Anal. Chem. 1083, 55, 446-450
446
volume fraction of the binary solvent containing the organic S i in the complex mixture 0 phase ratio Subscripts m and s represent mobile phase and solute, respectively.
wi
APPENDIX The discussion will be limited to ternary solvents. It is assumed that they are prepared by mixing the binary systems BS1 and BSz composed of water and the organic modifier SI and water and the organic modifier Sz,respectively. The volume fraction of Si in BSi is x i , the volume fraction of Si in the ternary is X i and the volume fraction of BSi in the ternary is win Taking into account the fact that w1 wz = 1, it can be written that
+
wlxl
= XI
(1 - w1)xz = xz
(17) (18)
If the composition of one of the binary systems is fixed (for instance xl), then the composition of the other binary mixture and the value of w1 can be calculated by using the following equations: w1
= XJXl
(19)
x2
= x1Xz/(x1-Xl)
(20)
Equation 20 precludes that x1>
x1
(21)
and x1
> X d ( 1 - XZ)
(22)
Registry No. MeOH, 67-66-1; MeCN, 75-05-8;THF, 109-99-9; dioxane, 123-91-1.
LITERATURE C I T E D Jandera, P.;Colin, H.; Guiochon, G. Anal. Chem. lD82, 54, 435-441. Colln, H.; Krstulovic, A.; Yun, 2.; Gulochon, Q. J . Chromatogr., In press. Colin, H.; Guiochon, Q,;Jandera, P. Chromatographla,in press. Snyder, L. R.; Klrkland, J. J. I n Introduction to Modern Liquid Chromatography", 2nd ed.; Wiley-Interscience: New York, 1979. Glaich, J. L.; Kirkland, J. J.; Squire, K. M. J. Chromatogr. lD80, 199, 57-79. "Handbook of Chemistry and Physics", 61st ed.; CRC Press: Boca Raton, FL, 1980. McCormlck, R. M.; Karger, B. L. Anal. Chem. ID81, 52,741-744. Colin, H.; Schmlner, J. M.; Guiochon, G. Anal. Chem. lD81, 53, 625-632.
RECEIVED for review February 26,1982. Resubmitted August 19, 1982. Accepted November 18, 1982.
Retention Behavior of Some Aromatic Compounds on Chemically Bonded Cyclodextrin Silica Stationary Phase in Liquid Chromatography Kazuml Fujlmura, * Teruhisa Ueda, and Telichi Ando Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Sakyo-ku, Kyoto 606, Japan
The retentlon behavior of some aromatic compounds on silica gels wlth chemically bonded cyclodextrin molecules has been studied. The capacity factor of the sample generally increased by virtue of the specific Interaction between cyclodextrin units and sample molecules. The effects of (a) the substituents and thelr relative positlon on the benzene or naphthalene ring, (b) the spacer length, (c) the mobile phase, and (d) the variety of cyclodextrin were examlned. The parameters of the mobile phase were the kind of organic solvent used and the water content. The effect of addltlon of cyclodextrin to the mobile phase was also Investigated under conventional reversed-phase hlgh-performance liquid chromatography conditions In order to confirm the existence of inclusion equilibrium between cyclodextrln and sample molecules.
