JP2008232802A - Manufacturing method of ods silica gel filler - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out a manufacturing method of an ODS silica gel filler more deactivated than the conventional types as a manufacturing method of an ODS silica gel filler. <P>SOLUTION: The manufacturing method of the ODS silica gel filler includes a primary end capping process using an amine compound, a secondary end capping process for using an amine compound different from the amine compound using in the primary end capping process and a process for performing additional end capping, using a tertiary amine in the ODS silica gel filler subjected to secondary end capping. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel process for producing ODS silica gel filler.

  Silica gel-based reverse phase chromatography (RPLC) is most commonly used with octadecylsilyl (ODS) silica gel packing, but irreversible secondary between elution and basic compounds in elution. Interaction has been a challenge since its development.

  Currently, it is well known that the irreversible secondary interaction between ODS silica gel filler and solute is caused by residual silanol after chemical modification. An example of an irreversible secondary interaction with the above has also been reported (Non-Patent Documents 1 to 3). Therefore, it is common to use an ODS silica gel filler using high purity silica gel with a low metal impurity content as a raw material for HPLC analysis of particularly highly coordinated compounds such as oxine copper and hinokitiol. As a result, the performance of the ODS silica gel filler has been dramatically improved, and it seems that there is no problem in the elution behavior of most coordination compounds.

  However, when performing highly sensitive analysis (microanalysis), irreversible secondary interactions may still occur in coordination compounds such as oxine copper and hinokitiol. Specific examples of such secondary interactions include coordinating compounds in the acidic region, especially irreversible secondary interactions of compounds represented by oxine copper and hinokitiol, and strongly basic compounds in the vicinity of neutrality. This is an irreversible secondary interaction.

As a countermeasure against such an irreversible secondary interaction, there is secondary silylation, that is, end capping, on the ODS silica gel filler. As a method of end capping, a method of silylation reaction with trimethylchlorosilane (TMCS) in the presence of an organic base such as pyridine at a toluene reflux temperature is carried out as a general method (Non-Patent Documents 4 to 6). Reaction (200 ° C. or higher) at a high reaction temperature (Non-Patent Document 7), gas phase reaction (SiC) in which a silylation reaction is performed in a sealed inert gas (Patent Document 1), reaction in an autoclave (Non-Patent Document 8) Patent Documents 2 to 3), reactions in supercritical fluids (Non-Patent Document 9, Patent Document 4), and other methods that require various reaction conditions have also been reported.
There is also a method including performing end capping using two or more different amine compounds as a reaction accelerator (Patent Document 5). In this method, it is said that end capping may be performed twice or more.

  The examination on the reaction temperature of the end capping, the reaction atmosphere, etc. has been carried out as described above, and some investigations have been carried out on the reaction reagent, for example, the silylating agent (Non-patent Document 10). Currently, almost no investigation has been conducted on these reaction reagents. In addition, the ODS silica gel filler produced by the currently reported end-capping method cannot be said to have been sufficiently deactivated despite the use of high-purity silica gel. There is also a situation. In addition, there is a problem that the operation is complicated, for example, a cleaning operation after end capping is required (Patent Document 5).

M. Verzele and C. Dewaele, Chromatographia, 18 (1984) 314. Y. Ohtsu, Y. Shiojima, J. Koyama, K. Nakamura, K. Kimata and N. Tanaka, J. Chromatogr., 481 (1989) 147 K. Kimata, N. Tanaka, and T. Araki, J. Chromatogr., 594 (1992) 87 C.H.Lochmueller, and D.B.Marshall, Analitica Chimica Acta, 142 (1982) 63 K. Jinno, S. Shimura, N. Tanaka, K. Kimata, J. C. Fetzer and W. R. Biggs, Chromatographia, 27 (1989) 285 N. Nagae, Y. Hatano and D. Ishii, Chromatography, 14 (1993) 45R Yoshihiro Sakai, Daigo Hamakawa, Suzue Ota, Abstracts of the 2nd LC Techno Plaza Lecture, BP2 (1997) 51 Y. Sakai and M. Hosoda, Chromatography, 17 (1996) 39 Takashi Hamada, Toshihide Ihara, Noriyuki Horimoto, Akira Nomura, 4th LC Techno Plaza Lecture Collection, BP2 (1999) 36 Keiji Tsuji, Separation of Separation Sciences '94, 21P (1994) D.W.Sindorf and G.E.Maciel, J. Am. Chem. Soc., 103 (1981) 4263 K.L.Miller and R.W.Linton, J. Chromatogr., 319 (1985) 9 N. Nagae, Chromatograhy, 15 (1994) 175 Supervised by Hiroshi Nakamura, “Liquid Black Dragon”, p. 32, Tsukuba Publishing Japan Society for Analytical Chemistry, Kanto Chapter, “High Performance Liquid Chromatography Handbook” Rev. 2 Japanese Patent No. 2611545 JP-A-6-174708 Japanese Patent Laid-Open No. 10-73579 Japanese Patent No. 2818857 JP 2003-172733 A

  Accordingly, an object of the present invention is to solve the above-mentioned problems and to find a simpler method for producing an ODS silica gel filler that has been inactivated as compared with conventional methods, as a method for producing an ODS silica gel filler. To do.

