CN104069840B - A kind of preparation method of reverse-phase chromatography silica filler - Google Patents

Abstract

The invention discloses a preparation method of a silica gel filler for reverse phase chromatography, which relates to a silica gel filler. Put the silicon balls in the centrifuge tube, add the glass water repellent to get the silicon ball-glass water repellant solution, seal the mouth of the centrifuge tube, oscillate for the first time, remove the upper glass water repellant after centrifugation, then add ethanol to the centrifuge tube, After the second oscillation, continue to centrifuge to remove the supernatant, and dry it to obtain the silica gel filler for reverse phase chromatography. The silica gel matrix filler is quickly modified by the glass water-repellent chemical coating method. The modified silica sphere filler has reversed-phase retention characteristics and can be used for reversed-phase high-performance liquid chromatography separation. The filler not only has good chromatographic separation performance, but also has excellent stability, and it still retains its original reversed-phase chromatographic performance under the conditions of soaking in 80% acetonitrile for 24 hours and baking at 80°C for 24 hours.

Description

A kind of preparation method of reverse phase chromatography silica gel packing

technical field

The invention relates to a silica gel filler, in particular to a preparation method of a reverse-phase chromatography silica gel filler.

Background technique

In high performance liquid chromatography (HPLC), reversed-phase chromatography packing is the most widely used reversed-phase stationary phase, and there are two main categories: bonded silica gel microspheres and organic polymer microspheres. Among them, the stationary phase based on silica gel is often used because of its high mechanical strength, good stability, easy regulation of the size and pore size of microspheres, and not being affected by various organic solvents. The chromatographic performance of the stationary phase based on silica gel is directly related to the physical and chemical properties of the silica gel surface.

Silanol groups -Si-OH are widely present on the surface of silica gel. Because of its good reactivity, it can be modified in different ways to obtain different types of chromatographic stationary phases. There are two commonly used modification methods, namely chemical bonding and polymer coating.

In the chemical bonding method, chlorosilane or alkoxysilane is usually used for silanization reaction to introduce functional groups, and then a small molecule silylating agent (such as trimethylchlorosilane or hexamethyltrisilamine) is used for end-capping , to eliminate residual silanol. In order to obtain a monolayer bonded phase, the silica gel, silylating reagent and solvent used in the reaction process must be strictly dehydrated, and the bonding reaction must be carried out at a relatively high temperature. At the same time, due to the existence of steric hindrance effect, it is impossible for silylating reagents with large molecular volume to fully react with the silanol groups on the surface of silica gel, and usually only 25% to 50% of the silanol groups undergo bonding reactions. The active part of the exposed silica gel surface will strongly adsorb analytes, especially strongly polar and alkaline substances, resulting in peak shape distortion and even irreversible adsorption. Moreover, in order to obtain a better separation effect, many separations require the use of acid, alkali, and salt buffer systems as the mobile phase. Under such elution conditions, the silylating reagent is easily lost, and the column life is also shortened.

The stationary phase prepared by modifying the surface of silica gel by polymer coating can overcome the disadvantages of the above-mentioned silica gel bonded phase. This method is mainly to wrap a thin polymer film on the silica gel matrix to effectively avoid the contact between the eluent and the analyte and the non-specific adsorption between the analyte and the silanol.

Contents of the invention

The purpose of the present invention is to overcome the problems of long reaction time, harsh conditions, cumbersome steps and difficult to guarantee reproducibility of silica gel bonded stationary phase, and provide a fast, simple and efficient preparation method of silica gel packing for reverse phase chromatography.

Concrete steps of the present invention are as follows:

Put the silicon balls in a centrifuge tube, add glass water repellant (Aquapel) to get a silicon ball-glass water repellant solution, seal the centrifuge tube mouth, shake for the first time, remove the upper layer of glass water repellent after centrifugation, and then pour it into the centrifuge tube Add ethanol, continue to centrifuge to remove the supernatant after the second oscillation, and dry to obtain the silica gel filler for reverse phase chromatography.

