JP2592519B2 - Reversed phase packing for open columns - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a reversed-phase packing material for open columns, and in particular, the inside of pores of silica gel is made hydrophobic while the outer surface is made partially hydrophilic. The present invention also relates to a reversed-phase packing material which is preferably used for open column chromatography and has improved affinity for polar solvents.

(Background Art) Conventionally, open column chromatography has been
The separation column can be adjusted simply by filling the chromatographic tube with the filler and allowing the developing solution to flow naturally, and the separation can be performed immediately afterwards. It has been widely used as a simple purification method for samples because it can be transferred to the preparative conditions of open column chromatography, and it does not require special equipment such as a liquid feed pump in addition to chromatographic tubes. ing.

Chromatographic techniques that can be applied to this open column chromatography can be broadly classified generally using a low-polarity developing solvent such as hexane, benzene, chloroform, and ethyl acetate to form a highly polar silica gel, alumina, Normal phase chromatography using a filler such as cellulose powder, and a highly polar developing solution such as methanol, acetonitrile, water, a buffer solution, or a mixture of a water-soluble organic solvent and water, and a low-polarity, hydrophobic Although there is reverse phase chromatography with high packing, most of the packing actually used for open column chromatography is normal phase packing. This is because the packing material used for normal phase chromatography is inexpensive, high stereoselectivity can be obtained in separation, the retention time can be easily adjusted by changing the composition of the developing solution, Is often volatile, and the reason is that sample processing after fractionation is easy.

By the way, when performing reversed-phase chromatography using a reversed-phase packing material in open column chromatography, normal-phase chromatography is not a problem, affinity with the developing solution is important, and it is used. The developer may be restricted.

That is, the types of developing solutions that can be applied to reverse phase chromatography are extremely wide, but developing solutions that are generally used are:
Usually, the composition is an aqueous solution containing an organic solvent at about 10 to 80%, and the separation and the retention time are adjusted by changing the concentration of the organic solvent. When a highly polar developing solution is used, a very serious problem that a phenomenon in which the filler floats in the developing solution occurs, and therefore, in the open column chromatography using the reversed-phase type packing material, the Therefore, the retention time and the separation cannot be freely adjusted, and thus the reversed-phase filler is avoided.

As described above, the cause of the floating of the reversed-phase packing material in the developing solution is the same as that of the low-polarity oil floating in the highly polar water, and the packing material for reversed-phase column chromatography is entirely used. Because the surface is constituted by introducing a hydrophobic group, the polarity is low, and by having high hydrophobicity, when the water content of the developing solution increases, the reversed-phase filler and This is because the affinity with the developing solution is reduced, and the filler does not adapt to the developing solution, but floats, which causes a problem that normal chromatography becomes difficult. For this reason, a method using a filler having a high polarity has been considered, but increasing the polarity of the filler decreases the hydrophobicity as a filler for reverse phase separation, and as a filler for separating a sample. Is inappropriate.

However, reversed-phase packings are less susceptible to the effects of moisture in the air during chromatography than normal-phase packings. Therefore, it has the characteristic that the range of samples to which separation can be applied is wide. When packing is used in a sealed column as in high performance liquid chromatography, reverse phase chromatography is more widely used than normal phase chromatography.

For this reason, there is a demand for the development of a packing material for an open column that can be applied to reverse phase chromatography separation capable of separating a wider range of samples and has no restriction on the developing solution.

(Problem to be Solved) Here, the present invention has been made in view of such circumstances, and a problem to be solved is that a wide range of developing liquid can be applied to the above-described reversed-phase filler. In order to make it a filler, it partially hydrolyzes hydrophobic groups on the surface of the filler to improve the affinity of the filler for highly polar solvents, that is, to make it hydrophilic, and it is suitable for open column chromatography. Is to make it applicable.

