JPH0378133B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel chemically modified cyclodextrin stationary phase and high performance liquid chromatography using the same. Cyclodextrins have a hydrophobic cavity with a certain size and shape, and are known to have the interesting feature of inclusion of a wide variety of guest compounds ranging from organic and inorganic compounds to ions. There is. The present inventors have focused on the ability to recognize the shape and size of molecules through the inclusion effect, and have been conducting research on its application to liquid chromatography. That is, first, aromatic amino acids were separated using a polyurethane resin obtained by crosslinking cyclodextrin with various diisocyanates. Y. Mizobuchi, M. Tanaka and T. Shono,
J. Chromatogr., 208; 35 (1981). Next, in order to improve the separation ability, a polyacrylamide gel chemically bonded with cyclodextrin was synthesized, and the isomers of benzene disubstituted products were separated. M.
Tanaka, Y.Mizobuchi, T.Sonoda and T.
Shono, Anal. Lett., 14(A4), 281 (1981). Kawaguchi,
Mizobuchi, Tanaka, Shono, 30th Annual Meeting of the Society of Analytical Chemistry 1A13
(1981). Nakae, Kawaguchi, Mizobuchi, Tanaka, Shono, 45th Spring Annual Meeting of the Chemical Society of Japan 1T38 (1982). Next, in order to shorten the separation time, a cyclodextrin stationary phase using silica gel as a support was synthesized, and various aromatic compounds were separated. Kawaguchi, Mizobuchi, Tanaka,
Shono, 30th Annual Meeting of the Society of Analytical Chemistry 1A13 (1981). Kawaguchi,
Mizobuchi, Tanaka, Shono, 45th Spring Annual Meeting of the Chemical Society of Japan
1T39 (1982). Or, summarize the results of separation using chemically bonded cyclodextrin stationary phase.
Recently reported. Kawaguchi, Nakae, Mizobuchi, Tanaka,
Shono, 43rd Analytical Chemistry Symposium 1C15 (1982). Thus, the present inventors have focused on the selective clathrate-forming ability of cyclodextrin and have been developing a packing material for liquid chromatography using cyclodextrin. As a result, it was revealed that polyacrylamide gel and silica gel, in which cyclodextrin is immobilized by chemical bonds, are very effective for separating aromatic compounds. However, when simply using a conventional stationary phase in which cyclodextrin is immobilized by chemical bonds, the following problems (1) to (3) mainly exist. (1) It is impossible to separate the m-form and p-form of dinitrobenzene. (2) It is impossible to separate the o- and m-forms of toluidine. (3) In order to elute compounds with carboxy groups such as benzoic acid derivatives, it is essential to mix a phosphate buffer with the eluent due to the elution time. Kawaguchi, Nakae, Mizobuchi, Tanaka, Shono, 43rd Analytical Chemistry Symposium 1C15 (1982). As a result of intensive research in view of the above problems, the present inventors have found that such problems can be solved by selecting a stationary phase fixed on silica gel as a conventional stationary phase in which cyclodextrin is fixed by chemical bonding. By modifying the hydroxyl groups of cyclodextrin immobilized by chemical bonds on the silica gel stationary phase with acetyl groups, and using a new chemically modified cyclodextrin stationary phase, all of these problems can be solved. This heading led to the completion of the present invention. That is, in the present invention, the hydroxyl group of cyclodextrin immobilized on silica gel is acetylated by chemical bonding. A novel chemically modified cyclodextrin stationary phase and high performance liquid chromatography using the same. In order to understand the essence of the present invention, a comparative study was conducted to see how the retention behavior changes compared to before acetylation by chemically modifying the hydroxyl group of cyclodextrin after fixing cyclodextrin to silica gel through chemical bonding. The results of the research conducted are as follows. That is, the conventional chemically bonded cyclodextrin stationary phase (α-en-or β-en-SuAPS-
S. Kawaguchi, Nakae, Mizobuchi, Tanaka, Shono, 43rd Analytical Chemistry Symposium 1C15 (1982). ) with acetic anhydride in pyridine to form a chemically bonded cyclodextrin stationary phase (Ic-α-en- or Ac-
β-en-SuAPS-S. ) can be obtained. The obtained stationary phase (Ac-α-en-or Ac-β-