In view of the above problems, the present inventors have surprisingly found that the ODS silica gel filler is further inactivated by changing the number of end capping and the reaction reagent from the conventional ones. As a result of research, the present invention has been completed.
That is, the present invention relates to the following inventions:
(1) Manufacturing method of ODS silica gel filler including:
-Primary endcapping with amine compounds,
-Secondary end capping using an amine compound different from the amine compound used for the primary end capping,
-Additional end-capping with tertiary amine to ODS silica gel filler with secondary end-capping.
(2) The production method, wherein the additional end capping is only tertiary end capping, and the amine compound used for the tertiary end capping is only a tertiary amine.
(3) The said manufacturing method with which tertiary end capping is performed at the temperature of 90 to 98.3 degreeC.
(4) The said manufacturing method whose amine compound used for secondary end capping is any one of a secondary amine and a tertiary amine.
(5) The said manufacturing method that the amine compound used for secondary end capping is only a secondary amine, and secondary end capping is performed at the temperature of 200 to 300 degreeC.
(6) The said manufacturing method whose amine compound used for secondary end capping is a secondary amine and a tertiary amine.
(7) The said manufacturing method whose amine compound used for primary end capping is only a secondary amine or a tertiary amine.
(8) The said manufacturing method whose ratio of the addition amount of the silylating agent used for secondary end capping and the addition amount of an amine compound is 1: 1-1: 10.
(9) The ratio of the peak height of the ODS silica gel filler before the additional end capping and the ODS silica gel filler after the additional end capping to the compound having basicity and coordination properties is 1: Said manufacturing method which is 2-1: 20.
(10) The ratio of the peak height to the basic compound of the ODS silica gel filler before secondary end capping and the ODS silica gel filler after secondary end capping is from 1: 2 to 1: 20. The manufacturing method as described above.
(11) An ODS silica gel filler produced by any one of the production methods described above.

The present invention has been made as a result of examining the reason why it is difficult to completely inactivate the ODS silica gel filler in terms of both the properties of silanol on the silica gel surface and the reactivity of the silylating agent as a reaction reagent.
Silanols currently present on the surface of silica gel are classified into 1) isolated silanol, 2) associated silanol, and 3) diol type silanol, based on 29Si CP / MAS NMR measurement results. However, among these silanols, associative silanols are said to be most reactive. (Non-patent document 12)

The reactivity of the silylating agent is as follows: “A representative of chlorosilanes is trimethylchlorosilane (TMCS), but in the case of silylation, a slight excess of tertiary amine (triethylamine, pyridine, trimethylamine) is used together. “The reaction proceeds well,” and “secondary silylamine, diethyltrimethylsilylamine (NEt ₂ -TMS), reacts quantitatively with equatorial OH groups at room temperature but does not react with axial OH groups”. Yes.

The present inventors examined the end-capping conditions in consideration of the properties of silanol on the silica gel surface and the reactivity of the silylating agent as a reaction reagent. As a result, the degree of inactivation of ODS silica gel filler can be reduced by modifying the type of reaction accelerator used for end capping, or by weighting end capping using a specific reaction accelerator. It has been found that it can be increased more remarkably.
For example, as will be described later, procainamide is obtained by reacting a secondary silylamine and TMCS / tertiary amine mixed solution of secondary silylamine and TMCS / tertiary amine with a secondary silylamine ODS silica gel filler. The elution behavior of solutes such as can be improved. In addition, by performing a three-step reaction using TMCS / tertiary amine as an end-capping agent for tertiary end-capping (final), even at a reaction temperature under the toluene reflux temperature, which has been difficult in the past, the reaction at each step Complete end capping is possible with a reaction of 3-5 hours).
In addition, for ODS silica gel fillers that have undergone primary end capping with TMCS / tertiary amine, Mightysil RP-18GP manufactured using this primary end capping product (secondary end capping at the secondary end capping stage) When high-temperature end-capping method using silylamine is used, end-capping with TMCS / tertiary amine gives an especially good quality ODS silica gel filler.

  That is, according to the present invention, additional end capping is performed on the ODS silica gel filler subjected to primary end capping and secondary end capping using a specific reaction accelerator, for example, final end capping is performed using TMCS. / By using tertiary amine, silanol can be eliminated to the limit. In other words, by applying the present invention, it is possible to produce a high-quality ODS silica gel filler.

(1) According to the method for producing an ODS silica gel filler of the present invention, an ODS silica gel filler having a high degree of inactivation of silica gel can be easily obtained.
(2) If the additional end capping is only tertiary end capping, and the amine compound used for the tertiary end capping is only a tertiary amine, an ODS silica gel filler having a high degree of inactivation of silica gel can be obtained. Obtained efficiently.
(3) Among the methods for producing the ODS silica gel filler of the present invention, the tertiary end capping is performed at a temperature of 90 ° C. to 98.3 ° C., so that the ODS silica gel can be safely filled without using harmful toluene. An agent is obtained.
(4) Among the methods for producing the ODS silica gel filler of the present invention, the degree of inactivation of silica gel, in which the amine compound used for secondary end capping is either a secondary amine or a tertiary amine. Higher ODS silica gel filler is more easily obtained.
(5) Among the methods for producing the ODS silica gel filler of the present invention, the amine compound used for secondary end capping is only a secondary amine, and the secondary end capping is performed at a temperature of 200 ° C. to 300 ° C. According to this, an ODS silica gel filler having a higher degree of inactivation of silica gel can be obtained.
(6) According to the method for producing the ODS silica gel filler of the present invention, the amine compound used for the secondary end capping is a secondary amine and a tertiary amine. A much higher ODS silica gel filler is obtained.
(7) Among the methods for producing the ODS silica gel filler of the present invention, when the amine compound used for primary end capping is only a secondary amine or only a tertiary amine, the degree of inactivation of the silica gel ODS silica gel filler with a higher C can be obtained more easily.
(8) Among the methods for producing the ODS silica gel filler of the present invention, the ratio of the addition amount of the silylating agent used for secondary end capping and the addition amount of the amine compound is 1: 1 to 1:10 According to this, an ODS silica gel filler having a higher degree of inactivation of silica gel can be obtained more easily.
(9) The ratio of the peak height of the ODS silica gel filler before the additional end capping and the ODS silica gel filler after the additional end capping to the compound having basicity and coordination properties is 1: Those having a ratio of 2 to 1:20 provide an ODS silica gel filler with an even higher degree of silica gel inactivation.
(10) Among the methods for producing the ODS silica gel filler of the present invention, the peak for the ODS silica gel filler before secondary end capping and the basic compound of the ODS silica gel filler after secondary end capping According to the ratio of the height ratio of 1: 2 to 1:20, the degree of inactivation of the silica gel is further increased especially for the elution of the basic and non-coordinating compounds. A much higher ODS silica gel filler is obtained.
(11) The ODS silica gel filler produced by any one of the production methods described above is an ODS silica gel filler in which the degree of inactivation of silica gel is higher than that of conventional products.