(Solution) In order to solve the above-mentioned problems, the present invention provides a method in which a hydrophobic organic group bonded to the inner and outer surfaces of a porous silica gel having pores is subjected to hydrolysis treatment in an acidic medium by a hydrolysis treatment. The porous silica gel is removed at a removal rate of 35% to introduce a hydrophilic group in place of the hydrophobic organic group, and the inside of the pores of the porous silica gel is made hydrophobic. Characterized in that the outer surface of the silica gel is partially made hydrophilic by the introduction of the compound, and a part of hydrophobic organic groups to be bonded to the outer surface is left. Mold filler is the gist of the invention.

(Specific Configuration) By the way, in order to obtain the reversed phase filler according to the present invention, first, a porous silica gel having ordinary pores is
Hydrophobic organic groups are bonded to the inner and outer surfaces by a reaction with a predetermined silylating agent.The hydrophobic organic groups bonded to and introduced into the silica gel are used as a filler for an open column. Examples thereof include an alkyl group such as a propyl group, an octyl group and an octadecyl group, a cyanoalkyl group such as a cyanopropyl group, and an aminoalkyl group such as an aminopropyl group. As the silylating agent that provides such a hydrophobic organic group, various known ones can be employed, and the reaction conditions are appropriately determined according to the selected silylating agent.

Next, the silica gel having the hydrophobic organic groups bonded to its inner and outer surfaces is subjected to a treatment operation in an acidic medium, generally in an aqueous acid solution, and the hydrophobic organic groups bonded to the outer surface of the silica gel are partially removed. Hydrolyzed,
By being removed at a removal rate of 5 to 35% (based on the hydrophobic organic groups bonded to the entire surface), a part of the desired hydrophobic organic groups bonded to the outer surface remained. A reversed phase filler is obtained. In addition, a hydrophilic group (silanol group) is formed on the outer surface of the silica gel by this hydrolysis.

Examples of the acid used for the treatment with the acidic medium include mineral acids (inorganic acids) such as hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid. If necessary, such an acidic medium may be silica gel. To increase the affinity with, with water,
A solvent such as methanol is mixed. In addition, as an operation method in the hydrolysis treatment, various known methods are appropriately adopted, for example, a reflux operation using a concentrated acid aqueous solution under vigorous stirring, and the like.

Then, after such a hydrolysis treatment, if necessary, a hydrophilic group (silanol group) on the outer surface of the silica gel is used.
Then, by introducing a hydrophilic group having a diol group or the like, partial hydrophilicity on the outer surface of the filler is enhanced.

By the way, as described above, the filler according to the present invention is one in which the hydrophobic organic groups bonded to the outer surface of the filler are partially removed by hydrolysis. 5 to 10% (based on hydrophobic organic groups), the affinity for polar solvents is markedly improved,
If the developing solution contains an organic solvent such as methanol or acetonitrile, it shows sufficient affinity and sedimentation properties, while the developing solution having extremely high polarity is used alone with water or contains a salt such as a buffer solution. With respect to the developing solution, particles floating in the developing solution may be observed in a filler having a removal rate of 5 to 10%. Therefore, when a high-polarity developing solvent is used, the degree of exfoliation of the outer surface is further reduced. Increase, preferably 25-
It is desirable to remove about 30%.

In the case of the partially hydrophilic reverse phase type filler as in the present invention, when the hydrophobic stationary phase (hydrophobic organic group) existing on the entire surface of silica gel is removed by more than 35%, the filler (silica gel) is removed. However, since the problem that the hydrophobicity of the silica gel is reduced and the solubility of the silica gel is promoted arises, the removal rate is in the range of 5 to 35%.

In short, in reversed-phase chromatography, the proportion of the stationary phase introduced into the silica gel as the filler determines the hydrophobicity of the filler and affects the retention time and the separation ability of the sample. When the introduced hydrophobic group is small, the hydrophobic interaction, which is a separation mechanism of reverse phase chromatography, decreases, and the retention time of the sample is shortened, so that sufficient separation may not be obtained.
For this reason, it is desirable to introduce as much stationary phase as possible in order to achieve an efficient separation in a short column, ie using a smaller amount of packing material. Therefore,
Also in the partially hydrophilic filler according to the present invention, unnecessarily exfoliation of the stationary phase causes a decrease in the hydrophobicity of the filler, which leads to a decrease in the separation ability based on the hydrophobicity, and therefore, is present on the outer surface portion. It is desirable that the hydrophobic group be left to such an extent that the affinity for the polar solvent is not impaired.

