g/ μ L that concentration controls, vibration after homogenate solution mixing, obtains homogenate；Intercept one section of elastic quartz capillary tube, grain size is pressed into capillary tube one end as exporting plunger slightly larger than the single through hole silicon ball of capillary inner diameter；Capillary tube with outlet plunger is fixed in centrifugal filling device, reservoir injects homogenate, weighs centrifugal, be fixed and fill mutually；After being centrifuged, take off populated capillary tube, insert a through hole silica gel particle as entrance plunger at capillary inlet end, obtain Nano filling capillary chromatography microtrabeculae.
Description
Technical field
The present invention relates to Nano filling capillary chromatography microtrabeculae to prepare, particularly relate to simple, quick, batch a kind of Nano filling capillary chromatography microtrabeculae preparation facilities preparing Nano filling capillary chromatography microtrabeculae and preparation method thereof.
Background technology
Flourish along with proteomics and metabolism group research, current chromatographic separation technology is difficult to the requirement meeting high-resolution, high selectivity and quickly analyzing, hence it is imperative that narrow spectrum chromatographic stationary phases new and effective, high.Nano material causes people's attention to its chromatographic applications gradually because of its homogeneous particle size distribution, high-specific surface area, abundant surface-modification chemistries and excellent stability.Research shows, no matter is as chromatograph packing material or enrichment material, and nano material has that separation efficiency height, bioaccumulation efficiency be high really, good stability and the advantage such as selectivity is single-minded.Therefore, how to effectively utilize Nano filling and become the important topic of chromatogram research.
It is known that chromatographic column is one of core component of high performance liquid chromatograph, it it is the core of chromatographic isolation.Capillary column is the one in liquid-phase chromatographic column, prepared by the elastic quartz pipes adopting internal diameter 50～300 μm more.Its less column volume can significantly reduce the consumption of column packing and mobile phase, and what be especially suitable for trace and trace samplings separates analysis.At present, the filler of capillary column is mainly micron particles, and the application of nano-scale particle capillary column is very few, and main cause is that Nano filling capillary column is prepared extremely difficult, greatly limit its application.Therefore, how to prepare the capillary column of nano-sized filler and become one of the key issue that can promote nanometer chromatographic material.Through investigation, the method preparing Nano filling capillary chromatographic column is mainly the filling of high-pressure homogenization wet method.This method needs the high-pressure pump equipment of at least 600bar, single is filled and be can only obtain a capillary post, fill difficulty length big, consuming time, filling speed heterogeneity, and owing to operating difficulties and the factor such as loaded down with trivial details cannot ensure the repeatability between post and post, these all greatly limit the preparation efficiency of Nano filling capillary column.
The applicant is the preparation method of a kind of capillary chromatographic column disclosed in Chinese patent CN102590402A, and the granular filler that will fill adds in acetone, prepares into homogenate；Grain size is pressed into capillary tube one end more than the single porous silicon ball of capillary inner diameter, then individual particle silicon ball is pushed in capillary tube as outlet plunger；By capillary inlet end and reservoir UNICOM, in reservoir, add homogenate be placed on centrifuge and be centrifuged, fix and order about lower entrance capillary column at gravity and centrifugal force；Another using the arrival end of capillary column populated for individual particle silicon ball press-in as entrance plunger；The capillary column prepared is preserved stand-by.
Chinese patent CN101923079A discloses a kind of mixed capillary chromatographic column with shower nozzle and preparation method thereof, by the capillary tube of different inner diameters and length by after pretreatment, the polymer integral material of different length is synthesized as needed in nozzle end, at capillary tube remainder after synthesis, homogenate dress post method is adopted to fill the high-efficient granule filler prepared, and change the kind filling filler according to demand, the size of granule, fill length and select to synthesize same material at tail end, and with the compacting in high pressure constant flow pump system of the reagent such as methanol after preparation completes, aging.Prepare chromatographic column after carrying out evaluation of chromatogram pillar, it is applied in liquid-mass chromatography system, can effectively solve traditional band shower nozzle packed column owing to post shower nozzle is along with causing because pressure is excessive in capillary chromatographic column use procedure that being partially filled with granular filler crushes, the problem in use deviate from or block capillary chromatographic column shower nozzle.
Summary of the invention
It is contemplated that overcome Nano filling capillary chromatography microtrabeculae preparation process complexity, manufacturing cycle length, prepare the problems such as equipment needed thereby costliness, the centrifugal force utilizing centrifuge is provided to replace the high hydraulic pressure of mechanical pump to carry out the Dispensable continuous filling of Nano filling, simple to operate, consuming time short, and support a kind of Nano filling capillary chromatography microtrabeculae preparation facilities of batch preparation and preparation method thereof.