The method for producing the ODS silica gel filler of the present invention includes:
(A) A step of performing an additional end capping using a tertiary amine on the ODS silica gel filler which has been subjected to at least secondary end capping, and (B) an amine compound different from the amine compound used for the primary end capping. A step of performing the used secondary end capping,
including.

  In the present invention, “primary end capping”, “secondary end capping” and “tertiary end capping” are respectively ODS silica gel fillers that have not undergone end capping, that is, the first silanization of silica gel that has not been silanized. It means end capping performed first, end capping performed first after the primary end capping, and end capping performed first after the second end capping. Also, “additional end capping” means any end capping further performed on ODS silica gel filler that has already been end capped.

Although the silica gel used for this invention is not limited, The thing of the specific surface area of 50-800 m < ² > / g which can perform chemical modification highly efficiently is used suitably. More preferably, it is 200-400 m < ² > / g. Further, particles having a particle diameter of 2 to 200 μm, more preferably 2 to 15 μm, and a pore diameter of 40 to 1000 mm, more preferably 40 to 200 mm are preferably used.
As the ODS group to be chemically bonded to the silica gel, any one having an octadecyl group such as a dimethyloctadecyl group, a methyloctadecyl group, and an octadecyl group can be used. As the silylating agent to be used, trimethylchlorosilane (TMCS), hexamethyldisilazane (HMDS), dimethylchlorooctadecylsilane (DMCODS), trimethylchlorosilane and the like are preferable.

  In the present invention, an amine compound is preferably used as a reaction accelerator for ODS formation and end capping. As the amine compound, any of primary, secondary and tertiary amines can be used. Examples of the primary amine include ethylamine, butylamine, and propylamine. Examples of the secondary amine include diethylamine (DEA), dibutylamine, diisopropylamine, morpholine, hexamethyldisilazane (HMDS), and the like. Examples of the tertiary amine include triethylamine (TEA), tributylamine, pyridine and the like. The secondary amine diethylamine, hexamethyldisilazane, tertiary amine triethylamine, pyridine and the like are particularly preferably used because they are liquid at normal temperature and easy to use.

  Two or more amine compounds can be used in combination. As such a combination, hexamethyldisilazane and pyridine or triethylamine, diethylamine and triethylamine, and the like are preferable, and a combination of hexamethyldisilazane and triethylamine is particularly preferable.

When an amine compound is used as a reaction accelerator in end capping, any method may be used for end capping. For example, when a secondary amine such as DEA is used as a reaction accelerator for ODS formation, trimethylchlorosilane was allowed to act in the presence of a secondary amine such as DEA or hexamethyldisilazane as a reaction accelerator in the primary end capping. Thereafter, it is exemplified that trimethylchlorosilane is allowed to act in the secondary end capping in the presence of a tertiary amine such as pyridine and TEA of different grades.
Also, for example, when TEA, which is a tertiary amine, is used as a reaction accelerator in the ODS reaction, trimethylchlorosilane was allowed to act as a reaction accelerator in the presence of a tertiary amine such as pyridine or TEA as primary end capping. Later, it is exemplified that trimethylchlorosilane acts as a secondary end capping in the presence of a secondary amine such as DEA or hexamethyldisilazane having a different grade.
For the ODS reaction according to the present invention, a known method such as the method described in Patent Document 5 can be used.

  As a method for end capping the silica gel filler, a known method using toluene or liquid paraffin as a solvent can be used. When hexamethyldisilazane is used as a reaction accelerator, it is preferable to use liquid paraffin as a solvent in order to perform end capping in a shorter time.

-About a process (A) A process (A) is a process of performing an additional end capping using a tertiary amine for the ODS silica gel filler which performed at least secondary end capping.

Among the methods for producing the ODS silica gel filler of the present invention, the number of additional end capping in the step (A) is not limited, but considering the production efficiency, the one in which the tertiary end capping is the final end capping is preferable.
The silylation accelerator used for the additional end capping is a tertiary amine, with TEA being particularly preferred. Therefore, it is particularly preferable to use tertiary end capping as the final end capping and TEA as the silylation accelerator.

In the production method of the present invention, the methods of primary end capping and secondary end capping are not limited as long as the types of amine compounds used for the respective end capping are not the same, and each of the single amine compounds as described above is used. In addition to a known method used in the method, an amine compound may be used in combination.
When a single amine compound is used in each of the primary end capping and the secondary end capping, it is preferable to use amine compounds of different grades, and the primary end capping is performed using a secondary amine, and the secondary end capping is performed. It is particularly preferred to carry out with a tertiary amine.

  When primary end capping is performed using a tertiary amine and secondary end capping is performed using a secondary amine, it is preferable to perform secondary end capping at a high temperature. Such temperature is typically 200 ° C to 300 ° C, preferably 220 ° C to 280 ° C, more preferably 230 ° C to 250 ° C.

  When the amine compounds are used in combination, the amine compounds to be used may be used by sequentially adding them, or all amine compounds may be mixed and used. It is preferable to use a combination of hexamethyldisilazane and triethylamine, and it is more preferable to sequentially add and use these amine compounds.

