Table 11. Comparison of Plate Heights for Three Different Modifications of the Bottom Surface of the Channel Surface modification
H , cm
Flow, cmis
Smooth Mylar surface 120-Grit scratches Random cut Mylar surface
0.16 0.32
0.105 0.097 0.100
0.35
flow pattern observed for a smooth glass plate column is illustrated in Figure 9A. The edge retardation is clearly demonstrated. The trailing streams of solute a t the edges clearly broaden the overall solute zone. An attempt was made to reduce the edge effect. The sample injection port was removed from the tapered end where solvent enters and placed at the point where the channel first attains its nominal breadth. In this configuration, the solute was isolated from the two edges by a stream of solute flowing past on each side. The solute zone was carried down the center of the channel occupying approximately one-fourth of the column breadth. The plate height was reduced nearly 20% by this modification. Results for several surface-modified columns with standard inlets are shown in Table 11. The first has a smooth Mylar surface on the bottom plate; this can be considered as the standard system. The channel with a Mylar strip 0.0254 mm thick down the center is not included in this table because it created such radical differences in the flow velocity between the center and edge regions that two completely resolved peaks were produced. The effect of the randomly cut Mylar surface is illustrated in Figure 9B. Erratic distortions in zone displacement are evident.
The results of these studies show very conclusively that any significant uneveness in the column surface will distort the flow pattern and contribute significantly to peak broadening. In a working system with an applied field, the effect would be expected to be even greater as the solute would be compressed into a layer very near the surface of the bottom plate.
LITERATURE CITED M. E. Hovingh, G. H. Thompson, and J. C. Giddings. Anal. Chem., 42, 195 (1970). E. Grushka, K. D. CaMweil, M. N. Myers, and J. C . Giddings in "Separation and Purification Methods", Vol. 2, E. S. Perry, C. J. Van Oss, and E. Grushka, Ed., Dekker, New York, N.Y., 1974, p 127. K. D. Galdwell, L. F. Kesner, M. N. Myers, and J. C.Giddings, Science, 176, 296 (1972). M. N. Myers, K. D. Caidweli, and J. C. Giddings, Sep. Sci., 9, 47 (1974). J. C. Giddings, F. J. F. Yang, and M. N. Myers, Anal. Chem., 46, 1917 (1974). F. J. F. Yang, M. N. Myers, and J. C. Giddings, Anal. Chem., 46, 1924 (1974). J. C . Giddings. F. J. F., Yang, and M. N. Myers, Sep. Sci., 10, 133 (1975). J. C.W i n g s , F. J. Yang, and M. N. Myers, Anal. Chem.,48, 1126 (1976). J. C . Giddings, K. D. CaMweli, and M. N. Myers, Macromolecules, 9, 106 (1976). J. C. Giddings, S e p . Sci., 1, 123 (1966). J. C. Giddings, J . Chem. Phys., 49, 81 (1968). J. C.Giddings, J . Chem. f d u c . , 50, 667 (1973). J. C.G i i n g s , Y. H.Ywn, K. D. Galdwell, M. N. Myers, and M. E. Hovingh, Sep. Sci., 10, 447 (1975). S. Krishnamurthy and R. S. Subrarnanian, Sep. Sci., 12, in press. J. C.Giddings, G. C. Lin, and M. N. Myers, Sep. Sci., 11, 553 (1976). J. C . Giddings, F. J. Yang, and M. N. Myers, J . Vim/., 21, 131 (1977). J. C. Giddings, Y,.,H. Ywn, and M. N. Myers, Anal. Chem., 47, 126 (1975). J. C. Giddings. Dynamics of Chromatography. Part 1. Principles and Theory", Dekker, New York, N.Y., 1965. J. C. Giddings, Sep. Sci., 6, 567 (1973).
RECEIVED for review April 1, 1977. Accepted June 6, 1977. This investigation was supported by National Science Foundation Grant CHE76-20870.
Separation of Aromatic Amine Isomers by High Pressure Liquid Chromatography with a Copper(l1)-Bonded Phase Francis K. Chow and Eli Grushka" Department of Chemistry, State University of New York at Buffalo, Buffalo, New York
Cu( 11) ion was coordinated to y-aminopropyltrlmethoxysiiane which was bonded to Partisii-IO. Six different groups of aromatic amine Isomers were separated by a column packed with such support using methanoVcyciohexane mobile phase. By controlling the polarity of mobile phase, each group of isomers can be separated in less than 8 mln. Resuits were compared with elution on a partlsli-10 column and on a NH2 column. I n most cases, the copper column gave the best resoiutlons* 'lots Of PKb Of the amines vs. log k'were The correlation Indicated that the interaction between Cu2+ and the was responsibie for the chromatographic behavior. The basicities of the amines most likely are not the only important parameter controlling the retention.