en-SuAPS-S. ) is shown in FIG. 1. The following two points are notable changes in retention behavior caused by acetylation. (1) It became possible to separate the isomers of dinitrobenzene and toluidine, which was impossible with β-en-SuAPS-S. See Figure 2. (2) Before acetylation, it was necessary to mix a phosphate buffer solution with the eluent to elute compounds with carboxyl groups, but after acetylation, elution with water became possible. These changes (1) and (2) are thought to be due to the fact that the hydroxyl groups of cyclodextrin were capped by acetylation and that the cyclodextrin internal pores became deeper. The essence of the present invention is to provide a chemically modified cyclodextrin stationary phase in which the hydroxyl group of cyclodextrin immobilized on silica gel by chemical bonding is chemically modified with an acetyl group, and a high performance liquid chromatography using the same. It shows that there is a graph. The invention particularly relates to all disubstituted benzenes such as dinitrilobenzene, cresol, toluidine, iodoaniline, nitroaniline, nitrophenol, nitrobenzoic acid, aminobenzoic acid, without exception to their respective o-, m- The present invention provides high-performance liquid chromatography that can easily separate p-isomers and a novel and extremely useful chemically modified cyclodextrin stationary phase for use therein, making a major contribution to this industry. There is. Examples are shown below. Example 1 Synthesis reaction formula of acetylated cyclodextrin stationary phase 1 Reference Cyclodextrin stationary phase (α-en- or β-en-
-SuAPS-S) 1.7g is placed in 10ml of dehydrated pyridine, 6ml of acetic anhydride is added, and then heated at 45°C for 6 hours. After filtering and washing thoroughly with methanol, heat at 80°C.
Dry under reduced pressure for 12 hours. The resulting stationary phase (Ac-α-en- or Ac-β-
The estimated structure of en-SuAPS-S is shown by the last structural formula of Reaction Formula 1. The synthesis reaction formula of the acetylated cyclodextrin stationary phase is shown below. Measurement method A 15 x 0.4 cm I.D. stainless steel column was used in all measurements. Other conditions were the same as in the case of the conventional cyclodextrin stationary phase in the comparative example. Chromatogram A typical chromatogram obtained using an acetylated cyclodextrin stationary phase is shown in Figure 4 (Ac
-α(β)-en-SuAPS-S). Ac−α−en−SuAPS
-S and Ac-β-en-SuAPS-S both elute with approximately symmetrical peaks, and good separation is obtained. Tables 1 and 2 show the retention times of various benzene disubstituted isomers in the acetylated cyclodextrin stationary phase. In this case as well, as with the cyclodextrin stationary phase, the retention time decreases as the methanol content in the eluent increases. Depending on the balance between separation time and resolution, the optimal eluent for isomer separation of these seven benzene disubstituted products is:
Ac-α-en-SuAPS-S is water, Ac-β-en-
It was determined by SuAPS-S to be methanol/water, 20/80. Under these conditions, Ac-β-en-SuAPS-S was able to completely separate isomers other than aminobenzoic acid and nitrobenzoic acid. In β-en-SuAPS-S, the m-form and p-form of dinitrobenzene and the o-form and m-form of toluidine are inseparable. Furthermore, when water was used as the eluent, aminobenzoic acid and nitrobenzoic acid could be completely separated into isomers. As a result, Ac
-β-en-SuAPS-S can be said to be optimal. Next, changes caused by acetylation will be described. Tables 3 and 4 show the retention times before and after acetylation in stationary phases with equal amounts of cyclodextrin immobilized. The results reveal the following changes. (1) Except for a few isomers, the acetylated cyclodextrin stationary phase has stronger retention. (2) The elution order may have changed due to acetylation. β-: Nitrophenol m, o, po,
m, p α-: Nitroaniline m, o, po,
m, p (Figure 5 a, b) (3) Before acetylation, when eluting compounds with carboxyl groups, it was essential to mix phosphate buffer into the eluent, but after acetylation, Elution became possible with water alone. (Figure 5c,
b). The above changes are thought to be due to the capping of the hydroxyl groups of cyclodextrin involved in hydrogen bonding by acetylation and the deepening of the cyclodextrin cavity.