Described Nano filling capillary chromatography microtrabeculae preparation facilities is provided with centrifuge and centrifugal filling device, and described centrifugal filling device is provided with detachable PEEK joint, PEEK interface, PTFE sleeve pipe, capillary tube and centrifuge tube；Described capillary tube is fixed on PEEK interface after inserting PTFE sleeve pipe；PEEK joint and PEEK interface are tightened with fixed capillary and are fixed on centrifuge tube；Centrifuge tube inserts in the rotor of centrifuge.
Described centrifuge tube can be provided with centrifuge tube lid.
The Nano microsphere that described centrifugal filling device can adopt particle diameter to be 150～950nm is as chromatograph packing material, and centrifugal filling device is closely coupled to fixed capillary microtrabeculae, thus efficiently quickly preparing Nano filling capillary chromatography microtrabeculae.
The preparation method of described Nano filling capillary chromatography microtrabeculae, comprises the following steps:
1) homogenate is prepared
Taking nanoparticle and be scattered in acetone, it is 1～50 μ g/ μ L that concentration controls, vibration after homogenate solution mixing, obtains homogenate；
2) oral pillar plug is prepared
Intercept one section of elastic quartz capillary tube, grain size is pressed into capillary tube one end as exporting plunger slightly larger than the single through hole silicon ball of capillary inner diameter；
3) fixing fill mutually
Capillary tube with outlet plunger is fixed in centrifugal filling device, reservoir injects homogenate, weighs centrifugal, be fixed and fill mutually；
4) entrance plunger is prepared
After being centrifuged, take off populated capillary tube, insert a through hole silica gel particle as entrance plunger at capillary inlet end, obtain Nano filling capillary chromatography microtrabeculae.
In step 1) in, described vibration can be swung and make homogenate solution tentatively mix by shake on the offensive, and then vibrate 5～30min in supersonic oscillations instrument, makes uniform filling be distributed in liquid.
The inventive method simple and effective, the Nano filling used is fixing mutually not by the restriction of the character such as material, size, structure, surface chemistry, prepared by the batch of the capillary chromatographic column being suitable for different inner diameters size.
Accompanying drawing explanation
Fig. 1 is the centrifugal filling device structural representation of Nano filling capillary chromatography microtrabeculae preparation facilities embodiment of the present invention.
Fig. 2 is Fig. 1 decomposing schematic representation.
Fig. 3 is SiO2Nano microsphere SEM phenogram.
Fig. 4 is SiO2Nano microsphere TEM phenogram.
Fig. 5 is alkyl modified SiO2Nano filling FTIR phenogram.In Figure 5, curve a is SiO2, curve b is SiO2-C8。
Fig. 6 is alkyl modified SiO2Nano filling capillary chromatography microtrabeculae post bed SEM phenogram.
Fig. 7 is SiO2The chromatogram of Au-S-C18 Nano filling capillary chromatography microtrabeculae enrichment SRIF-A.In the figure 7, curve a is 5 μ g/mL SRIF-A former states, and curve b is 5 μ g/mL SRIF-A pregnant solutions.
Fig. 8 is SiO2The chromatogram of Au-S-C18 Nano filling capillary chromatography microtrabeculae enrichment ovalbumin.In fig. 8, curve a is 10 μ g/mL ovalbumin former states, and curve b is 80 μ L10 μ g/mL ovalbumin short column 1 pregnant solutions, and curve c is 80 μ L10 μ g/mL ovalbumin short column 1 pregnant solutions, and curve d is 80 μ L10 μ g/mL ovalbumin short column 1 pregnant solutions.
Detailed description of the invention
Following example will the present invention is further illustrated in conjunction with accompanying drawing.
Referring to Fig. 1 and 2, described Nano filling capillary chromatography microtrabeculae preparation facilities embodiment is provided with centrifuge and centrifugal filling device, and described centrifugal filling device is provided with detachable PEEK joint 1, PEEK interface 2, PTFE sleeve pipe 3, capillary tube 4 and centrifuge tube 5；Described capillary tube 4 is fixed on PEEK interface 2 after inserting PTFE sleeve pipe 3；PEEK joint 1 and PEEK interface 2 are tightened with fixed capillary and are fixed on centrifuge tube 5；Centrifuge tube 5 inserts in the rotor of centrifuge (being not drawn in fig 1 and 2).
Described centrifuge tube 5 can be provided with centrifuge tube lid 51.
The Nano microsphere that described centrifugal filling device can adopt particle diameter to be 150～950nm is as chromatograph packing material, and centrifugal filling device is closely coupled to fixed capillary microtrabeculae, thus efficiently quickly preparing Nano filling capillary chromatography microtrabeculae.
The preparation method embodiment of Nano filling capillary chromatography microtrabeculae of the present invention given below.