The addition amount of the silylating agent and the addition amount of the amine compound used for each end capping are not limited as long as the inactivation of the silica gel is promoted. The ratio between the addition amount of the silylating agent and the addition amount of the amine compound is preferably 1: 1 to 1:10, more preferably 1: 2 to 1: 9, and most preferably 1: 2.3. ~ 1: 4.5.
The method of additional end capping is not limited, and known methods such as a toluene reflux method, a high temperature liquid phase method, a gas phase method, and a supercritical method can be used. The additional end capping can also be performed using a hydrocarbon such as n-heptane as a solvent at a temperature below 100 ° C., which is a temperature below the boiling point of the solvent. Therefore, the production method using the step (A) can be carried out at a much lower temperature than the conventional method, for example, the toluene reflux method carried out at a temperature of around 110 ° C. Such temperature is preferably 90 ° C to 98.3 ° C, more preferably 90 ° C to 98 ° C, and most preferably 97 ° C to 98 ° C.

-About a process (B) A process (B) is a process of performing the secondary end capping using the amine compound different from the amine compound used for the primary end capping. “Different amine compounds” mean one or more types of amine compounds that are not the same, and there may be commonly used amine compounds. For example, the secondary amine alone and the combined use of the secondary amine and the tertiary amine are different amine compounds.
In the method of the present invention, the method of primary end capping and the kind of reaction reagent are not limited, and known methods using each of the above single amine compounds can be used, and amine compounds can be used in combination. You can also. In primary end capping, it is preferable to use an amine compound in combination, and it is particularly preferable to use a secondary amine in combination. The most preferred combination is a combination of HMDS and DEA.

  Further, when the primary end capping is performed using a tertiary amine and the secondary end capping is performed using one or more secondary amines, it is preferable to perform the secondary end capping at a high temperature. . Such temperature is typically 200 ° C to 300 ° C, preferably 220 ° C to 280 ° C, more preferably 230 ° C to 250 ° C.

Combinations of amine compounds used in step (B) are preferably different in class from each other, more preferably a combination of secondary amine and tertiary amine, and a combination of hexamethyldisilazane and triethylamine. Is most preferred.
Combinations of the amine compounds used in the step (B) may be used by sequentially adding them, or all may be mixed and used. It is more preferable to sequentially add and use these amine compounds.

  As described above, the addition amount of the silylating agent and the addition amount of the amine compound used for each end capping are not limited as long as the inactivation of the silica gel is promoted, and also in the step (B). The total addition amount of the amine compound is preferably larger than the addition amount of the silylating agent. Therefore, the ratio between the addition amount of the silylating agent and the total addition amount of the amine compound is preferably 1: 1 to 1:10, more preferably 1: 2 to 1: 9, and most preferably 1: 2.3 to 1: 4.5.

The step (A) or (B) in the method for producing the ODS silica gel filler of the present invention increases the peak height with respect to the solute of the filler,
(1) The peak height of the ODS silica gel filler before the additional end capping in step (A) and the ODS silica gel filler after the additional end capping, with respect to the compounds having basicity and coordination properties. Ratio and / or (2) ODS silica gel filler before secondary end capping in step (B) and ODS silica gel filler after secondary end capping and compounds having basic and coordinating properties Those having peak height ratios of 1: 2 to 1:20, respectively, are preferred.

Although the present invention also relates to an ODS silica gel filler, the production method of such ODS silica gel filler is not limited as long as it is a production method including the above steps (A) and (B), and other methods are known methods. Is used.
In addition, the present invention has an advantage that acid cleaning of ODS silica gel after end capping, which is essential in the prior art, is unnecessary.

The ODS silica gel filler produced by the production method of the present invention is packed in a column and used for HPLC measurement. The column material may be stainless or resinous such as polyethylene, polypropylene or polystyrene.
There is no particular limitation on the substances that can be measured with the ODS silica gel filler according to the present invention. For example, golf course pesticides such as oxine copper, thiuram, ashram, mecoprop, benzulide, carbamate pesticides such as aldicarb and methiocarb, basic substances such as benzylamine and procainamide, organic acids such as benzoic acid, sorbic acid and dehydroacetic acid And various substances such as esters thereof, caffeine and phenol. Advantageously, it can be particularly preferably applied to oxine copper, which has conventionally been difficult to analyze with good reproducibility.

  Next, the present invention will be described in more detail with reference to examples. However, these examples are merely examples of the present invention, and do not limit the present invention in any way.

[Example 1]
1. Experiment 1-1 New End Capping of ODS Filler For the end capping study, ODS-DD and ODS-DTp, which were fillers subjected to different primary end capping, were used. That is, ODS-DD is a primary end-capped filler with secondary silylamine, and ODS-DTp is a primary end-capped filler with TMCS / tertiary amine. did.
We also examined the results of endcapping Mightysil RP-18GP with TMCS / tertiary amine to confirm the difference in reactivity.

1-1-1. Synthesis method of primary end-capping filler ODS-DD
High purity silica gel was dried for 3 hours at 120 ° C. (particle size 5 [mu] m, an average pore diameter of 12 nm, a specific surface area of 350m ² / g) reacting diethylamine dimethylchlorosilane octadecyl silane and (DMCODS) a secondary amine (DEA) promote ODS silica gel (ODS-D) synthesized by reaction as an agent was used as a raw material, and HMDS was used as an end capping agent for primary end capping, and the reaction was carried out at a reflux temperature of toluene (110 ° C.) in the presence of DEA.

1-1-2 Primary end-capping filler ODS-DTp synthesis method
ODS silica gel synthesized by reacting DMCODS with DEA, a secondary amine, as a reaction accelerator on high purity silica gel (particle size 5μm, average pore size 12nm, specific surface area 350m2 / g) dried at 120 ° C for 3 hours (ODS-D) was used as a raw material, TMCS was used as an end-capping agent for primary end-capping, and reacted in the presence of pyridine as a tertiary amine at a toluene reflux temperature (110 ° C.).

  Subsequently, secondary end capping and tertiary end capping were performed, and the same end capping method was used for both ODS-DD and ODS-DTp, and the results were compared.

