JPH0737970B2 - Packing material for reversed-phase liquid chromatography - Google Patents

Description

TECHNICAL FIELD The present invention relates to a packing material for reversed phase liquid chromatography, which comprises a powder coated with a silicone resin.

[Prior art] At present, analysis and preparative purification techniques using high-performance liquid chromatography have been remarkably developed, and some companies are pursuing purification using a liquid chromatograph plant which is further enlarged. It is being appreciated. In such a situation, the quality of the packing material used to fill the column is
It goes without saying that it is important in analysis and preparative purification.

At present, about 80% of analysis and preparative purification by high performance liquid chromatography is performed by reverse phase liquid chromatography. Among them, silanol groups (Si-
Many applications have been reported using ODS-silica gel (C ₁₈ type) packing material in which dimethyloctadecylchlorosilane is chemically bonded to (OH group).

In addition, as a filler for reversed-phase liquid chromatography, C ₈ type dimethyl octyl trichlorosilane silanol group of silica gel is chemically bonded, C ₄ types dimethyl butyl chlorosilane is chemically bonded, trimethylchlorosilane Known are the C ₁ type chemically bonded to phenyl type and the phenyl type chemically bonded to triphenylchlorosilane.

[Problems to be Solved by the Invention] However, in the above-mentioned conventional filler for chemical bond reverse phase liquid chromatography, the reaction between the silanol group and the alkylchlorosilane is not completed and the silanol group is
10 to 20% remains. The number of residual Suranoru groups, descending order of the molecules, i.e., it is said that C ₁₈ type> C ₈ type> C ₄ type> C ₁ type common in order to become a serious problem in the reverse-phase liquid chromatography There is. That is, when the residual silanol group is present, strong interaction with the polar substance occurs, so that the peak of the polar compound becomes broad during analysis and preparative separation, and the tailing phenomenon occurs. In addition, the residual silanol group causes irreversible adsorption with a compound having a large polarity, which accelerates deterioration of the packed column. Furthermore, due to the presence of residual silanol groups and the presence of siliceous substances, the acid resistance and alkali resistance are weak, and the pH range of mobile phase solvents that can be used is limited to pH 2 to 7 in the case of conventional packed columns. There are problems. Therefore, a part of the conventional packed column was treated again with trimethylchlorosilane (TMS) or the like in order to block residual silanol groups after the reaction with dimethyloctadecylchlorosilane or dimethyloctylchlorosilane. There are also things. However, the residual silanol groups cannot be completely eliminated even by TMS treatment. That is, there is still no high-quality reversed-phase liquid chromatography packing material containing no residual silanol groups.

Even if the silanol groups could be completely blocked by TMS, the problem of weak acid resistance and alkali resistance cannot be solved because the exposed siliceous material still exists.

The object of the present invention is to completely eliminate the influence of silanol groups,
It is an object of the present invention to provide a column packing material for reversed-phase liquid chromatography, which has a sharp peak of a polar compound, is excellent in acid resistance and alkali resistance, and can be used for a long period of time.

In view of these circumstances, the inventors of the present invention have conducted extensive studies and as a result, have found that the influence of residual silanol groups is significantly reduced by a resin capsule-type filler that utilizes low-temperature plasma irradiation, which is essentially different from the conventional chemical bond filler. The present invention has been completed by finding that the amount can be made extremely small, and further, the resistance to acid and alkali can be strong.

[Means for Solving Problems] That is, according to the present invention, a surface treatment with an organosiloxane obtained by irradiating a powder with a low temperature plasma together with one or more kinds of the organosiloxane represented by the following (a) is performed. It is a packing material for reversed-phase liquid chromatography, which comprises a resin capsule type powder.

The present invention will be described in more detail below.

The organosiloxane (hereinafter referred to as silcon) used in the present invention is represented by the following formula.

(A) (R ₁ R ₂ SiO) m (R ₃ R ₄ R ₅ SiO _0.5 ) n (wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each a hydrogen atom or a carbon number of 1 to 30) Represents hydrocarbon groups up to
Two hydrogen atoms are not bonded to the same silicon atom. Further, the hydrocarbon group may be substituted with at least one halogen atom. m is an integer of 1 to 250, and n is 0 or 2. More specifically, it is a linear organosiloxane represented by the following formula (b) or a cyclic organosiloxane represented by the following formula (c).