The particle diameter of the silicon spheres can be 100nm～30μm; the ratio of the silicon spheres, glass waterproofing agent and ethanol can be (5μg～100g):(5μL～50mL):(5μL～50mL), wherein, the silicon spheres Calculated by mass, glass waterproofing agent and ethanol are calculated by volume, all silicon balls are immersed in glass waterproofing agent, all fillers can be immersed in ethanol; the first oscillation can be placed in an ultrasonic oscillator for 5-120 minutes ; The second oscillation can be oscillated in an ultrasonic oscillator for 5-10 minutes; the drying temperature can be 40-80° C., and the drying time can be 30-180 minutes.

The present invention adopts the glass waterproofing agent (Aquapel) chemical coating method to quickly modify the silica gel matrix filler, and the modified silica ball filler has the characteristic of reverse-phase retention, and can be used for reverse-phase high-performance liquid chromatography separation.

The present invention utilizes the polymer coating method to quickly and easily modify the silica gel matrix filler, and utilizes the commercialized glass waterproofing agent (Aquapel) (main chemical composition: naphtha 90% to 100%, C10-12 alkane/naphthene) to treat the silica gel, Thereby obtaining reversed-phase high-performance liquid chromatography filler. This kind of filler not only has good chromatographic separation performance, but also has excellent stability. It still retains its original reversed-phase chromatographic performance under the conditions of soaking in 80% acetonitrile for 24 hours and baking at 80°C for 24 hours.

Description of drawings

Fig. 1 is a scanning electron microscope (SEM) characterization diagram of a small column bed coated with a glass waterproofing agent with 350nm SiO ₂ filler.

Fig. 2 is the chromatogram of the enrichment of three kinds of polypeptide mixtures in the glass waterproofing agent coated 350nm SiO ₂ filler column.

Fig. 3 is a chromatogram of the enrichment of three kinds of polypeptide mixtures after the 350nm SiO ₂ filler column coated with glass water repellent was soaked in 80% ACN for 24 hours.

Fig. 4 is a chromatogram of the enrichment of the three polypeptide mixtures after the glass waterproofing agent coated 350nm SiO ₂ filler column was baked at 80°C for 24 hours.

Fig. 5 is an energy spectrum (EDS) characterization diagram of a glass waterproofing agent coated with a 10 μm silica gel filler.

Fig. 6 is a chromatogram of benzene series separated by a chromatographic column coated with a glass water repellant and filled with 10 μm silica gel.

Fig. 7 is a chromatogram of protein mixtures separated by a chromatographic column coated with a glass waterproofing agent and a 10 μm silica gel filler.

detailed description

Example 1:

Weigh 150mg of _SiO2 nanoparticles with a particle size of 350nm and place them in a 5mL centrifuge tube, add 1mL of Aquapel, seal the centrifuge tube mouth, place in an ultrasonic oscillator and vibrate for 20min, then centrifuge, remove the supernatant, add 2mL of ethanol and sonicate for 5min, Centrifuge to remove the ethanol solution in the upper layer, place in an oven and bake at 50°C for 3 hours, then seal and store.

Chromatographic retention performance evaluation of packing materials:

Take an appropriate amount of the above-mentioned Aquapel-treated _SiO2 nanoparticles and disperse them in ethanol at a concentration of 10 mg/mL. After oscillating in an ultrasonic oscillator for 10 min, they are centrifuged and filled to prepare two solid-phase extraction columns with a length of 5 mm, and demonstrate their effect on Solid-phase extraction and enrichment effects of three peptide mixtures of somatostatin acetate, angiotensin acetate and oxytocin at a concentration of 10 μg/mL. Figure 1 is a column bed scanning electron microscope (SEM) image of Aquapel coated with _350nmSiO2 nanometer filler column, and Figure ₂ is a chromatogram of the enrichment of three polypeptide mixtures by Aquapel coated with 350nmSiO2 nanometer filler column. As can be seen from Figure ₁ , Aquapel coated with 350nm SiO ₂ nanoparticles did not change its original particle size and uniformity. Excellent enrichment effect.