Embodiment 1: alkyl modified SiO2The preparation of Nano filling capillary chromatography microtrabeculae
A alkyl modified SiO2The synthesis of Nano filling
Dehydrated alcohol, water are mixed in addition round-bottomed flask with ammonia with the volume ratio of 47: 9: 14, magnetic agitation mix homogeneously, add the tetraethyl orthosilicate of above-mentioned 1/14 volume under stirring.Airtight stirring reaction 6～10h at a certain temperature；Above-mentioned half the amount tetraethyl orthosilicate of volume, dehydrated alcohol, water etc. are added afterwards under stirring, continue reaction 6～10h, ageing 1～2h, centrifugal water is washed till few 3 times, ethanol is washed till few 3 times, dry 4～6h for 110 DEG C, by room temperature temperature programming to 600～650 DEG C in the Muffle furnace of 16h inherence after cooling.Fig. 3 and 4 are the SiO of synthesis2Nano filling scanning electron microscope (SEM) and transmission electron microscope (TEM) figure, it can be seen that synthesized Nano filling size uniformity.Take the SiO of above-mentioned synthesis2Microsphere is scattered in toluene (mass ratio is 1: 449), disposable addition is dissolved with the n-octytriethoxysilane (mass ratio is 4.8: 1.7) of triethylamine, 110～120 DEG C of condition magnetics are stirred at reflux the SiO that can obtain alkyl modified after 15～17h, water and ethanol are washed till few 3 times respectively2Nano filling.Fig. 5 is FTIR phenogram, 2850cm-1And 2920cm-1There is CH at place2Absworption peak, illustrate that C8 is successfully bonded in SiO2Surface.
The preparation of b Nano filling capillary chromatography microtrabeculae
(1) homogenate is prepared
Take the SiO that 14mg n-octytriethoxysilane is modified2Nano filling is scattered in 1mL acetone, and hand vibration makes homogenate solution tentatively mix, and then vibrate 5min in supersonic oscillations instrument, makes uniform filling be distributed in acetone.
(2) oral pillar plug is prepared
Intercept internal diameter 100 μm, the elastic quartz capillary tube that external diameter is 365 μm 4, each root length degree is 5cm, is placed in the centrifuge tube filling 110 μm of through hole silica gel particles of diameter capillary tube one end, raps and makes single through hole silica gel particle press-in capillary tube one end as outlet plunger bottom centrifuge tube.
(3) fixing fill mutually
Being separately fixed in centrifugal filling device by above-mentioned 4 capillaries (centrifuge tube used herein is 15mL centrifuge tube), inject 120 μ L homogenates, weigh centrifugal in reservoir, arranging rotating speed is 12000r/min, and centrifugation time is that 15min is filled with.
(4) entrance plunger is prepared
After being centrifuged, take off populated capillary tube, respectively insert a through hole silica gel particle as entrance plunger at its arrival end.
(5) scanning electron microscope (SEM) characterizes post bed structure
On 4 Nano filling capillary chromatography microtrabeculaes of above-mentioned preparation, one section of 5mm post bed of each intercepting is fixed on the sign carrying out surface sweeping Electronic Speculum on L-type metal platform, post bed pattern such as Fig. 6.It can be seen that Nano filling capillary chromatography microtrabeculae prepared by centrifugal fill method has excellent post bed homogeneity.
Embodiment 2:SiO2The preparation of Au-S-C18 Nano filling capillary chromatography enrichment pillar
aSiO2The preparation of Au-S-C18 nuclear shell structure nano filler
First, by a certain amount of SiO prepared according to embodiment 12Nano microsphere is scattered in 10mL1: 1 methanol and in the solution of water, prepares the SnCl of 90mL0.03M2Solution mixes with above-mentioned nanoparticle dispersion liquid, reacts 45min under magnetic stirring, is centrifuged after washing 8 times and is scattered in 4mL water；Secondly, 1.5mL1g/100mL gold chloride is joined dissolved with 25mgK2CO3100mL water in, at least dark reaction 24h, formed Ageing solution.50mL Ageing solution adds 1mLSiO2Sn2+Dispersion liquid, hydroxylamine hydrochloride solution to the reactant liquor being added dropwise over suitable concn under magnetic stirring is sapphirine, ageing 1h again after reaction 8～10h, overnight dries for 50～70 DEG C after washing at least 6 times.Finally, appropriate SiO is taken2Au Nano microsphere is dissolved in 5ml dehydrated alcohol, prepares a certain amount of 20mM stearyl mercaptan solutions, mixes by a certain percentage, reacts 24h and can obtain SiO under magnetic stirring2Au-S-C18 Nano filling.
bSiO2The preparation of Au-S-C18 Nano filling capillary chromatography microtrabeculae and evaluation
SiO2The fill method of Au-S-C18 Nano filling capillary tube enrichment pillar is similar to embodiment 1, and taken capillary pipe length is 1cm, and in fixing phase filling process, capillary tube is fixed on 1.5mL centrifuge tube, and the little column length of enrichment prepared is 4mm.Pillar adopts Liquid Chromatography mode to carry out the evaluation of concentration effect after preparing.