(In the formula, R ₆ , R ₇ and R ₈ may be the same or different from each other and are selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a phenyl group and a halogen group, but the hydrogen atoms are the same silicon atom. X is 1 to 250 and y is a numerical value of 3 to 250. The smaller the number of x in formula (b) and the number of y in formula (c), the smaller. , Because it has a low boiling point and volatilizes a large amount to be adsorbed on the powder,
In particular, the trimer in the formula (c) is most preferable because it is easy to polymerize due to its steric property. However, even those having a high boiling point can be added and treated as liquid.

The present invention is selectively applied from any one of these or a combination of two or more thereof. Generally, the compounding amount of the silicone used is preferably 1.0 to 40% by weight based on the powder. If it is less than 1.0% by weight, the holding power as a filler is weak, which is not preferable, and if it exceeds 40% by weight, the pores are crushed by an excessive amount, which is not preferable.

The powder used in the present invention is any powder used as a carrier for general liquid chromatography, for example, silica gel, alumina, glass beads such as porous glass, zeolite, titanium dioxide, iron oxide, graphite, etc. Even those not used in the reverse phase system can be used because the silicone coating can be easily formed by low temperature plasma irradiation.

However, spherical or crushed silica gel having pores of 50 to 500 A, a large surface area, and a particle diameter of 3 to 500 μm is preferable.

Next, in the present invention, a mixture of silicone and powder is irradiated with a low temperature plasma, and the plasma used at this time can be generated by corona discharge, glow discharge or the like in the low frequency and high frequency regions. The irradiation atmosphere may be any of vacuum, nitrogen, oxygen, hydrogen, argon, helium, and air. The treatment time is preferably carried out within a range that does not adversely affect the powder, and an appropriate treatment time can be selected depending on the type of gas used, the output of plasma, the type of powder to be treated, etc., but is preferably 20 Seconds to 60 minutes.

Specifically, low temperature plasma irradiation may be performed using, for example, a practic line (manufactured by Sankyo Electric Industry Co., Ltd.).
The practicable line is suitable for treatment in the air as it is or in the presence of a specific gas in contact with the powder.

The molecular weight of the silicone resin formed on the powder surface using the above low-temperature plasma irradiation is 150,000 or more. There is no means to confirm that the molecular weight is 150,000 or more. That is, in the case of silicone, as it is polymerized by polymerization, it becomes difficult to dissolve in water or an organic solvent, and it is not possible to extract the resin to measure the molecular weight, and the state of being coated on the powder surface It is also impossible to measure the molecular weight of the resin in.

Therefore, in order to indirectly estimate the molecular weight, chloroform was extracted in a state where the polymerization was not sufficiently performed, and the extract was extracted with GP.
The maximum molecular weight of the extracted resin was 150,000 when analyzed by C (gel permeation chromatography) and the molecular weight of the extracted resin was determined in terms of polystyrene. Therefore, it can be said that the molecular weight of the resin sufficiently polymerized to a state where it is not extracted with chloroform is 150,000 or more.

The powder coated with this silicone resin can be used as a packing material for reversed-phase liquid chromatography even if it is directly packed in a column. When R ₁ is a methyl group, trimethylchlorosilane is chemically bonded to silica gel. It has a holding power comparable to the conventional C ₁ type.

In addition, a packing material having stronger retention than the above-mentioned C ₁ type packing material [for example, C ₁₈ type (ODS-silka gel), C ₈
Type or phenyl type] is used in most cases. The inventors of the present invention also obtain a packing material for reversed-phase liquid chromatography having a retention capacity comparable to that of the above-mentioned reversed-phase packing material by using methylphenylsilicone, methyloctylsilicone, etc. for these types of packing materials as well. I was able to.

EXAMPLES Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this.

Example 1 10 g of spherical silica gel powder having a pore size of 100 A and a particle size of 5 μm
And cyclic silicone [in the formula (c), R ₆ = CH ₃ , R ₇ = C
H ₃ and y = 3] 2.0 g were mixed so as to be uniform, and a practic line (manufactured by Sankyo Denki Co., Ltd.) was used, while flowing 200 ml / min of nitrogen, at a low frequency (5 kHz), Plasma was irradiated for 10 minutes under the condition of an output of 30 W to obtain coated spherical silica gel.

Using a packer and a pump, 3 g of the obtained coated powder, an inner diameter of 4.
A 6 mm and 25 cm long stainless steel column was packed by the equilibrium slurry method to prepare a packed column. Connect this column to a high-speed chromatograph and
% Methanol was flown at a flow rate of 1 ml / min, a standard mixture of uracil, methyl benzoate, toluene and naphthalene was injected, and a chromatogram was obtained by detection at 254 nm using a UV detector. As a result, the retention time of each peak and the number of theoretical plates were as follows.