Chromatographic retention stability evaluation of packing materials:

1) Inject 80% acetonitrile (ACN) into the 2 SPE small columns prepared above, store them sealed at room temperature for 24 hours, and then test their effects on three kinds of somatostatin acetate, angiotensin acetate and oxytocin with a concentration of 10 μg/mL. The solid-phase extraction and enrichment effect of the polypeptide mixture, the chromatographic separation spectrum after enrichment is shown in Figure 3.

2) Place the two SPE cartridges prepared above in an oven at 80°C for 24 hours, and then test their solid-phase effects on the three polypeptide mixtures of somatostatin acetate, angiotensin acetate and oxytocin at 10 μg/mL. The extraction and enrichment effect, the chromatographic separation spectrum after enrichment is shown in Figure 4.

Comparing Figures 2 to 4, it can be proved that the Aquapel coated 350nm SiO ₂ nano-filler column still retains its reversed-phase chromatographic performance well even after being soaked in 80% ACN for 24 hours and baked at 80°C for 24 hours. It proves that Aquapel coated with 350nm SiO ₂ nano filler has excellent stability and can effectively avoid the problem of stationary phase loss.

Example 2:

Weigh 300mg particle size 10μm pore size Put the silicon ball filler in a 5mL centrifuge tube, add 1mL Aquapel, seal the centrifuge tube mouth, place it in an ultrasonic oscillator for 1h and centrifuge, remove the supernatant, add 2mL ethanol and ultrasonicate for 5min, centrifuge, remove the upper ethanol solution, place Bake in an oven at 60°C for 3 hours and then seal and store.

Take an appropriate amount of the above-mentioned Aquapel-treated silica sphere filler and disperse it in ethanol, the concentration is controlled at 50 mg/mL, shake it in an ultrasonic oscillator for 10 minutes, and then fill it with high-pressure homogenate to prepare a capillary short column with a length of 9 cm and an inner diameter of 100 μm, and verify its Separation effect on benzene series (thiourea, toluene, mixture of ethylbenzene and propylbenzene) and protein mixture (BSA, cytochrome C, transferrin and ovalbumin). Figure 5 is the energy spectrum (EDS) characterization diagram of Aquapel coated with 10 μm silica gel filler, Figure 6 is the chromatogram of benzene series separated by Aquapel coated with 10 μm silica gel filler chromatography column, Figure 7 is the separation of protein by Aquapel coated 10 μm silica gel filler chromatography column Chromatogram of the mixture. It can be seen from Figure 6 and Figure 7 that the 10 μm silica gel-filled chromatographic column coated with Aquapel can well separate the highly hydrophobic benzene series and protein mixture. Combined with the energy spectrum characterization in Figure 5, it can be seen that the 10 μm silica gel filler coated with Aquapel has a relatively high carbon content. Reverse-phase chromatographic separation is possible.

Claims (6)

1. a preparation method for reverse-phase chromatography silica filler, is characterized in that concrete steps are as follows:

Silicon ball is placed in centrifuge tube, silicon ball-Aquapel glass water repellent solution is obtained after adding Aquapel glass waterproofing agent, the sealing centrifuge tube mouth of pipe, 1st vibration, centrifugal rear removing upper strata Aquapel glass waterproofing agent, then in centrifuge tube, add ethanol, continue centrifugal removing supernatant after the 2nd vibration, after oven dry, namely obtain reverse-phase chromatography silica filler.

2. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that the particle diameter of described silicon ball is 100nm ~ 30 μm.

3. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, it is characterized in that the proportioning of described silicon ball, Aquapel glass waterproofing agent, ethanol is for (5 μ g ~ 100g): (5 μ L ~ 50mL): (5 μ L ~ 50mL), wherein, silicon ball is calculated in mass, Aquapel glass waterproofing agent, ethanol are calculated by volume, all silicon balls are all immersed in Aquapel glass waterproofing agent, and the submergence of all fillers equal energy in ethanol.

4. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described 1st vibration is for being placed in supersonic oscillations instrument vibration 5 ~ 120min.

5. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described 2nd vibration is for the 5 ~ 10min that vibrates in supersonic oscillations instrument.

6. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, it is characterized in that the temperature of described oven dry is 40 ~ 80 DEG C, the time of oven dry is 30 ~ 180min.