1-1-3 Secondary end capping 25 g each of ODS silica gel fillers ODS-DD and ODS-DTp after completion of primary end capping are put into a 1 liter four-necked flask, and 300 ml of dehydrated toluene is added and stirred.
To this, 10 ml of HMDS and 10 ml of TMCS are sequentially added, and finally 40 ml of triethylamine (TEA) is added, followed by heating at a toluene reflux temperature (110 ° C.) for 3 to 4 hours.
The reaction is cooled, collected by filtration and washed with 200 ml of tetrahydrofuran (THF). Then, it is washed twice with 300 ml of methanol water (50%) and then with methanol.
Dry at 120 ° C. to obtain a reaction product. These fillers are referred to as ODS-DDTth and ODS-DTp-Tth, respectively.

1-1-4 Tertiary end capping Take 25 g of ODS-DDTth and ODS-DTp-Tth, whose secondary end capping has been completed in 1-1-3, into 1 liter four-necked flasks, add 300 ml of dehydrated toluene, and stir. . TMCS 10ml and TEA 20ml are added to this, and it is made to react at toluene reflux temperature (110 degreeC) for 3 to 4 hours.
The reaction is cooled, collected by filtration and washed first with 200 ml of tetrahydrofuran (THF).
Then, it is washed twice with 300 ml of methanol water (50%) and then with methanol. Dry at 120 ° C. to obtain a reaction product. These fillers are referred to as ODS-DDTth-Tt and ODS-DTp-Tth-Tt, respectively.

1-1-5 Endcapping of Mightysil RP-18GP
Using ODS-DTp as a raw material, secondary silylamine (HMDS / TMCS) as an end-capping agent, 25g of Mightysil RP-18GP produced by high-temperature end-capping is put into a 1-liter four-necked flask, and 300 ml of dehydrated toluene is added. And stir. TMCS 10ml and TEA 20ml are added to this, and it is made to react at toluene reflux temperature (110 degreeC) for 3 to 4 hours.
The reaction is cooled, collected by filtration and washed with 200 ml of tetrahydrofuran (THF). Then, it is washed twice with 300 ml of methanol water (50%) and then with methanol.
Dry at 120 ° C. to obtain a reaction product. This filler is called Mightysil RP-18GP-Tt.

1-2 Column packing The synthesized packing material is packed into a column for evaluation of end capping.
The column packing method (Non-Patent Document 14) is shown below.
Weigh 1.7 g of packing material, add 13 ml of chloroform and 13 ml of THF, and ultrasonically disperse in an ultrasonic bath for 5 minutes (when column is 4.6 mmΦ × 150 mm).
Using the column packing mode of liquid chromatograph (Hitachi L-6100), methanol aqueous solution (80%) is packed as a pressurized solvent.
The number of theoretical plates was confirmed, and a column with a theoretical plate number of 85,000 plates / m or more was used.

1-3 Evaluation of packing materials 1-3-1 Evaluation of end-capping The packed columns are Mightysil RP-18GP batch tests, Test II (Procainamide test), Test IV (Oxin copper test), Test V. (Basic substance containing pyridine) and Test VI (hinokitiol test: injection amount: 1 μl) were used to evaluate the end-capping quality.

1-3-2 Micro-coordinating compound test In order to confirm the remaining silanol more strictly, the elution condition of trace amount (ng order) of oxine copper was measured under the elution condition near pH 4.5, and the quality of end capping was examined. did.
Elution condition Eluent: Acetonitrile / 20 mM KH ₂ PO ₄ = 40/60, Flow rate: 0.5 ml / min.,
Detection wavelength: UV240nm, injection volume: Oxin copper (2ppm solution) 2.5μl

2. Results and Discussion 2-1 Elution behavior of basic compound procainamide Fig. 1 shows the results of elution behavior of procainamide in each filler after tertiary end capping.

Regarding the elution of procainamide (second peak), a process of reacting in the presence of TEA using a mixture of TMCS and HMDS as a silylating agent in all secondary end capping reaction systems (with secondary silylamine and TMCS / tertiary amine mixed solution), but the elution behavior was good in all cases.
The fact that the reaction was performed in the presence of TEA in the TMCS / HMDS mixture as the end-capping agent this time revealed that the reaction between TMCS and organic amines had a problem in elution of basic compounds. This is caused (Patent Document 5). In particular, when procainamide was used as a sample, a highly tailored chromatogram was obtained. This chromatogram is the result of using a filler (ODS-TDT) in which primary end capping was performed with TMCS and DEA end capping agents and secondary end capping was performed with TMCS and TEA end capping agents. It was revealed that when HMDS was added to this end-capping agent (TMCS / TEA) and the reaction was performed, the elution situation of procainamide was improved as shown in FIG. 2-b).
This result also revealed that HMDS plays an important role in improving the elution of basic compounds.

  For the ratio of peak height of uracil and procainamide (results under the conditions measured this time) (Table 1), ODS-DDTth- is a tertiary end-capped product of ODS-DD that is primary end-capped with secondary silylamine. Tt is 2.0, and ODS-DTp-Tth-Tt, the tertiary end-capping product of ODS-DTp endcapped with TMCS / pyridine (tertiary amine), is 1.7, and ODS-DDTth-Tt is better. It was. Uracil is a compound that does not interact with silanol.

  On the other hand, Mightysil RP-18GP-Tt, a high-temperature end-capped ODS silica gel filler with secondary silylamine, has a peak height ratio of procainamide / uracil of 2.0, and ODS-DTp-Tth-Tt using the same raw material. The same chromatogram was obtained (Table 1). That is, when secondary silylamine was subjected to high temperature end capping as secondary end capping after TMCS / tertiary amine primary end capping, it was possible to obtain results equivalent to ODS-DDTth-Tt. The chromatogram of Mightysil RP-18GP-Tt is shown in FIG.