Example 2 10 g of spherical silica gel powder having a pore size of 100 A and a particle size of 5 μm
And cyclic silicone [in the formula (c), R ₆ = C ₄ H ₉ , R ₇ =
CH ₃ and y = 5] 2.5 g were mixed so as to be uniform, and a practic line (manufactured by Sankyo Denki Co., Ltd.) was used, while flowing 200 ml / min of argon, and at a low frequency (5 kH
z), plasma irradiation was performed for 10 minutes under an output of 30 W to obtain coated spherical silica gel.

Using a packer and a pump, 3 g of the obtained coated powder, an inner diameter of 4.
A 6 mm and 25 cm long stainless steel column was packed by the equilibrium slurry method to prepare a packed column. Connect this column to a high-speed chromatograph, and use 70 as the mobile phase.
% Methanol was flown at a flow rate of 1 ml / min, a standard mixture of uracil, methyl benzoate, toluene and naphthalene was injected, and a chromatogram was obtained by detection at 254 nm using a UV detector. As a result, the retention time of each peak and the number of theoretical plates were as follows.

Example 3 10 g of spherical silica gel powder having pores of 100 A and a particle size of 5 μm
And linear silicone [in the formula (b), R ₆ = CH ₃ , R ₇
= Those _{C 6 H 17, R 8 =} H and x = 3 to 10] were uniformly mixed to 2.5g, using plasma ashing apparatus LTA-4Sn-type (Yanagimoto Seisakusho Co.), High frequency (13.56MHz), output 20W through nitrogen 200ml / min to maintain vacuum degree of 1.0mmHg
Plasma was irradiated for 10 minutes under the conditions to obtain coated spherical silica gel.

Using a packer and a pump, 3 g of the obtained coated powder, an inner diameter of 4.
A 6 mm and 25 cm long stainless steel column was packed by the equilibrium slurry method, the packed column was connected, 70% methanol as a mobile phase was run at a flow rate of 1 ml / min, and a standard mixture of uracil, methyl benzoate, toluene, and naphthalene was used. Was injected and detected at 254 nm using a UV detector to obtain a chromatogram. As a result, the retention time of each peak and the number of theoretical plates were as follows.

Example 4 10 g of spherical silica gel powder having a pore size of 100 A and a particle size of 5 μm
And linear silicone [in the formula (b), R ₆ = CH ₃ , R ₇
= Things _{C 18 H 37, R 8 =} H and x = 3 to 10] were uniformly mixed to 3.0 g, with a plasma asher LTA-4Sn-type (Yanagimoto Seisakusho Co.), A high frequency (13.56MH) was passed through argon nitrogen 200ml / min to maintain the vacuum degree 1.0mmHg.
z), plasma irradiation was performed for 10 minutes under the condition of output 20 W to obtain coated spherical silica gel.

Using the obtained coated powder 3g packer and pump, the inner diameter 4.6m
A stainless steel column of m and a flow rate of 25 cm was packed by the equilibrium slurry method to prepare a packed column. Connect this column to a high-speed chromatogram, and use 70% as a mobile phase.
Methanol was flown at a flow rate of 1 ml / min, a standard mixture of uracil, methyl benzoate, toluene and naphthalene was injected, and detection was carried out at 254 nm using a UV detector to obtain a chromatogram. As a result, the retention time of each peak and the number of theoretical plates were as follows.

Example 5 Crushable silica gel powder 10 having pores of 120A and a particle size of 10 μm
g and linear silicone [in the formula (c), R ₆ = HC ₃ , R ₇
= C ₁₈ H ₃₇ , R ₈ = CH ₃ and x = 10 to 30] 3.0 g are mixed so as to be uniform, and a practic line (manufactured by Sankyo Denki Co., Ltd.) is used to obtain 200 ml of helium / Plasma was irradiated for 10 minutes at a low frequency (5 kHz) and an output of 30 W while flowing min to obtain coated spherical silica gel.

Using a packer and a pump, 3 g of the obtained coated powder, an inner diameter of 4.
A 6 mm and 25 cm long stainless steel column was packed by the equilibrium slurry method to prepare a packed column. Connect this column to a high-speed chromatograph and use it as a mobile phase.
% Methanol was flown at a flow rate of 1 ml / min, a standard mixture of uracil, methyl benzoate, toluene and naphthalene was injected, and a chromatogram was obtained by detection at 254 nm using a UV detector. As a result, the retention time of each peak and the number of theoretical plates were as follows.

Comparative Example 1 In this example, the effects of the silanol groups of the resin capsule type filler according to the present invention and the conventional chemically bonded filler were compared.