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[Table] The following relates to conventional cyclodextrin stationary phases for reference purposes. A comparative experimental example is shown. 1 Synthesis method (see reaction formula 2) Silica gel with a carboxyl group at the end (SuAPS-S) synthesized by the usual method ⁽¹⁾
5g of mono[6-deoxy-6-[N-(2-
aminoethyl)〕amino〕−α−cyclodextrion(α
-en) ⁽²⁾ or mono[6-deoxy-6-[N-
(2-aminoethy)〕amion〕−β−
Disperse 2 g of cyclodextrin (β-en) ⁽³⁾ in 100 ml of 0.15M PH5.5 phosphate buffer (adjust to PH5.5 with IN HCl after dissolving α-en or β-en),
While stirring with a motor at room temperature, 1-
ethyl-3-(3-dimethylainopropyl)
Carbodiimide is added in the order of 1 g, 0.5 g, and 0.3 g at 2 hour intervals. After stirring for 24 hours,
Filter, wash well with deionized water, then
After washing with methanol, dry under reduced pressure at 80°C for 12 hours. (1) Methods in Enzymology, 34 , 59 (1974) (2) LDMelton and KNSlessor,
Carbohyd.Res., 18 , 29 (1971) (3) Y.Matsui, T.Yokoi and K.Mochida,
Chem. Lett., Oct., 1037 (1971) The synthetic reaction formula leading to immobilization of α- or β-cyclodextrin on silica gel is shown below. 2 Measurement method A stainless steel column of 15 x 0.4 cm I.D. (for cyclodextrin stationary phase) or 30 x 0.4 cm I.D. (for SuAPS-S used as a control) was used, and the equilibrium slurry method was used. Filling was carried out by The equipment used is as follows. Detector: Applied spectroscopic equipment UV detector UVILOG-7 Pump: Kyowa Seimitsu KHD-W-52 Damper: Kyowa Seimitsu KHP-UI-130A Injector: Kyowa Seimitsu KDI The eluent was water/methanol or 0.15M phosphate buffer ( PH4.7, 5.5, 6.8), and the mixing ratio (v/v) is 100/0, 90/10, 80/20, 70/
It was set to either 30 or 50/50. The flow rate is 1
ml/min (0.55ml/min only for amino acid separation)
min). All samples for separation were mixed solutions of 0.2mM each, and 20μ of each was injected. 3. Experimental Results An example of isomer separation of disubstituted benzene using a cyclodextrin stationary phase is shown in FIG. 6 (chromatogram of nitroaniline isomers). As can be seen from this chromatogram, good separation was achieved with the cyclodextrin stationary phase. Tables 5 to 7 show the retention times of various benzene disubstituted isomers. The following characteristics can be found from these results. (1) The inclusion effect of cyclodextrin plays a major role in retention. In ΓSuAPS-S (Table 8), the column length is twice as long, and even with water as the weakest elution condition, the separation is not very good, but the cyclodextrin stationary phase shows strong retention. Separation is also good. ΓWhen the inclusion equilibrium constants of cyclodextrin were measured for some solutes, the numerical order for the isomers matched the elution order. (2) The retention becomes weaker as the methanol content in the eluent increases. (Tables 5 and 6) The fact that the inclusion power of Γ cyclodextrin decreases with increasing methanol concentration,
This can be explained by its antiphase behavior. (3) Nitrophenols, especially carboxylic acids, which have solutes whose retention becomes weaker due to the presence of ions. (Table 7) (4) Retention time changes depending on the pH of the eluent. (Table 7) (5) Compounds with similar structures can be separated. (Figure 7: Amino acid chromatogram) 2 to 5 amino acids that cannot be separated by Γ SuAPS-S are well separated. The optimal eluent for isomer separation of the eight disubstituted benzenes used in this experiment is as follows. α-en-SuAPS-S: 0.15M PH5.5 phosphate buffer. Dinitrobenzene m- and p- are not separated. The longest time is 74 minutes for p-iodoaniline. β-en-SuAPS-S: Methanol/0.15M
PH5.5 phosphate buffer 30/70. dinitrobenzene m
-, p- and toluidine o-, m- were not separated. The longest time is 25 minutes for p-iodoaniline. In addition, in the case of β-cyclodextrin stationary phase,
Separation of phenols and aromatic alcohols (Chapter 8)
), it can also be used for the separation of aromatic carboxylic acids (Fig. 9), and the separation of monosubstituted naphthalenes is also possible (naphthol, naphthalene carboxylic acid, sodium naphthalene sulfonate).