Interaction with metal ions have been used advantageously in liquid chromatography. Helfferich ( I ) , in 1961, discussed the concept of ligand-exchange chromatography. Since then many separations have been achieved using ligand-exchange chromatography with Cu(II), Co(II), Ni(II), Cd(II), Zn(II), etc. attached to the solid support. Walton (2, 3 ) has recently 1756
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
74274
reviewed the technique quite extensively. The solid supports commonly used in ligand exchange are resins with sulfonate, carboxYla% Or iminoacetic groups which coordinate the metal ions. A larger number of compounds have been separated by ligand-exchange ckromatography; for example, D,L-aminO acids (4-81, amino Sugars (9,l o ) , aziridine and ethanolamine ( 1 1 ) , and alkyl amines ( I 2 ) . In cases a counter ligand, such as phase in Order to "37 must be present in the the solutes. Thermodynamic and kinetic studies of ligandexchange c ~ o m a ~ o g r a p can h y be found in the literature (viz., 8, 13, 14). Although the method is very selective, the use of ion-exchange resins can pose some difficulties, the major one of which is the bead,s lack of mechanical strength, As a consequence, the operating pressures are restricted and retention times can be long. Silica gel, which is mechanically strong, has been impregnated with metal ions in order to achieve selective separations. Guha and Janak (14) have reviewed the use of metals in the chromatographic separation of organic compounds. Much work has been done with silver ions (viz., 15-17 and references therein). Kunzru and Frei (18)have studied the use of Cd2+impregnated silica gel for the separation of
aromatic amines. Yasuda (19,20) has reported the Rf values of aromatic amines on TLC loaded with several metal salts. The metal ions impregnated on silica gel can be leached out by the mobile phase and, as a consequence, the retention times and the separations may change with time. Although the mobile phase can include the metal ions, such procedure can lead t o detection interferences especially when Cu2+is used in conjunction with a UV detector. Ligand-exchange chromatography on silica gel using a basic mobile phase is also difficult because of instability of the support in such a medium. Since solute interactions with metal ions can result in selective separations, it is desirable to design a system where the metal is held tightly by the support. The possibility of using a permanently bonded moiety to bind a metal cation suggests itself. The present work reports initial results using copper coordinated to an alkylamine which is bonded to silica gel. Karger and his co-workers are currently pursuing a similm line of investigation (21). Fritz and King (22) have recently reported the extraction of trace amounts of Cu(I1) by passing a solution through a column containing propylamine bonded to silica gel. Since amine columns are common, and since they seem to bond copper ions, it was decided that such a system is ideal for initial studies of the effects of bonded copper on the chromatographic behavior of some aromatic amines. There are several reasons for choosing aromatic amines as the test solutes. (1) T h e -NH2 group in aromatic amines is about lo6 less basic than in aliphatic amines. Consequently, these solutes would not deplete Cu(I1) from the column. (2) Ring substitutions have great effects on the properties of the aromatic amines. Therefore, the elution order of these compounds can give a better insight into the retention mechanisms. (3) Aromatic amines have been separated previously (18-20, 23, 24) so that comparisons with other works can be made. (4) Aromatic amines are of practical value and are often used as intermediates in various industrial products such as dyestuffs, drugs, and perfumes. (5) Many biologically and medicinally important compounds such as amino acids, muccopolysaccharides, etc. are known to form complexes with Cu(11). The separation of the aromatic amines can help in designing a system for the separation of' the above compounds.