Further, when a chromatographic filler using silica gel as a carrier is used as in the present invention, it is necessary to always consider the stability of the carrier. That is, silica gel is a relatively hardly soluble carrier as long as the solvent used is not alkaline, but a slight solubility poses a problem when passing through a solvent as in chromatography. This can also be understood from the fact that when a mobile phase containing water is sent to a silica gel column for normal phase, silica gel gradually dissolves, causing a decrease in the number of theoretical plates and a split in a peak. In contrast, reversed-phase fillers such as _C18- type fillers can be used stably even in the use of water-containing solvents. From the viewpoint of the stability against dissolution of the silica gel, it is preferable that the ratio of removing the hydrophobic group is small, and it is considered that the reduction in the exposed portion of the silica gel caused by the separation of the stationary phase contributes to the stability of the filler.

(Examples) Hereinafter, some examples of the present invention will be shown to clarify the present invention more specifically. However, the present invention imposes some restrictions by the description of such examples. It goes without saying that you don't receive anything.

In addition, in addition to the following examples, the present invention, in addition to the above-described specific description, various changes, corrections, and modifications based on the knowledge of those skilled in the art without departing from the spirit of the present invention. It should be understood that improvements can be made.

Parts and percentages shown below are based on weight unless otherwise specified.

Example 1 A porous silica gel having a particle size of 230 to 400 mesh was heated and dried at 150 ° C. for 3 hours under reduced pressure of 200 mmHg in advance.

Next, 10 parts of the obtained silica gel was added to octadecyldimethylchlorosilane as a silylating agent.
The reaction was carried out by refluxing for 6 hours in toluene with 3 parts of pyridine using 3 parts of pyridine as a catalyst, and the octadecyl group was introduced to the entire inner and outer surfaces (inner and outer surfaces of the pores) of the silica gel. ODS (octadecyldimethylsilylated) silica ( _C18 type filler) was synthesized.

Then, the obtained COS silica: 10 parts, 11N hydrochloric acid:
The mixture was put into 200 parts, and a hydrolysis reaction was carried out under a heating temperature of 100 ° C. while stirring vigorously (about 1000 rpm).

To check the removal status of hydrophobic organic groups (ODS) in this hydrolysis operation, silica gel was sampled out every hour and wet packed into a stainless steel column with an inner diameter of 4.6 mm and a length of 150 mm. After filling by the method, a change in the capacity ratio of toluene: k 'was measured using a solvent having a composition of methanol: water = 60: 40 (volume ratio) as a mobile phase. Shown in the figure.

In addition, since the evaluation conditions of the filler were measured under reversed-phase conditions, the k 'value of toluene tends to decrease as the octadecyl group that expresses the hydrophobicity of the filler is removed. From the change in the capacity ratio, the removal rate of the silylating agent can be estimated.

That is, as is apparent from FIG.
It is believed that the hydrolysis reaction on the outer surface of the filler is largely completed within about or about 5 hours, since it rapidly decreased by time and then decreased very little thereafter. Therefore, in the present invention, in order to leave the octadecyl group on the outer surface of the silica gel, it is necessary to adopt a condition within 5 hours under the hydrolysis conditions.

Example 2 Regarding the silica gel obtained in Example 1 in which the octadecyl group was introduced to the entire inner and outer surfaces and the silica gel in which the octadecyl group of the outer surface was partially hydrolyzed and removed, the respective carbon contents were determined. Upon measurement, the results shown in Table 1 below were obtained.

As a result, the carbon content of the filler whose surface was partially exfoliated by hydrolysis was reduced by only 5% as compared with the filler having an octadecyl group introduced on the front surface, and relatively many octadecyl groups remained. It was confirmed that it was doing.