Cyclodextrins (CD's), which are known to be cyclic oligosaccharides consisting of six or more a-(1,4)-linked D-glucopyranose units, form inclusion complexes with a variety of organic molecules both in the solid state and in an aqueous solution. The stability of such inclusion complexes is, in general, most closely related to the fitness of the size of guest molecules to that of the cavity of cyclodextrin units, although many other factors such as van der Waals forces, dipole-dipole interaction, hydrogen bonding, and hydrophobic interaction
also play a role in determining the ease of complex formation (1-4). Many attractive features of cyclodextrins, such as found in covalent or noncovalent catalysis and enantiomeric catalysis, arise from these specific interactions between cyclodextrin units and guest molecules (1-4). The recent application of cyclodextrins as models for enzymes is also based on this specific inclusion property. In the field of chromatography, much attention is now being paid to the use of cyclodextrins as an additive in the mobile phase and/or the stationary phase bonded to a suitable support. Three review articles dealing with this subject have been published recently (5-7). In an attempt to use cyclodextrin as a stationary phase, several kinds of polymer gels have been prepared (6, 7). These polymer gels, however, require a long analytical time because of their low mechanical strength, and hence the application of these gels to modern high-performance liquid chromatography (HPLC) seems to be difficult. This paper describes a preparation of silica gels with chemically bonded cyclodextrins and the retention behavior of some aromatic compounds on these bonded silica gels. EXPERIMENTAL SECTION Reagents and Materials. a- and P-cyclodextrinsused were purchased from Nakarai Chemicals Co. (Kyoto, Japan). All aromatic compounds and silanes were of the highest quality available and were purchased from various suppliers. These
0003-2700/83/0355-0446$01.50/0C2 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
reagents were used with!out further purification. The silica gel used was a highly micro~porousPolygosil60-5 (Macherey-Nagel, Duren, West Germany). Methanol, acetonitrile, and tetrahydrofuran were of HPLC quality. Water was purified by means of ion-exchange followed by a Milli-Q water purification system (Millipore Corp., MA). Preparation of Silica Gels with Chemically Bonded Cyclodextrin. Diamino Type. Six grams of silica gels, which had been dried in vacuo at 1.50 "C for 6 h, was added to 150 mL of a 10% solution of [3-[(2~aminoethyl)amino]propyl]trimethoxysilane in dry toluene. The slurry formed was refluxed under nitrogen for 20 h with continuous stirring. The diamine-modified silica gels thus obtained were filtered, washed with toluene, acetone, and methanol in this order, and then dried in vacuo at 100 "C for 2 h.
( 4 - S 1-0- S i - ( C H Z13 NH,
>
NH2 + T s O G CH~CH~'
Two grams of a- or 6-CD was dried in a vacuum desiccator overnight and was treated with 4.0 g of p-toluenesulfonyl chloride in dry pyridine at room temperature for 6 h, to tosylate the primary hydroxyl groups. The resulting cyclodextrin tosylate, after being dried in vacuo at 50 "C for 2 h, was added to the slurry prepared from 2 g of the diamine-modified silica gels and 100 mL of dry pyridine. This mixture was continuously stirred at 70 "C for 40 h, to give diamina type silica gels with chemically bonded cyclodextrins. They were filtered, washed with pyridine, acetone, and methanol, and dried in vacuo at 60 "C for 2 h. Amino Type. The modified silica gels of the amino type were prepared in the same way as those of the diamino type, except for using (3-aminopropy1)trimethoxysilanein place of [3-[(2aminoethyl)amino]propyl]trimethoxysilane.
:(:-
pyridine
7 O o C , 4011
a
:i
T
?-Si- 0-Si-(C H2)3 "-6)
-
1
Apparatus, A Waters Associates Model 6000 solvent delivery system, equipped with a Rheodyne 7105 sample injector,was used in conjunction with a Jasco variable-wavelength detector, UVIDEC-100-11. The columns (4 mm i.d. X 20 cm or 15 cm) were packed by the modified viwcosity method, using a Chemco Model 24 slurry packing apparatus (Chemco Scientific Co. Ltd., Osaka, Japan) at ca. 500 kg/cm2. Procedure. Aromatic compounds were dissolved in methanol t o give a concentration of 1 wg/mL. The amount of the sample injected was usually of the order of nanograms. Experiments were carried out at room temperature. The flow rate was 0.7 mL/min, and the detector was operated at 254 nm. The mobile phase was filtered through a 0.7-pm membrane filter and degassed before use.
RESULTS AND DISCUSSION Effects of Addition of Cyclodextrin in the Mobile Phase on Retention. Cyclodextrins form inclusion complexes with various organic mcllecules in aqueous solutions. The solubility in water is 14.5 g/100 mL for a-CD and 1.85 g/100 mL for p-CD. In most organic solvents, their solubility is very small except for a limited number of solvents, such as dimethylformamide, pyridine, dimethyl sulfoxide, etc. Moreover, the existence of water is essential for the formation of inclusion complexes be tween cyclodextrin units and guest molecules. These facts imply that an aqueous solution must be selected as the mobilte phase in liquid chromatography.