EXPERIMENTAL Apparatus. The liquid chromatograph consisted of a Milton Roy (Riviera Beach, Fla.) mini pump model 133 and an LDC (Riviera Beach, Fla.) UV detector model 1285 operated at 254 nm. A Gilson (Middletown, Wis.) fluorimeter model Fl/B was also used as a detector. The excitation filter had a maximum transmittance at 360 nm. The emission was passed through a filter with a 400-nm cut off. The stainless steel columns dimensions were 25 cm X 0.21 cm i.d. Reagents. Partisil-10 was obtained from Whatman Inc. (Clifton, N.J.); y-aminopropyltrimethoxysilane was purchased from Silar (Waterlake, N.Y).All the aromatic amines used in this study were brought from various sources and used without further purification. The mobile phase was reagent grade methanol which was distilled before use. The cyclohexane modifier was used without further purification. Procedure. The column packing was prepared as follows: 1 0 7 ~ y-aminopropyltrimethoxysilanein tolune was added to Partisil-10, which had been dried in a vacuum oven at 180 "C, and the mixture was then refluxed for 6 h. The surface coverage of the bonded propylamine, computed from C, H, N analysis and the method suggested by Unger (25) was found to be 3.2 pmol/mL. The column was then packed using the slurry technique (26). The Cu2+was bonded to the amine by passing CuSO, (1 X 10 M) dissolved in dry methanol through the packed column. The
'
amount of Cu(1I) on the column was determined by washing the column with 150 mL of HC1 solution (pH 1.5) (22) and titrating the copper as discussed by Belcher and Nutten (27). The injection was done with a 10 pL syringe. The amounts injected were 1-2 pg of each solute. The mobile phases were methanol or cyclohexane/methanolsolutions. The chromatograms are given in terms of absorbance units, or relative intensity when the fluorimeter was used. Data were obtained on silica gel and bonded amine columns in addition t o the bonded copper column. The effect of the copper concentration in the mobile phase on the fluorescence spectra of the aniinonaphthalenes was not studied.
RESULTS AND DISCUSSION The amount of Cu bonded to the amine was found to be 0.23 kmol/m2. Since the ratio of the amine to Cu(I1) is about 10 to 1, it is clear that not all the -NH2 groups were coordinated to copper. The reasons for such low Cu coverage are not known to us a t present, but they are undoubtedly due to the fact that the bonded alkyl amine has only one group capable of coordinating the copper, and to the solvation effect of the methanol. Initial attempts used water and buffered solution as the mobile phases, since Fritz and King (22) indicated that the Cu(I1) remains on the propylamine a t p H greater than 5 . However, it was found that the use of such mobile phases did leach the Cu(I1) out of the column. I t should be pointed out, however, that the amount of copper loaded on the column in the present study is much higher than that reported by Fritz and King (22). Also, Fritz and King did not pass the mobile phase continuously; rather they stopped the flow once the metal ions were trapped. Cu(I1) ions could be added to the mobile phase in order to compensate for their loss. However, the concentration of copper ions needed to replace the loss could not be used because of the large absorbance of such a solution a t 254 nm. Because of these restrictions, the work described here made use of organic mobile phases. Over the period of this study (about two months), the capacity ratios were constant to within experimental errors. One of the most important sources of error, it is felt, was due to the inability to reproduce the composition of the mobile phase after it had been changed. The experimental evidence seems to indicate that no copper was leached from the column t o any appreciable extent when organic solvents were used as mobile phases. No attempts were made to measure quantitatively