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【table】 [Brief explanation of drawings]

Figure 1 shows the stationary phase (Ac-α-en- or Ac-β-
represents the estimated structure of en-SuAPS-S). However, Ac
−α−en−SuAPS−S, m=12, n=
5. For Ac-β-en-SuAPS-S, m=
14, n=6. Figure 2 a and b are respectively Ac-
The figure shows high performance liquid chromatograms of dinitrobenzene isomers obtained using β-en-SuAPS-S and β-en-SuAPS-S under the following separation conditions. Column: 15cm x 0.4cmI.D. Eluent: (a) Methanol/Water = 20/80 (b) Methanol/PH5.5 phosphate buffer = 30/70 Flow rate: 1ml/min Measurement wavelength: 254nm Sample: 0.2 mM, 20μ Figures 3a and b represent the structures of α-CD (α-cyclodextrin) and β-CD (β-cyclodextrin), respectively, shown in the predicted structures of Figure 1. Figure 4 a and b are Ac-α-en-SuAPS, respectively.
-S and Ac-β-en-SuAPS-S were used.

Represents a typical high performance liquid chromatogram of the formula. Figure 4 c and d are Ac-α-en, respectively.
-SuAPS-S and Ac-β-en-SuAPS-S were used.

Represents a typical high performance liquid chromatogram of the formula. Eluent: (a) (c) water (b) methanol/water = 20/80 Measurement wavelength: 254 nm Figure 5 a and b are Ac-α-en-SuAPS-
Using S and α-en-SuAPS-S

Represents the high performance liquid chromatogram of [formula]. Figures 5c and d are Ac-β-en-, respectively.
Using SuAPS-S and β-en-SuAPS-S

Represents the high performance liquid chromatogram of [formula]. Eluent: (a) (b) (c) water (d) methanol/PH5.5 phosphate buffer = 30/70 Measurement wavelength: 254 nm Figure 6 a, b and c are SuAPS-S, α-
Figure 2 depicts high performance liquid chromatograms of nitroaniline isomers using en-SuAPS-S and β-en-SuAPS-S. Eluent: (a) Water (b) PH5.5 phosphate buffer (c) Methanol/PH5.5 phosphate buffer = 30/70 Measurement wavelength: 254 nm Figure 7 a and b are SuAPS-S and β, respectively −en
-High performance liquid chromatogram of the following six amino acids using SuAPS-S is shown. amino acid: Eluent: (a) Water (b) PH6.8 phosphate buffer Measurement wavelength: 205 nm Figure 8 a and b are SuAPS-S and β-en, respectively.
- Phenols and aromatic alcohols using SuAPS-S [

[Formula] represents the high performance liquid chromatogram of n=0, 1, 2, 3]. Eluent: (a) Water (b) Methanol/PH6.8 phosphate buffer = 30/70 Measurement wavelength: 205 nm Figure 9 a and b are SuAPS-S and β-en, respectively.
-Aromatic carboxylic acids using SuAPS-S [

[Formula] represents the high performance liquid chromatogram of m=0, 1, 2, 3, 4]. Eluent: (a) water (b) methanol/PH6.8 phosphate buffer = 30/70 Measurement wavelength: 205 nm.

Claims (1)

[Claims] 1. The hydroxyl group of cyclodextrin immobilized on silica gel by chemical bonding is acetylated.
Novel chemically modified cyclodextrin stationary phase. 2. The hydroxyl group of cyclodextrin immobilized on silica gel by chemical bonding is acetylated.
High performance liquid chromatography using a novel chemically modified cyclodextrin stationary phase.