5 5-
04
0 Concn of
45
6-CD
08 wt%
447
448
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
Table I. Comparison of Capacity Factors ( h ' )and Separation Factors (a)on Six Stationary Phases diamino type
k' 1
k',
non bondeda a-CD bonded p-CD bondeda
0.42 1.32 1.50
0.42 1.36
amino type
k' 1
k' 2
0.35
1.41 1.09
nonbondedb a-CD bonded p-CD bonded
a211
1.00
k'm
k' 0
aolm
C content, wt %
0.33 0.64
0.34
0.73
4.6 5.4 5.4
1.10
1.39
1.03 1.14 1.26
"211
k'nl
k',
aolm
C content, wt %
0.35 1.63
1.00
0.27
0.27
1.00
1.16
0.82
1.12
1.27
1.17
0.73 0.88
1.06
1.20
3.1 4.9 5.0
1.03 1.83
2.75
Column, 4 mm i.d. X 20 cm; mobile phase, methanol/water (20:80); flow rate, 0.7 mL/min. Column, 4 mm i.d. X 1 5 cm; mobile phase, methanol/water (20:80); flow rate, 0.7 mL/min. k I L ,k ' > ,k ' m , and k ' , denote the capacity factors of 1-and 2-naphthylamine and m- and o-nitroaniline, respectively. a
k'
log
1
k'r------
1: B
A
CH30H
A
B CH3CN
-0 2
B
A
THF
Flgure 2. Effect of organic solvents on retention of 1-naphthylamine 0)4 (0,0 )and 2-naphthylamine (0, m): column: (A) diamine (0, mm i.d. X 20 cm; (B) diamino-P-CD (0,D) 4 mm i.d. X 20 cm; mobile phase, organic solvent/water (20:80);flow rate, 0.7 mL/min.
gels of the diamino type (diamino-a-CD) and of the amino type (amino-a-CD),is most noteworthy, because it suggests that the stationary phase of this type is very promising for the separation of various compounds, even though more detailed study would be necessary to elucidate the effect of such factors as the type of modification, the chain length of the spacer, and the content of cyclodextrin. On the basis of the data in Table I, all subsequent experiments were carried out by using a diamino-P-CD column. Effect of Organic Solvents a n d Their Content i n Mobile Phase. The results of preliminary experiments concerning the elution conditions showed that none of the sample molecules could be eluted from a diamino-P-CD column within a reasonable time by water alone, indicating that an aqueous organic eluent is essential for the present system. Figure 2 shows the effect of organic solvents in the mobile phase on the retention of 1- and 2-naphthylamines on a diamino-p-CD and a diamine columns. The ratio of the organic solvent to water (20:80) was kept constant. If methanol was used as the organic solvent, the capacity factors of naphthylamines on the diamino-P-CD column were much greater than those on the diamine column and, moreover, the selectivity of the diamino-P-CD column was sufficiently high to resolve naphthylamine isomers under the conditions used. When acetonitrile or tetrahydrofuran (THF) was used, however, the capacity factors of the samples on the diamino-p-CD column were nearly equal to those on the diamine column, and neither of the two columns exhibited a high selectivity. Since it may be assumed that exchange occurs between molecules of the sample and molecules of the organic solvent or water during the process of inclusion complex formation, the above-described results show that methanol molecules are replaced by sample molecules more easily than are molecules of acetonitrile or THF. Therefore, a methanol/water mixture was chosen as a mobile phase in all subsequent experiments. Effect of Water Content i n Mobile Phase. The content of water in the mobile phase also affected the values of capacity factor on the diamino-P-CD column. Figure 3 shows a plot of the logarithm of the capacity factors of 1- and 2-
I
0
20 LO 60 Water content ,
80
100
Vi.v l v
Figure 3. Effect of water content (X) in mobile phase on retention of 0 )and 2-naphthylamine (0, U): column, diamine 1-naphthylamine(0, (0, 0)4 mm i.d. X 20 cm, diamino-P-CD (0,W) 4 mm i.d. X 20 cm; mobile phase, methanoVwater ((100 - X ) : X ) ;flow rate, 0.7 mL/min. ( a )
(bi
( C !