the amount of Cu(I1) in the column as a function of time. The mobile phase (2% MeOH in hexane) used by Kunzru and Frei ( 1 8 ) was also tried. T h e resulting retention times were much too long to be of practical value. I t seems that the interaction between the aromatic amines and the Cu(I1) is much stronger than that between the solutes and Cd. For these reasons, it was decided to use methanolic mobile phases. Initial experiments were run using 1- and 2-aminonaphthalene as solutes, in conjunction with a fluorimeter. The two aminonaphthalenes were not separated on the bonded amine or silica gel columns using 10% cyclohexane in methanol as mobile phase. However, on the bonded copper column, these compounds were separated from one another and from anthracene, as is shown in Figure 1. The k'values of the aminonaphthalenes as a function of the amount of cyclohexane in the mobile phase are given in Tables 1-111. The use of a fluorimeter as a detector allowed a closer investigation of the effect of the copper on the retention, since copper solutions (concentration larger than M Cu) which absorb strongly in the UV region of 254 nm do not fluoresce. Using a plain silica gel column, and a 20% cyclohexane in methanol mobile phase containing lo2 M CuS04, the aminonaphthalenes were resolved. Then, when the same mobile phase without Cu(I1) was used, the resolution deteriorated ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
1757
Table I. Capacity Ratios at Various % Cyclohexane/MeOH, Cu Columns [ 30%]
[O%I
Compounds o-Toluidine rn-Toluidine p-Toluidine o-Methoxyaniline p-Methoxyaniline 2,4-Dimethylaniline 2,5-Dimethylaniline 3,4-Dimethylaniline o-Chloroaniline rn-Chloroaniline p-Chloroaniline o-Nitroaniline rn-Nitroaniline p-Nitroaniline
p Kb 9.58 9.30 8.92 9.52 8.70
11.3 10.5 10.0
14.2 11.5 13.0
1-Aminonaphthalene 2-Aminonaphthalene
log k '
k'
0.20 0.60 0.94 0.27 1.33 0.26 0.26 0.94 0.10 0.27 0.35 0.16 0.16 0.16
-0.70 -0.22 -0.027 -0.57 0.12 -0.58 -0.58 -0,027
0.33 0.75 0.83
-0.48 -0.12
0.49 0.49 2.25 0.12 0.37 0.60 0.17 0.33 0.33
-0.31 -0.31 0.35 -0.92 -0.43 -0.22 -0.76 -0.48 -0.48 [ 30%]
0.39 0.87
-0.41 -0.060
-1.00
-0.57 -0.46 -0.79 -0.79 -0.79 [lo%]
0.20 0.60
10.0
9.7
[38%]
log k'
k'
-0.70 -0.22
k'
log k'
0.36 1.10 1.82 0.52 3.59 0.45 0.27 4.82
-0.08
0.11
0.46 0.98 0.21 0.39 0.39
-0.44 0.075 0.26 -0.28 0.56 -0.35 -0.57 0.68 -0.95 -0.34 -0.0037 -0.68 -0.41 -0.41 [ 38701
0.39 1.04
-0.41 0.017
Table 11. Capacity Ratios at Various % Cyclohexane/MeOH, Partisil-10 Columns [ 30761
[O%I
Compounds o-Toluidine m-Toluidine p-Toluidine o-Methoxyaniline p-Methoxyaniline 2,4-Dime thylaniline 2,5-Dimethylaniline 3,4-Dimethylaniline o-Chloroaniline rn-Chloroaniline p-Chloroaniline o-Nitroaniline rn-Nitroaniline p-Nitroaniline
PKb 9.58 9.30 8.92 9.52 8.70
k'
0.075 0.075 0.32 0.17 0.75 0.17 0.17 0.42
11.3
0.10
10.5
0.10 0.10 0.025 0.025 0.025
10.0
14.2 11.5 13.0
log
/2'
-1.12 -1.12 -0.49 -0.77 -0.77 -0.77 -0.37 -1.00 -1.60 -1.60 -1.60
0.23 0.40 0.63
-0.64 -0.40 -0.20
0.31 0.31
-0.60 -0.42 -0.21 -0.62 0.13 -0.57
0.12 0.12 0.12
-0.51 -0.51 -0.96 -0.92 -0.92 -0.92
0.25 0.38 0.61 0.24 1.35 0.27 0.27 0.51
0.10 0.10 0.10
-1.0 -1.0 -1.0
[ 20701
0.11 0.11
0.14 0.15 0.15 0.15 [ 30%]
log k '
k'
log k '
k'
0.21 0.21
-0.67 -0.67
0.20 0.20
-0.70 -0.70
0.20 - 0 . 7 0 0.20 - 0.70
log k '
-0.57
-0.29 -0.96 -0.96 -0.85 -0.82 -0.82 -0.82 [38%] k'
log k '
0.15 -0.81 0 . 1 5 -0.81
Table 111. Capacity Ratios at Various % Cyclohexane/MeOH, NH, Column [OR1
IT I '
0
__iL 1 -L
O
Flgure 1. Separation of aminonaphthalenes on the Cu column. Mobile phase: 10% cyclohexane/methanoi; flow rate 1.2 mL/min.; detector: fluorimeter. (1) Anthracene, (2) 1-arninonaphthalene, (3) 2-aminonaphthalene
as the Cu(I1) was washed from the column, and finally the resolution was completely lost. This study leads to two 1758
log k '
0.80
-1.00 -1.00
I
AN.
k'
k' 1-Aminonaphthalene 2-Aminonaphthalene
.