SRIF-A (buying spontaneous work biological engineering (Shanghai) limited company) and ovalbumin (buying from Sigma) are configured to respectively solution that concentration is 5 μ g/mL and 10 μ g/mL as sample, collect pregnant solution after blowing the steps such as concentration under 1mL/min, 40min5%～80%ACN gradient condition, carry out the separation of high performance liquid chromatography through the centrifugal loading of 12000r/min, the centrifugal eluting of 12000r/min, nitrogen, carrying out ultraviolet detection under 214nm wavelength, chromatographic isolation is as shown in FIG. 7 and 8.It can be seen that through SiO2After the concentration of Au-S-C18 Nano filling capillary tube enrichment pillar, the chromatograph response value of SRIF-A and ovalbumin is all obviously enhanced.
Claims (4)
1. the preparation method of a Nano filling capillary chromatography microtrabeculae, it is characterized in that adopting a kind of Nano filling capillary chromatography microtrabeculae preparation facilities, described a kind of Nano filling capillary chromatography microtrabeculae preparation facilities is provided with centrifuge and centrifugal filling device, and described centrifugal filling device is provided with detachable PEEK joint, PEEK interface, PTFE sleeve pipe, capillary tube and centrifuge tube；Described capillary tube is fixed on PEEK interface after inserting PTFE sleeve pipe；PEEK joint and PEEK interface are tightened with fixed capillary and are fixed on centrifuge tube；Centrifuge tube inserts in the rotor of centrifuge；
Said method comprising the steps of:
1) homogenate is prepared
Taking nanoparticle and be scattered in acetone, it is 1～50 μ g/ μ L that concentration controls, vibration after homogenate solution mixing, obtains homogenate；
2) oral pillar plug is prepared
Intercept one section of elastic quartz capillary tube, grain size is pressed into capillary tube one end as exporting plunger slightly larger than the single through hole silicon ball of capillary inner diameter；
3) fixing fill mutually
Capillary tube with outlet plunger is fixed in centrifugal filling device, the detachable PEEK joint of centrifugal filling device injects homogenate, weighs centrifugal, be fixed and fill mutually；
4) entrance plunger is prepared
After being centrifuged, take off populated capillary tube, insert a through hole silica gel particle as entrance plunger at capillary inlet end, obtain Nano filling capillary chromatography microtrabeculae.
2. the preparation method of a kind of Nano filling capillary chromatography microtrabeculae as claimed in claim 1, it is characterised in that described centrifuge tube is provided with centrifuge tube lid.
3. the preparation method of a kind of Nano filling capillary chromatography microtrabeculae as claimed in claim 1, it is characterised in that described centrifugal filling device adopt particle diameter be the Nano microsphere of 150～950nm as chromatograph packing material, centrifugal filling device is closely coupled to fixed capillary microtrabeculae.
4. the preparation method of a kind of Nano filling capillary chromatography microtrabeculae as claimed in claim 1, it is characterized in that in step 1) in, described vibration is that shake on the offensive swings and makes homogenate solution tentatively mix, and then vibrate 5～30min in supersonic oscillations instrument, makes uniform filling be distributed in liquid.
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| CN107228911A (en) * | 2017-05-08 | 2017-10-03 | 瑞谱信（厦门）科技有限公司 | Ultrashort chromatogram microtrabeculae for biological sample quick separating and preparation method thereof |
| CN110646553B (en) * | 2019-11-05 | 2024-04-26 | 兰州东立龙信息技术有限公司 | Centrifugal filling column loading machine |
| CN112823840A (en) * | 2019-11-21 | 2021-05-21 | 中国科学院大连化学物理研究所 | Method for manufacturing capillary packed column plunger by photonic crystal fiber and application |
| CN112146956B (en) * | 2020-09-24 | 2023-06-20 | 中国科学院重庆绿色智能技术研究院 | Glass needle tip nano-hole filling device based on vacuum method and using method thereof |
| CN113237969A (en) * | 2021-04-21 | 2021-08-10 | 南京大学 | Preparation of monodisperse mesoporous silicon nano chromatographic packing with center-through and radial aperture and application of packing in chromatographic separation |
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| US6095202A (en) * | 1998-04-15 | 2000-08-01 | Research Foundation State University Of New York | Method and device for packing capillary columns |
| CN102590402A (en) * | 2012-03-28 | 2012-07-18 | 厦门大学 | Preparing method of capillary tube chromatographic columns |
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