k' I k'
k' I o*
r i O A
B
M A
B
d O
' M
A
B
Figure 4. Retention behavior of benzene derivatives on diamine (A) and diamino-P-CD (B) columns: column size, 4 mm i.d. X 20 cm; moblle phase, methanoVwater (20:80);flow rate, 0.7 mL/min.
naphthylamines against the contents of water in the mobile phase. On the diamine column, the logarithms of the capacity factors were nearly constant within the range of the contents of water investigated. On the diamino-p-CD column, however, an increase in the content of water was accompanied by an increase in the capacity factor as well as in the separation factor. This finding is in accord with the fact that water is indispensable for the formation of inclusion complexes between cyclodextrin units and guest molecules. Retention Behavior of Benzene Derivatives. Figure 4 lists the capacity factors of some benzene derivatives, measured on diamine and diamino-p-CD columns using a methanol/water (20:80) mixture as eluent. As had been expected, all the samples tested showed much larger capacity factors as well as separation factors on the diamino-6-CD column than on the diamine column. The magnitude of the capacity factor on the diamino-p-CD column, which should reflect the magnitude of the equilibrium
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983 Nitrotoluene
r-----l1
k’
4
Iodatohene
Xylene
k’1
1 k’ - 1
I
3.0
3.0
7
4 /// I
I
R = A
B
A
3
449
6
A
Flgure 5. Retention behavior of nitrotoluene, iodotoluene, and xylene isomers on diamine (A) and diamino-P-CD (B) columns. Chromatographic conditions are the same as those given in Figure 4. Nitroanill n e
Iodoaniiine
-H
-NH2
-CN
-NO2
-CY3 -N(CH$Z-OH
Figure 7. Retention behavior of 1- (0, 0 ) and 2-substituted (0, m) naphthalenes on diamine (0, 0) and diamino-P-CD ( 0 ,m) columns. Chromatographic conditions are the same as those given in Figure 4.
Toiuldine
40
P-
i
/
i
Ao-
/
/ I
0-
0-
A
3
i
i
A
E
0A
B
Figure 6. Retention behavior of nitroaniline, iodoaniline, and toluidine isomers on diamine (A) alnd diamino-P-CD (e) columns. Chromatographic conditions are the same as those given in Figure 4.
constant for the inclusion complex formation, is highly dependent on the number and the kind of the substituents; the introduction of a nitro, hydroxyl, or iodo substituent into benzene has an effect of increasing the capacity factor, while that of a methyl or amino substituent decreases it. The effect of the nitro group may be explained by the fitness of its size to the cyclodextrin cavity, and that of the hydroxyl group, by the formation of hydrogen bond with the secondary hydroxyl group on the rim of the cyclodextrin cavity (1-4,6-8). The increase in capacity factor of iodophenol, which is most probably due to the hydrophobicity of the iodo group, is in contrast with the decrease in capacity factor of aniline, caused by the greater hydrophilicity of the amino group (1-4). The k’-value-increasing effect of the nitro and the iodo groups and the k ’-value-decreasing effect of the methyl and the amino groups are clear from Figures 4c, 5, and 6, respectively; the introduction of the former group into phenol, toluene, or aniline, especially a t their 4-position, results in an increase in k’, whereas the introduction of the latter group leads to a substantial decrease in K’. As Figure 4b shows, the introduction of two methyl groups at 1 and 6 positions OS phenol brings about a decrease in k’. This indicates the existence of the effect of steric hindrance in complex formation. The larger k’ values of para-disubstituted benzenes than those of meta or ortho isomers are also probably related with the absence of steric hindrance in the former compounds. Tlhe above-mentioned retention behavior of benzene derivatives on diamino-P-CD bonded phase was quite different from that observed on conventional alkylbonded reversed phase; for example, when a column of 4 mm i.d. x 20 cm, packed with LiChrosorb RP-8, and a methanol/water (1:1) mixture was used, the elution orders of the compounds corresponding to Figure 4a-c were aniline 5 phenol benzene < nitrobenzene < toluene, phenol < o-cresol < 2,6-xylenol, and 0- C m- = p-iodophenol, respectively, and
-
I
,
I
0
2
4
v -
0
2
4
6
8
1
0
>
6
,
8
Time, minutes
Figure 8. Separation of nitroaniline (a) and cresol (b) isomers on diamino-P-CD column: column size, 4 mm i.d. X 20 cm; mobile phase, methanoVwater (20:80); flow rate, 0.7 mL/min.