log k '
-0.12
[lo%]
L
[ 38%]
k'
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
Compound o-Toluidine rn-Toluidine p-Toluidine o-Methoxyaniline p-Methoxyaniline 2,4-Dimethylaniline 2,5-Dimethylaniline 3,4-Dimethylaniline o-Chloroaniline rn-Chloroaniline p-Chloroaniline p -Ni troaniline m-Nitroaniline o-Nitroaniline Benzene o-Phenyldiamine 1-Aminonaphthalene 2-Aminonaphthalene
pKb 9.58 9.30 8.92 9.52 8.70
[ 34%]
k'
log k'
k'
log k '
11.5
0.60
14.2
0.60 0.35
-0.62 -0.62 -0.62 -0.68 -0.68 -1.06 -1.06 -1.06 -0.46 -0.46 -0.54 -0.22 -0.22 -0.22 -0.46
0.12 0.12 0.12 0.12 0.12 0.079 0.079 0.079 0.19 0.19 0.19 0.23 0.31 0.38
-0.92 -0.92 -0.92 -0.92 -0.92
13.0
0.24 0.24 0.24 0.21 0.21 0.087 0.087 0.087 0.35 0.35 0.29 0.60
11.3 10.5 10.0
9.5 10.0 9.7
-1.10
-1.10 -1.10
-0.72 -0.72 -0.72 -0.64 -0.51 -0.42 0.11 -0.96
0.00
0.00
0.40
-0.40 0.23 -0.64 -0.40 0.23 -0.64
0.40
important conclusions: (1) the Cu(I1) is essential for the separation, and (2) the presence of the bonded amine is needed to yield a stable copper column, a t least with nonaqueous
3
I 0
1
0
4 d
m
I Lpp-_
0
5
~~~~
IO
I5
MIN.
Figure 2. Separation of dimethylanilines on the Cu Column. Detector: UV at 254 nm, 0.32 AUFS. Mobile phase: 38% cyclohexane/methanol; flow rate 0.68 mL/min. (1) Benzene, (2) 2,4-dimethylaniline, (3) 2,5-dimethylaniline, (4) 3,4-dimethylaniline
mobile phases. A similar study would be impossible to carry out on the amine column since the copper would bond to the amine. Possible explanations for the separation of the aminonaphthalenes lie in the geometry and basicity of the molecules. T h e 2-aminonaphthalene is the more linear and more basic of the two compounds. The steric hindrance of the fused ring system is minimized and, as a consequence, interaction between the Cu(I1) and the amine group on the solute is more likely to occur. This results in the longer retention times of the 2-aminonaphthalene. In pure silica gel or in bonded amine columns, the methanol in the mobile phase competes successfully with the solutes for the active sites on the support, and as a result no resolution occurs. T h e capacity ratios of isomers of toluidine, chloroaniline, nitroaniline, dimethylaniline, and methoxyaniline are given in Tables 1-111. Table I contains data obtained on the bonded copper, Table I1 shows data on the silica gel column, and Table I11 gives the retention behavior on a bonded amine column. The various mobile phase compositions are also shown in the Tables. The pKb’s of the solutes (for aqueous solution), are indicated when available. The hold-up time in the bonded copper column and on the silica gel column was obtained using benzene which eluted before all other solutes. Most likely the elution volume of the benzene is slightly larger than that of the void volume, and therefore the capacity ratios of the solutes on the Cu(I1) and the silica gel columns, as reported in Tables I and 11, are somewhat smaller than the true k’ values. Nevertheless, the trends depicted in Tables I and I1 are indicative of the chromatographic behavior of the isomeric aromatic amines on these columns. With the bonded amine column, the retention time of benzene was longer than several of the aromatic amines. Hence, with this column the capacity ratio was calculated relative to o-phenyldiamine which had the shortest elution time of all solutes tried. Examples of some separations are shown in Figures 2-4, for the dimethylanilines, the chloroanilines, and a complex mixture of several aromatic amines. No attempts were made to optimize the resolutions. It should be pointed out that the separation of some of these compounds was accomplished previously, albeit under different conditions. The aim of the present study is to show the influence of the metal ion. A comparison of the data in Table I with those in Tables 11and I11 shows that the separations in Figures 2-4 are not possible in the absence of Cu(I1). The isomers of the toluidine and of the chloroaniline have different k’values on the bonded copper column a t 100% methanol. In the silica gel or the bonded amine column, a t least two isomers of each group have the same k’s when 100%
----
__
-
-
5
0
IO
MIN
Figure 3. Separation of chloroanilines on the copper column. Detector: U V at 254 nm, 0.32 AUFS. Mobile phase: 38% cyclohexane/methanol; flow rate: 0 92 mL/min. (1) Benzene, (2) o-chloroaniline, (3) mchloroaniline, (4) p-chloroaniline 3
I
/
\
L
\
~.-),
i 0
5
IO
I
I5
~
-I--~ --L
20
23
__
MI N Figure 4. Separation of some aromatic amines on the copper column.