The fillers prepared in Examples 1 to 5 were used as the filler of the present invention. As a control, a commercially available ODS-silica gel (C
₁₈ types) 6 packing columns were used. Similarly to each commercially available column, 3 g of each packing material of the present invention was packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 25 cm by the equilibrium slurry method to prepare a packed column. Connect each column to a high performance liquid chromatograph and use 15% as mobile phase.
Acetonitrile was passed through the column at a flow rate of 1 ml / min, and a pyridine / phenol test was performed at a chart speed of 5 cm / min. Pyridine phenol tests and analyzes a mixture of pyridine and phenol by high performance liquid chromatography, in which the respective retention times (T _R), was calculated T _R (pyridine) / T _R (phenol) value, As the value exceeds 1 and increases, the number of residual silanol groups increases, and the influence on polar substances increases. At the same time, the degree of tailing of the elution peak of pyridine was evaluated in 5 grades from the largest one, and the results are shown in Table 1 together. Further, the results of both the pyridine / phenol test and the tailing of the pyridine elution peak were comprehensively evaluated on the basis of three grades of A, B and C, and shown in Table 1. As a result,
The capsule-type reversed-phase liquid chromatography packing material according to the present invention is compared with a commercially available chemical-bonding packing material.
The effect of the silanol group was very small, and it was demonstrated that the performance was excellent.

Comparative Example 2 In this example, the alkali resistance of the resin capsule type filler according to the present invention and the conventional chemical bond type filler was compared.

The fillers prepared in Examples 1 to 5 were used as the filler of the present invention. As a control, 6 kinds of commercially available ODS-silica gel (C ₁₈ type) packed columns provided in a form filled with a conventional chemical bond type packing material were used.
Similarly to each commercially available column, 3 g of each packing material of the present invention was packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 25 cm by the equilibrium slurry method to prepare a packed column. Connect each column to a fully automated high performance liquid chromatograph and use Britton Robinson buffer as the mobile phase.
Flow 70% methanol adjusted to pH 10 at a flow rate of 1 ml / min,
A naphthalene standard material was injected and a chromatogram was obtained.
This operation was repeated fully automatically to determine the continuous operation time in which the retention time of the peak of the naphthalene chromatogram was shortened by 2 minutes. It is said that a commercially available chemically-bonded packed column gradually deteriorates and loses its retentivity when it is allowed to flow at pH 8 or higher for a certain period of time or longer.

The results are shown in Table-2. As expected, the resin-capsule type packing material column according to the present invention has been proved to endure continuous operation for a considerably long time and to be excellent in alkali resistance as compared with a commercially available packing column.

[Effects of the Invention] The packing material for reversed phase liquid chromatography obtained by the present invention has various excellent properties as compared with the conventional packing material for chemically bonded reverse phase liquid chromatography.

That is, in the conventional chemical bond type filler, as described above, the residual silanol groups are present in an amount of about 10 to 20%, and therefore, the elution of polar substances (particularly basic substances) is greatly affected. On the other hand, the filler obtained by the present invention is a resin capsule type and can substantially eliminate the influence of silanol groups.

Further, it is possible to prevent the tailing phenomenon in the elution of the polar substance, which is a drawback of the conventional chemical bond filler.

Furthermore, since the silica gel is covered with a uniform silicone resin layer in this filler, it has a particularly strong alkali resistance,
It can be used for a long time between pH 2 and 12.

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Claims (4)

[Claims] 1. Reversed-phase liquid chromatography using a resin capsule powder surface-treated with an organosiloxane, which is obtained by irradiating a powder with a low temperature plasma together with one or more of the organosiloxanes represented by (a) below. Filler. (A) (R ₁ R ₂ SiO) m (R ₃ R ₄ R ₅ SiO _0.5 ) n (wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each a hydrogen atom or a carbon number of 1 to 30) Represents hydrocarbon groups up to
Two hydrogen atoms are not bonded to the same silicon atom. Further, the hydrocarbon group may be substituted with at least one halogen atom. m is an integer of 1 to 250, and n is 0 or 2. ) 2. The packing material for reversed phase liquid chromatography according to claim 1, wherein the low temperature plasma irradiation is low temperature plasma irradiation generated by corona discharge or glow discharge in a low frequency or high frequency region. . 3. Low temperature plasma irradiation under vacuum, nitrogen, oxygen,
The packing material for reverse phase liquid chromatography according to any one of claims (1) to (2), which is carried out in an atmosphere in the presence of hydrogen, argon, helium, or air. 4. The total coating amount of the organosiloxane represented by (a) is 1.0 to 40% by weight based on the powder, and any one of claims (1) to (3). The packing material for reverse phase liquid chromatography according to item 1.