hence only partial separations could be achieved, except for a mixture of phenol, o-cresol, and 2,6-xylenol. The elution orders of the compounds corresponding to Figures 5 and 6 on a similar reversed-phase system as described above were o5 p - < m-nitrotoluene, 0- 5 m- p-xylene, p- < m- < onitroaniline, o- ir m- 5 p-iodoaniline, and 0- < m- < ptoluidine, respectively, and besides, none of the iodotoluene isomers could be eluted within 1 h. Retention Behavior of Naphthalene Derivatives. As is shown in Figure 7, the k‘values of all naphthalene derivatives tested on a diamino-P-CD column are three times or more as large as those observed on a diamine column. The magnitude of the increase in k’values of naphthalene derivatives on a diamino-p-CD column is greater than that of benzene derivatives. This fact demonstrates that naphthalene molecules fit the 0-CD cavity better than benzene derivatives. Figure 7 also reveals that 2-substituted isomers show larger k’values than 1-substituted isomers. This suggests that there is a larger steric hindrance for the inclusion complex formation between P-CD and 1-substituted naphthalene derivatives than 2-substituted isomers. It seems that all 1-substituted naphthalene derivatives tested, except 1-naphthol, show smaller k’ values than naphthalene itself, whereas 2-substituted isomers, except 2-naphthonitrile, show larger k ’values than naphthalene. This suggests a larger steric hindrance being present for inclusion of 1-substituted naphthalenes into the P-CD cavity than for inclusion of 2-substituted naphthalenes. The larger k’ values for 1-and 2-naphthol may be attributed to the formation of hydrogen bonding similar to those in the case of phenol, and the small k’values for 1- and 2-naphthonitrile as compared with naphthalene may be attributed to the high electron
-
450
Anal. Chem. 1983, 55, 450-453
0 2
4
6
8
10
0 2 4 Time,
6
8
0
2
4
6
8
101214
minutes
Figure 9. Separation of naphthylamine (a),N,Ndimethylnaphthylamine (b), and nitronaphthalene (c) isomers. Chromatographic conditions are
the same as those given in Figure 8, except for mobile phase for (b), methanoVwater (40:60). density of the cyano group as well as of the inside of the cyclodextrin cavity (1). In a reversed-phase system, however, no pairs of isomers such as shown in Figure 7 could be separated by a column of LiChrosorb RP-8 and methanol/water (1:l). Chromatographic Separation. The performance of the P-CD-bonded column was tested by attempting a chromatographic separation of the isomers of some typical compounds, and satisfactory results were obtained. Though it is generally difficult to separate nitroaniline isomers by a conventional RP-HPLC system if the pH of the eluent is not adjusted, they could be separated completely on a diamino-0-CD column by using a mixture of methanol/water (20:80) as eluent without adjusting the p H of the eluent, as shown in Figure 8a. Cresol isomers could also be separated easily by the present system as shown in Figure 8b, though the RP-HPLC system failed to separate the ortho and the meta isomers. The order of elution of monosubstituted benzene isomers on the CD-bonded column is generally ortho < meta < para
and is the reverse of the one observed when alkyl-bonded silica and aqueous organic solvent containing cyclodextrin are used as stationary and mobile phase, respectively. It reflects that the sorption is based on the inclusion complex formation. An exception is the order of elution of nitroaniline isomers, which is meta < ortho < para on a CD-bonded column and different from the one (para < meta < ortho) observed when cyclodextrin is used as eluent. The reason for this apparent anomaly is not clear at present. For naphthalene derivatives, each pair of isomers could be separated successfully except for naphthol and naphthonitrile isomers. A diamine column or a conventional alkyl-bonded silica column could not achieve the separation of these isomers. As Figure 9 shows, 2-substituted isomers are, without exception, eluted after 1-substituted isomers, demonstrating the easier formation of inclusion complexes of the former with P-cyclodextrin. In conclusion, an attempt was made successfully to bond cyclodextrin molecules chemically on the surface of porous silica gels for HPLC. The silica gels thus obtained showed a characteristic property of cyclodextrin of forming inclusion complexes with various organic compounds, just as cyclodextrin itself does in aqueous solution. Registry No. Cyclodextrin, 12619-70-4; 0-cyclodextrin, 7585-39-9;1-naphthylamine,134-32-7;a-cyclodextrin,10016-20-3.