Detector: U V at 254 nm, 0.32 AUFS. Mobile phase: 38% cyclohexane/methanol; flow rate: 0.64 mL/min. (1) Benzene, (2) ochloroaniline,(3) o-nitroaniline,(4) m-nitroaniline,(5)o-methoxyaniline, (6) p-chloroaniline, (7)m-toluidine, (8)p-toluidine, (9) p-methoxyaniline methanol is used as the mobile phase. The Cu(I1) column did not separate two of the nitroanilines, the least basic solutes, even with 38% cyclohexane/methanol mobile phase. Interestingly, however, using a mobile phase of similar composition, the bonded amine column did show a different k ‘ value for each of the nitroaniline isomers. The retention behavior of the amines can be explained in terms of their basicities and steric hindrences (18,24,28). The methanol, which is present in large quantities (between 62% and loo%), can compete with the aromatic amines for the hydrogen bonding sites on the Partisil-10 column. The interaction between the lone pair of electrons on the nitrogen (electron donor) and the Cu(I1) (electron acceptor) in the copper column is stronger than the interaction of the methanol and the metal ion and, therefore, separations of the amines are possible. This is obviously an oversimplification since, as the data show, substituents and their position in the ring alter the retention significantly. An examination of Table I shows that compounds from different classes of isomers having similar pKh values can have different retention values. With the toluidines, chloroanilines, ANALYTICAL CHEMISTRY, VOL. 49, NO. 12,
OCTOBER 1977
1759
10.0-
PKb
9.5
-
90
12.0;
-
8.5
80
-15
-1.0
-0.5
0
0.5
l!O
.
11.0
J
Log k' Figure 5. Correlation of pK, of the toluidines to log k'. Cu column (-) and Partisil-10 column (---); ( 0 )para, (A)meta, ( W ) ortho. (A) 38 YO cyclohexane/methanol,(6)0 % cyclohexane/methanol,(C) 30 % cyclohexane/methanol, (D) 30 % cyclohexane/methanol, (E) 38 % cyclohexane/methanol, (F) 0 % cyciohexane/methanol
/
105
I I
10'0
9.5
1
1
L -2.0
-1.5
-1.0
-05
0
0.5
Log k' Figure 6. Correlation of pKb for t h e chloroanllines to log k ' . Cu column (-), and Partisil-10 column (---). ( 0 )para, (A)meta, ( W ) ortho. (A)
38 YO cyclohexane/methanol,(6)30% cyclohexane/methanol,(C) 0% cyclohexane/methanol, (D) 38% cyclohexane/methanol, (E) 30% cyclohexane/methanol, (F) 0 YO cyclohexane/methanol
and nitroanilines no solute had a k'value which is larger than the para isomers at all mobile phase compositions and on both the Cu(I1) and silica gel columns. Young and McNair (23) reported linear correlations between the pKb and log k ' for the isomers of several classes of aromatic amines. Figures 5-7 show some plots of PKb vs. log k'on the copper and Partisil-10 columns, and in one instance on the bonded NH2 column. Although in general, when retention differences occur, there is a trend of increasing k' with increasing basicity, it is not 1760
-2.0
, -1.5
, -1.0
,
,
,
Qd
0
0.5
1.0
Log k' Figure 7. Correlation of pK, of the nitroanilines to log k'. Cu column (-), Partisil-10 (- - - ) and -NH, column (- - - -). ( 0 )meta, (A)para, (I ortho. ) (A) 0% cyclohexane/methanol. (6) 38% cyclohexane/
-
methanol. (C) 30 YO cyclohexane/methanol. (D)34% cyclohexane/methanol. (E) 0% cyclohexane/methanol. (F) 38% cyclohexane/methanol. (G)30% cyclohexane/methanol. (H) 0 YO cyclohexane/methanol
4
12'0
10.51
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1 2 , OCTOBER 1977