Example 3 Using the partially hydrophilic reverse phase filler (hereinafter abbreviated as PHS) obtained in Example 2 by partially hydrolyzing the surface silylating agent (C ₁₈ ) with a hydrochloric acid catalyst. Performing an evaluation on the affinity of such a filler for a solvent,
The results are shown in Table 2 below. Also, as a comparison,
General reversed phase packing material (C ₁₈ type, C ₈ type, C ₁ type filler) used to evaluate in the same manner. Incidentally, such a C ₁₈ type filler is silica gel wherein the octadecyl group is introduced on the entire surface which was not subjected to hydrolysis, also C ₈ type, C ₁ type filler,
Each was synthesized using octyldimethylchlorosilane and trimethylchlorosilane as silylating agents.

And as an evaluation method of these fillers, methanol-containing water prepared by changing the ratio of water:
Each of the above-mentioned various fillers was added, and the filler was shaken to uniformly disperse the filler. After that, the mixture was allowed to stand, and the sedimentation state of each filler was observed.

As apparent from the results, C ₁₈ type, C ₈ type, in type C ₁ of each filler, the methanol content lower than 60%, i.e. the content of water is increased than 40%, the filling It turned out that the agent floated with respect to the solvent and could not be used as a filler for column chromatography.

On the other hand, the partially hydrophilic reversed phase filler (PHS) shows a complete affinity for water that does not contain methanol at all, so a very polar solvent must be used. It was confirmed that it can be used favorably as a packing material for the reversed-phase column chromatography used.

Example 4 The column chromatography of theobromine and theophylline was studied using the partially hydrophilic reversed-phase packing material used in Example 3. As an evaluation method, the theobromine and theophylline were simultaneously injected into a packed column for analysis and evaluation, and the results are shown in FIG. 20% methanol was used as a developing solution.

Generally, theobromine and theophylline are drugs having relatively high hydrophilicity, and their molecular structures are very similar, and the only difference between the two samples is the presence or absence of one methyl group. When column chromatography separation is applied to the difference between the methyl groups, which are hydrophobic groups, recognition by normal-phase chromatography is difficult, while reverse-phase chromatography, which has a high ability to recognize hydrophobicity, is difficult.
Due to the high hydrophilicity of the sample, a developing solution having an organic solvent concentration at which the filler does not float is not retained by the filler, and is difficult to separate by column chromatography.

However, as is evident from the results in FIG. 2, the analysis using the partially hydrophilic reversed-phase filler according to the present invention showed that theobromine and theophylline could be separated.

(Effects of the Invention) As is clear from the above description, in the filler according to the present invention, the hydrophobic organic groups bonded to the inner and outer surfaces of the porous silica gel are converted to 5 by hydrolysis in an acidic medium. ~
Since the silica gel has been removed at a rate of 35% and the outer surface of the silica gel has been partially hydrophilized, even when using a developing solution containing no organic solvent in open column chromatography, the packing No suspension could be caused, and all mobile phases usable in reversed-phase high-performance liquid chromatography became applicable. In addition, since the hydrophobic groups remain on the inner surface of the silica gel, the filler also has sufficient hydrophobicity, thereby making it possible to apply reverse phase chromatography.

[Brief description of the drawings]

FIG. 1 is a graph showing a change with time of a capacity ratio of toluene: k ', showing a progress state when an octadecyl group is hydrolyzed and removed using hydrochloric acid as a catalyst. FIG. 5 is a chromatogram of theobromine and theophylline when the partially hydrophilic reversed-phase filler according to Example 1 is used.

Claims (1)

(57) [Claims] 1. The method according to claim 1, wherein the hydrophobic organic groups bound to the inner and outer surfaces of the porous silica gel having pores are removed at a removal rate of 5 to 35% by a hydrolysis treatment in an acidic medium. While a hydrophilic group is introduced in place of the organic group, and the inside of the pores of such porous silica gel is made hydrophobic, the outer surface of the silica gel becomes partially hydrophilic by the introduction of the hydrophilic group. A reversed-phase packing material for an open column, characterized in that a hydrophobic organic group to be bonded to an outer surface thereof is partially left.