LITERATURE CITED (1) Bender, M. L.; Komiyama, M. "Cyclodextrin Chemistry"; Springer-Ver-
lag: New York, 1978. (2) Cramer, F . ; Hettler, H. Naturwissenshaften 1967, 5 4 , 625-632. (3) Bergeron, R. J. J . Chem. Educ. 1977, 5 4 , 204-207. (4) Saenger W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362. (5) Smolkov6-Keulemansov& E.; Krqsl, S. J . Chromatogr. 1980, 184, 347-361. (6) Hinze, W. L. Sep. f u r i f . Methods 1981, IO, 195-237. (7) SmolkovB-Keulemansov6, E. J . Chromatogr. 1962, 251, 17-34. (8) Gelb, R. I.; Schwartz, L. M.; Cardelino, B.; Fuhrman, H. S.;Johnson, R. F.; Laufer, D. A. J . A m . Chem. SOC.1961, 703, 1750-1757.
RECEIVED for review August 2, 1982. Accepted November 4, 1982.
Determination of Alkylmercury in Seawater at the Nanogram per Liter Level by Gas Chromatography/Atmospheric Pressure Helium Microwave-Induced Plasma Emission Spectrometry Koichi Chlba, Kazuo Yoshida, Klyoshl Tanabe,' Hlroki Haraguchl, * and Keiichlro Fuwa Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 713, Japan
The determination of alkylmercury compounds has been Investigated by atmospheric pressure hellurn mlcrowave-lnduced plasma emisslon spectrometry combined with gas chromatography. The detection llmits for CH,HgCI, C,H,HgCI, and (CH,),Hg were 0.09, 0.12, and 0.40 pg/L, respectively, In the case of peak height analysls, and the dynamic ranges for these compounds were more than 4 orders of magnltude. Furthermore, they could be detected separately by the above GC/MIP system. I n addition, the present method has been applied to the determination of alkylmercury compounds In seawater, where the preconcentratlon of the seawater samples was performed with a benzene cysteine extractlon.
Present address: National Institute for Public Health, Minato-
ku, Tokyo 108, Japan.
In recent years, a sensitive and accurate determination of organomercury compounds has been required in biological and environmental fields. These mercury compounds are more toxic than metallic mercury, and they may cause serious illness in extremely polluted areas. I t has been reported that organomercury compounds are significantly concentrated in fish ( 1 4 3 , predominantly as methylmercury compounds ( 2 , 7). The syntheses of methylmercury compounds by microorganisms in freshwater sediments have been investigated by some workers (8, 9). There are, however, only a few reports on the determination of methylmercury compounds in seawater (10, 11). This may be because the concentrations of organomercury 'compounds in seawater are generally much lower than those of inorganic mercury compounds. The most widely accepted method for the determination of methylmercury compounds is based on the Westoo's pro-
0 1983 American Chemical Society 0003-2700/83/0355-0450$01.50/0