clear whether a linear relationship does exist, at least in the systems investigated here. Because of the small k ' values obtained here, the behavior depicted in Figures 5-7 gives, at best, the trend of the data. The effect of the mobile phase composition is also of interest. The behavior of the k'values of the aminonaphthalenes as a function of the percent cyclohexane in methanol is a case in point. While with silica gel, k ' is almost a constant as the amount of cyclohexane increases, the reverse is true on the Cu(I1) column. The rate of increase of the two isomers on the Cu(I1) column is different, indicating improved separation at the highest percent of cyclohexane. Keeping in mind the small k'values, the data in Table I seem to indicate that with the exception of the nitroanilines, the least basic solutes, the k'value of the para isomer on the copper column, in general shows the greatest rate of change as a function of the mobile phase composition. The peak shape and column efficiencies seem to be a function of the mobile phase composition and the nature of the stationary phase. At zero percent cyclohexane, all solutes gave sharp and symmetrical peaks on the Cu(I1) column. However, using 38% cyclohexane/methanol mobile phase, the most basic amines, namely p-toluidine, p-methoxyaniline, and 3,4-dimethylaniline, gave broad tailing peaks. T h e para isomers of the less basic anilines still exhibited sharp peaks a t the above mobile phase. An example of this behavior is shown in Figures 2 and 3. Although the pKb of 3,4-dimethylaniline is not given here, the data seem to indicate that it is the most basic of all the amines studied. The efficiencies as measured by the plate height, in general, were much better on the silica gel and amine columns. This was most pronounced with the more basic aromatic amines. For example,
the plate height as measured with p-toluidine using 38% cyclohexane in MeOH mobile phase, was ten times higher on the copper column than on the amine colume (Hvalue of 6.0 m m and 0.62 mm, respectively). The capacity ratios of p toluidine, of course, were not the same on the two columns. For the least basic isomers, the efficiencies were about the same on both columns. The interaction between the Cu(I1) ion and the most basic amines, is different, it seems, than the interaction with the rest of the amines. The broad peaks tend to indicate that the rate of Cu-basic amine complex formation and/or dissociation is slow. With the silica gel and the bonded amine columns, sharp and symmetrical peaks resulted from all solutes. I t is clear that the bonded copper does provide a n active site which interacts with aromatic amines to yield separations. T h e selectivities are unlike those occurring on silica gel surfaces or in bonded amine columns. I t seems that the basicity of the aromatic amine, although important, is not the sole factor which determines the retention on the bonded Cu(I1) column. Steric effects undoubtedly play an important role in the interaction with the copper. A similar conclusion was reached by Kunzru and Frei (18)in their studies on the Cd-impregnated silica gel. T h e present Cu(I1) column cannot be used with aqueous solvents for reasons outlined previously. A more stable Cu(I1) column can be achieved by using a ligand which will bind copper ions more strongly than the propylamine. Leyden and his co-workers (viz. 29) have described the extraction of Cu2+ from aqueous solution by bifunctional moieties, bonded to silica gel. Initial studies in our laboratory show that Cu2+ bonded to a bidentate molecule is much more stable in such systems. Subsequent publications will describe the preparation and properties of metal ions which are strongly bonded to the chromatographic support.