  In addition, from the value of the peak height ratio of procainamide and uracil shown in Table 1, the elution behavior of procainamide is the result of secondary end capping when tertiary end capping is performed with TMCS / tertiary amine. It is shown that it exceeded slightly.

2-2 Regarding Elution Status of Pyridine FIG. 4 shows the results of improvement in elution of pyridine, which is another basic compound.
As for elution of pyridine (second eluting peak), ODS-DDTth-Tt (Fig. 4a)) is similar to ODS-DTp-Tth-Tt shown in Fig. 4b), as is the elution behavior of procainamide. Good results were shown. That is, similar to the results of the elution test of procainamide, better elution can be achieved for the elution of the basic compound by using HMDS in the primary end capping.

  In addition, with Mightysil RP-18GP-Tt, a chromatogram equivalent to that of ODS-DDTth-Tt was obtained in the same manner as in the case of procainamides with respect to the elution behavior of pyridine (FIG. 5).

2-3 Elution behavior of trace amount oxine copper with respect to elution of oxine copper In order to confirm the elution behavior of oxine copper, a test was conducted in the case of an absolute amount of 5 ng. The reason is that when the injection amount is 100 ng, a relatively good chromatogram is obtained except for ODS-DD which is a primary end-capping product, and it is difficult to clarify the performance of the filler according to the present invention. Because it was thought. For this reason, the test was performed using an eluent of 20 mM KH ₂ PO ₄ / acetonitrile = 60/40 at pH 4.5 where the sample injection amount was changed to 5 ng and the elution of the coordinating compound was a severe condition. .
The results are shown in FIG.

It was found that the elution of oxine copper showed a sharper peak even when the amount of ODS-DDTth-Tt (FIG. 6a)) was small, similarly to the elution of the basic compound (FIG. 6b)).
Moreover, the elution behavior of oxine copper changed greatly depending on the end capping state. In other words, at the stage of secondary end capping, although almost no oxine copper was eluted in both ODS-DDTth-Tt and ODS-DTp-Tth-Tt (Fig. 7), Excellent elution behavior as shown in 6 was achieved.
On the other hand, in Mightysil RP-18GP, the elution behavior of oxine copper was remarkably improved (FIG. 8).

  As described above, in any packed column, the elution behavior of oxine copper was greatly improved by reacting TMCS / tertiary amine in the tertiary end capping.

From these results, the elution behavior of oxine copper is affected by both irreversible secondary interactions with silanols that have secondary interactions with basic compounds and silanols with irreversible secondary interactions with coordination compounds. It became clear to do. This is because oxine copper is a nitrogen-containing coordination compound and also exhibits basicity. At the same time, the compound is used as an evaluation substance for residual silanol, and any of the irreversible secondary interactions described above is used. It can be said that the compound was found to be a very important compound that serves as an index for confirmation.
In addition, the ODS silica gel filler synthesized by the production method developed this time does not need to be washed with a phosphoric acid acidic solution, which is necessary for conventional ODS silica gel fillers. It was also found that oxine copper can be eluted even under conditions.

2-3-2 Effect of reaction reagent addition amount on oxine copper elution On the other hand, the elution behavior of oxine copper was different depending on the addition amount of the end capping agent used in the secondary end capping step. That is, in particular, out of the amount of TMCS, HMDS and TEA mixed solution used for secondary end capping, it affected the elution behavior of oxine copper.
FIG. 9 shows the result. Fig. 9-a) shows the results of a reaction in which TMCS is 40 mM, HMDS is 25 mM, and TEA is 70 mM with respect to 25 g of filler. Fig. 9-b) is TMCS of 80 mM and HMDS is 50 mM with respect to 25 g of filler. It is the result of the reaction which added 140 mM of TEA. FIG. 9-c) shows the results of the reaction in which TMCS was added at 80 mM, HMDS at 50 mM, and TEA at 280 mM.
These results indicate that not only the addition amount of the silylating agent but also the addition amount of organic amine TEA, which is a reaction accelerator, greatly affects the results. By comparing FIG. 9-b) and c), the addition amounts of TMCS and HMDS as silylating agents are the same, but when the addition amount of TEA as an organic amine is doubled, the oxine copper is also relatively good. The elution behavior is shown, indicating that the results are very different.
Therefore, in this examination, the reaction reagent which added the organic amine addition amount more than twice the amount with respect to the silanizing agent was used.

2-4 Concerning elution of hinokitiol Hinokitiol is an evaluation substance used for confirming the elution status of coordination compounds, like oxine copper, but unlike oxine copper, it is not a nitrogen-containing compound and does not show basicity. Since it is a compound, it is a compound that makes it possible to measure the elution behavior as a purely coordinating compound.
The chromatogram of hinokitiol after tertiary end capping is shown in FIG.
Concerning the elution behavior of hinokitiol, ODS-DTp-Tth-Tt showed better results, contrary to the elution status of basic compounds and oxine copper.

FIG. 11 shows the elution behavior of hinokitiol after primary end capping and secondary end capping. In the primary end capping stage, hinokitiol was eluted, though not completely, in the reaction route starting from ODS-DTp (FIG. 11b-1), which was end capped with TMCS / tertiary amine. On the other hand, hinokitiol was not eluted at all in the filler ODS-DD (FIG. 11a-1) subjected to primary end capping with secondary silylamine.
In addition, after secondary end capping, both ODS-DDTth and ODS-DTp-Tth showed almost the same chromatogram (FIGS. 11a-2) and b-2)).
These results show that end capping using TMCS / tertiary amine is most effective in improving the dissolution of coordinating and non-basic compounds.
In addition, in the secondary end capping, the reaction of TMCS / tertiary amine occurs because the mixture of HMDS and TMCS reacts in the presence of TEA, which is a tertiary amine.