LITERATURE CITED (1) F. G. Helfferich. Nature(London), 189, 1001 (1961). (2) H.F. Walton, S e p . furif. Methods, 4, 189 (1975). (3) H. F. Walton, Ion-exchange and solvent extractions, a series of advances“, Vol. 4, J. A, Marinsky and Y. Marcus, Ed., 1973,p 121. (4) V. A. Davakov, S. V. Rogozhin, A. V. Semechkin, and T. P. Sachkova, J . Chromatogr., 82, 359 (1973). (5) V. A. Davankov, S.V. Rogozhin, A. V. Semechkin, and T. P. Sachkova, J . Chromatogr., 93, 363 (1974). (6) R. V. Snyder and R. J. Angelici, Mech. Methods Enzymol., 3, 468 (1957). (7)J. Seematter and G. Brushmiller, J. Chem. Soc., Chem. Commun., 1277 (1972). (8) A. V. Semechkin, S. V. Rogozhin, and V. A. Davankov. J . Chromfogr., 131, 65 (1977). (9) F. W. Wagner and S. L. Shepherd, Anal. Biochem., 41, 314 (1971). (10) J. Navratil, E. Murgia, and H. F. Walton, Anal. Chem., 47, 122 (1975). (11) K . Shimomura, J-J. Hsu. and H. F. Walton, Anal. Chem., 45, 501 (1973). (12) J. D. Navratil and H. F. Walton, Anal. Chem., 47, 2443 (1975). (13) M. Doury-Berthcd, C. Poitrenaud, and B. ‘rremillon. J . Chromatogr.. 131, 73 (1977). (14) 0.K. Guha and J. Janak, J . Chromato:gr., 68, 325 (1972). (15) R. R. Heath, J. H. Tumiinson, R. E. Doolittle, and A. T. Provaaux, J . Chromatogr. Sci., 13, 380 (1975). (16) R. Aigner, H. Spitzy, and R. W. Frei, A,qa/. Chem., 48, 2 (1976). . R. W. Frei. J . Cktromatwr. Sci.. 14. 381 11976). 117) R. Aioner. H. S D ~ Vand (l8i D. Kinzru and R.’W. Frei, J . Chbmatcigr. S c i , 12, 191 (1974) (19) K. Yasuda, J . Chromatogr., 60, 144 (1971). (20) K. Yasuda, J . Chromatogr., 72, 413 (1972). (21) E. L. Karger, Northeastern University, private communication, 1977. (22) J. S. Fritz and J. N. King. Anal. Chem.. 48, 570 (1976). (23) P. R. Young and H. M. McNair, Anal. Chem., 47, 756 (1975). (24) P. R. Young and H. M. McNair, J , Chromatogr., 119, 569 (1976). (25) K. K. Unger, N. Becker, and P. Roumeliotis. J . Chromatogr., 125, 115 11R76) _,. (26) L. R. Snyder and J. J. Kirkland, “Modern Liquid Chromatography”, Wilev-Interscience. New York. N.Y.. 1974. (27) R. Belcher and A. J.’ Nutten, “Laboratory Manual of Quantitative Inorganic Analysis“, Butterworth. London, 1955,Chap. 3, p 244. (28) L. R. Snyder, “Principles of Adsorption Chromatography”, Marcel Dekker, Inc., New York, N.Y.. 1968. (29) D. E. Leyden and G. H. Luttrell. Anal. Chem., 47, 1612 (1975). ~
RECEIVED for review May 23, 1977. .4ccepted ,July 19, 1977. We thank NIH for supporting the present work under grant GM-20846-02.
Determination of Tetraethyllead in Gasolines by High Performance Liquid Chromatography T. C. S. RUO, M. L. Selucky, and 0. P. Strausz” Hydrocarbon Research Center, University of Alberta, Edmonton, Alberta, Canada
A high-performance liquid chromatographic (HPLC) method for the determination of tetraethyllead (TEL) in gasollnes has been developed. The method is based on the separation of TEL from other UV absorbing material on silica gel and quantiiicetion of the UV detector response. TEL concentrations corresponding to as little as 0.03 pg Pb In the sample (corresponding to 0.01 g Pb/imp. gal) could be determined quantitatively. The response of other alkylleads (tetramethyllead (TML) and mixed alkylleads (TMLTTEL)) differs appreciably from that of TEL, precluding the use of this method for unknown samples. However, the analysis can be done In 5 min using commercially available equipment and can be used in all cases where the type of alkyllead present In the gasoline is known.
A number of methods has been suggested for the determination of tetraethyl- or total lead in gasolines. They
T6G 2G2
comprise wet processes (1, Z ) , polarography ( 3 ) , gas chromatography (4-6), x-ray spectrometry (7), and atomic absorption (8) or methods using an atomic absorption spectrometer as detector ( 4 , 5 , 8). Except for chromatographic methods, they have been introduced as ASTM standards. Wet methods are tedious and time consuming, since they require conversion of T E L to P b salts which are either determined gravimetrically, titrimerically, or spectrophotometrically. Also, polarography requires the same sample pretreatment and can be classified 8 s a wet method. Gas chromatography using conventional detectors is complicated for aromatic base gasolines because of peak overlaps and special columns must be used for retarding the aromatic hydrocarbons (6). Atomic absorption requires sample stabilization (8). Thus, of all the conventional methods, only x-ray spectrometry does not require sample pretreatment. Combination of gas chromatographic separation with an atomic absorption detector has been demonstrated to give reasonable repeatability ( 4 ) . The method is limited, however, ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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