  The result of MightysilRP-18GP-Tt is shown in FIG. End-capping with TMCS / tertiary amine showed a better chromatogram of hinokitiol than Mightysil RP-18GP. In other words, the quality of Mightysil RP-18GP is considered to be greatly influenced by the state of primary end capping. By performing the tertiary end capping as described above, a coordination compound having no basicity such as hinokitiol. As a result, even better quality can be obtained.

2-5 Comparison with other companies As shown above, it has been found that the end capping of ODS silica gel fillers has different performance depending on the silylamine to be reacted. It has been found that end-capping is possible even with a wet reaction.
At present, it can be said that there are many ODS silica gel fillers that have no problem with elution of basic compounds, but there are few measures for the elution of coordination compounds, and there are many fillers that are actually incompletely endcapped. This is shown in FIG. 13 (elution behavior of oxine copper) and FIG. 14 (elution behavior of hinokitiol).

These are chromatograms measured using GL Sciences's Intosyl ODS-3 and Shiseido's Capsule Pack C18 MG-II (particle size 5 μm, 150 mm × 4.6 mmΦ). Although there is no problem with the elution behavior, there is a defect in the elution of the coordinating compound (FIGS. 13 and 14).
One of the reasons for this is the recognition that irreversible secondary interactions of coordination compounds are caused by metal impurities contained in the filler, and most studies have been conducted on silanols that have secondary interactions with coordination compounds. It is thought that there is no background.
Therefore, in the conventional high-purity silica gel, it is difficult to remove the irreversible secondary interaction with the coordinating compound unless the end capping is finally performed reliably.

2-6 Causes of Irreversible Secondary Interaction Based on the results described so far, residual silanol of ODS silica gel filler, basic compound, and irreversible secondary interaction of coordinating compound occur. The cause is considered as follows, for example.
There is no doubt that the silica gel surface is composed of silanol (-Si-OH) and siloxane (-Si-O-Si-). Silanol is a geminal silanol (twin) as shown in the figure below. Silanol), isolated silanol, and associative silanol (single silanol) (Non-Patent Document 11). When this is classified in terms of three-dimensional structure, geminal silanol and associative silanol become equatorial silanol, and isolated silanol becomes axial silanol.

シリカゲル表面に存在するシラノール（非特許文献１５）

Silanol present on silica gel surface (Non-patent Document 15)

  In addition, alkylsilylation of silica gel is said to react from a highly reactive associative silanol (Non-patent Document 12), and this reaction mechanism is shown in the figure below. This reaction mechanism shows an example of reaction of an equatorial-type associative silanol. As a result, when the associative-type silanol is endcapped, it shows that an axial-type silanol (isolated silanol) appears.

シラノールのアルキルシリレーションの反応機構（非特許文献１２）

Reaction mechanism of alkyl silylation of silanol (Non-patent Document 12)

That is, when ODS silica gel in which a large amount of associative silanol remains is endcapped with secondary silylamine, the isolated silanol remains unreacted, which results in irreversible two-way irreversibility with the coordinating compound. It is thought to cause the next interaction. However, judging from the phenomenon that the irreversible secondary interaction with the basic compound is reduced, the basic compound has an irreversible secondary interaction with the associated silanol.
Endcapping using TMCS / tertiary amine, another method widely used in alkylsilylation, includes isolated silanols, but the silylation reaction proceeds, but irreversible secondary interactions with basic compounds. It is believed that the reaction with the acting silanol is less than when it is endcapped with a secondary silylamine.
Therefore, taking advantage of these characteristics of the end-capping agent, TMRS / tertiary amine can be used after removing silanol which has irreversible secondary interaction with basic compound as much as possible using at least two different amine compounds. By performing end capping, the inactivation of the ODS silica gel filler can be achieved to the limit even at the toluene reflux temperature.

3. Conclusion The method for producing a packed column using the new end capping according to the present invention is based on the reaction mechanism of silanol and silylamine, for example, by performing the third end capping and making the number of times of end capping different from that of the conventional method. This is a synthetic method that can be applied regardless of the particle size of silica gel, in which capping is performed using an amine compound different from the amine compound used for the primary end capping.
By applying the production method of the present invention, it is possible to improve the quality of conventional products, that is, to manage a very small amount of oxine copper that has been difficult to manage, and to make quantitative measurement difficult. It is possible to quantify the low-concentration region of coordinating antibacterial agents such as oxophosphoric acid, which can be commercialized as a new high-quality end-capping product.

  The present invention contributes greatly to the development of packed columns and other related industries.

It is a chromatogram which shows the elution behavior of procainamides after tertiary end capping. a) ODS-DD-Tth-Tt, b) ODS-DTp-Tth-Tt Eluent: pH 7.6, 20 mM phosphate buffer / methanol = 60/40, flow rate: 0.5 ml / min. column temperature: 40 ° C , Detection: UV254nm 0.16AUFS (peaks are uracil, procainamide, acetylprocainamide in order) It is a chromatogram which shows the difference in the elution behavior of procainamide by the difference in an end capping agent. a) ODS-TDT, b) ODS-DTp-Tth eluent: pH 7.6, 20 mM phosphate buffer / methanol = 60/40, flow rate: 0.5 ml / min, column temperature: 40 ° C, detection: UV254nm 0.16AUFS (The peaks are uracil, procainamide, and acetylprocainamide in order) It is a chromatogram which shows the elution behavior with respect to procainamides of Mightysil RP-18GP-Tt. Eluent: pH 7.6, 20 mM phosphate buffer / methanol = 60/40, flow rate: 0.5 ml / min. Column temperature: 40 ° C, detection: UV254 nm 0.16 AUFS (peaks are uracil, procainamide, acetylprocainamide in this order) It is a chromatogram which shows the pyridine elution behavior after end capping. a) ODS-DD-Tth-Tt, b) ODS-DTp-Tth-Tt Eluent: acetonitrile / H ₂ O = 30/70, flow rate: 1 ml / min., column temperature: 40 ° C., detection: UV254nm 0.16AUFS (From the beginning, uracil, pyridine, aniline, o-toluidine, N-methylaniline, 2-ethylaniline, 3-nitroanisole, N, N-dimethylaniline) It is a chromatogram which shows the elution behavior of pyridine of Mightysil RP-18GP-Tt. Eluent: acetonitrile / H ₂ O = 30/70, flow rate: 1 ml / min. Column temperature: 40 ° C., detection: UV254 nm 0.16 AUFS (peaks are uracil, pyridine, aniline, o-toluidine, N-methylaniline from the beginning. 2-ethylaniline, 3-nitroanisole, N, N-dimethylaniline)

It is a chromatogram which shows the elution situation of trace amount oxine copper. a) ODS-DDTth-Tt, b) ODS-DTp-Tth-Tt eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5 ml / min. column temperature: 40 ° C., detection: 240 nm, sample Injection amount: 5ng It is a chromatogram which shows the elution behavior of oxine copper after secondary end capping. a) ODS-DDTth, b) ODS-DTp-Tt Eluent: 20 mM KH ₂ PO ₄ / Acetonitrile = 60/40, Flow rate: 0.5 ml / min. Column temperature: 40 ° C., detection: 240 nm, sample injection amount: 5 ng It is a chromatogram which shows the elution situation of trace amount oxine copper. a) Mightysil RP18GP-Tt, b) Mightysil RP-18GP eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5 ml / min. column temperature: 40 ° C., detection: 240 nm 0.01 AUFS Sample injection volume: 5ng It is a chromatogram which shows the elution result of oxine copper by the difference in the addition amount of a reaction reagent. a) Reaction with TMCS 40 mM (5 ml), HMDS 25 mM (5 ml), TEA 70 mM (10 ml) b) Reaction with TMCS 80 mM (10 ml), HMDS 50 mM (10 ml), TEA 140 mM (20 ml) c) TMCS 80 mM (10 ml) ), Reaction with HMDS 50 mM (10 ml), TEA 280 mM (40 ml) Eluent: 20 mM phosphoric acid / acetonitrile = 95/5, flow rate: 1.0 ml / min, column temperature: 40 ° C., detection: 250 nm, sample: uracil, Oxin copper, caffeine (in order of elution) (Inject 5 μl of sample solution containing 20 μg / ml of oxine copper) It is a chromatogram which shows the elution situation of hinokitiol after tertiary end capping. a) ODS-DDTth-Tth, b) ODS-DTp-Tth-Tt Eluent: 20 mM KH ₂ PO ₄ / Acetonitrile = 60/40, Flow rate: 0.5 ml / min. Column temperature: 40 ° C., Detection: UV320 nm 0.16 AUFS Injection volume: 0.5 μg

It is a chromatogram which shows the elution behavior of hinokitiol after primary end capping and secondary end capping. a) -1 ODS-DD, a) -2 ODS-DDTth, b) -1 ODS-DTp, b) -2 ODS-DTp-Tth Eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5ml / min. Column temperature: 40 ° C, detection: UV320nm 0.16AUFS, injection volume: 0.5μg It is a chromatogram which shows the comparison of the elution behavior of hinokitiol of Mightysil RP-18GP-Tt and Mightysil RP-18GP. a) Mightysil RP-18GP-Tt, b) Mightysil RP-18GP eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5 ml / min. column temperature: 40 ° C., detection: UV320 nm 0.16 AUFS, injection Amount: 0.5μg It is a chromatogram chromatogram which shows the elution behavior of oxine copper of the other company's column. a) GL Science Inertsil ODS-3, b) Shiseido Capcell Pak C18 MG-II Eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5 ml / min. column temperature: 40 ° C., detection: 240 nm 0.01 AUFS, sample injection volume: 5ng It is a chromatogram which shows the comparison of the elution behavior of hinokitiol of the other company column. a) GL Science Inertsil ODS-3, b) Shiseido Capcell Pak C18 MG-II Eluent: 20 mM KH ₂ PO ₄ / acetonitrile = 60/40, flow rate: 0.5 ml / min, column temperature: 40 ° C., detection: UV 320 nm 0.16 AUFS, injection volume: 0.5 μg

Claims (11)

Method for producing ODS silica gel filler comprising the following steps:
-Primary endcapping with amine compounds,
-Secondary end capping using an amine compound different from the amine compound used for primary end capping, and-Additional end capping using tertiary amine to the ODS silica gel filler that has undergone secondary end capping. Process.   The production method according to claim 1, wherein the additional end capping is only tertiary end capping, and the amine compound used for the tertiary end capping is only a tertiary amine.   The manufacturing method of Claim 1 or 2 with which a tertiary end capping is performed at the temperature of 90 to 98.3 degreeC.   The manufacturing method in any one of Claims 1-3 whose amine compound used for a secondary end capping is any one of a secondary amine and a tertiary amine.   The production method according to claim 4, wherein the amine compound used for secondary end capping is only a secondary amine, and the secondary end capping is performed at a temperature of 200C to 300C.   The manufacturing method in any one of Claims 1-3 whose amine compound used for secondary end capping is a secondary amine and a tertiary amine.   The manufacturing method of Claim 6 whose amine compound used for a primary end capping is only a secondary amine or only a tertiary amine.   The manufacturing method in any one of Claims 1-7 whose ratio of the addition amount of the silylating agent used for secondary end capping and the addition amount of an amine compound is 1: 1-1: 10.   The ratio of peak height to basic and coordinated compounds of the ODS silica gel filler before additional end capping and the ODS silica gel filler after additional end capping is 1: 2-1 : The manufacturing method in any one of Claims 1-8 which is 20.   The ratio of peak height to basic compound of ODS silica gel filler before secondary end capping and ODS silica gel filler after secondary end capping is 1: 2 to 1:20. The manufacturing method in any one of Claims 6-8. The ODS silica gel filler manufactured by the manufacturing method in any one of Claims 1-10.

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