Anal. Chem. 1985, 57,997-1005
997
Effects of Solvent Composition and Temperature on the Separation of Anilines with Silica, Amino, and Diamine Bonded Phase Columns C. Allen Chang* a n d Chen-Shi Huang Department of Chemistry, University of Texas a t El Paso, El Paso, Texas 79968-0513
By the use of extrathermodynamic relationships, Le., linear free energy relationship and linear AH-pK, relationship, a retention mode change induced by the change of mobile phase composition for the liquid chromatographic Separations of several substituted aniilnes using amlno and diaminebonded phase columns Is demonstrated. The change Is not obvious when a silica column is used. Possible interaction modes are proposed at a molecular level as static views of the retention process involving hydrogen bonding as the Interaction force.
Although it is believed that the majority of liquid column chromatographers practice their separations using bonded phase columns with a reversed-phase mode, normal-phase separations such as liquid-solid adsorption cannot be overlooked. In general, liquid-solid adsorption chromatography has exceptional potential for the control of the selectivity of separations of organic position isomers and some water-insoluble polymers (I,2). However, very little is known concerning their retention mechanisms a t the molecular level. Recently, Kiselev and co-workers have published several papers concerning the selectivity in liquid adsorption chromatography (3). A classification of several variants according to the main types of intermolecular interactions that determine retention has been discussed ( 4 ) . In particular, it has been stated that for the separation of polar compounds, the retention may be determined by specific solute-stationary phase and mobile phase interactions, especially when high concentrations of polar eluent are added. This is consistent with our recent discovery that a linear relationship is observed between AH values of the retention w d pKb values of several polar solutes, i.e., chloroanilines and nitroanilines, using several polar bonded phase columns (5). Because linear free energy relationships are also observed for these systems, the specific interactions are likely to take place through the lone pair electrons of the aromatic amino groups of chloroanilines and nitroanilines. In this paper we wish to report a systematic study on the effects of solvent composition and temperature on the separation of several substituted anilines (Le., 0-,m-, and ptoluidines, chloroanilines,and nitroanilines) using silica, amino, and diamine bonded phase columns (BPC). The eluents are 2-propanol/n-heptane mixtures a t various proportions. Extrathermodynamic relationships are applied in this study in order to gain more information concerning the retention behavior. EXPERIMENTAL SECTION Apparatus. A Micromeritics (Norcross, GA) Model 7500 liquid chromatography system was used. This system was equipped with a Model 750 solvent delivery system, a Model 752 ternary solvent mixer, a Model 731 column compartment with a universal sample injector and a variable temperature controller from ambient to .~
-. .. .. .
150 "C, and a Model 786 variable wavelength (200-600 nm) detector with a deuterium lamp. A Waters (Milford, MA) HPLC system equipped with two Model 6000 A solvent delivery systems, a Model 660 solvent programmer, and a Model 440 absorbance detector (254 nm) was also used. Pressure-Lok series C-160 (Precision Sampling Corp., LA) 10-pL and 25-pL syringes were used. Chromatograms were recorded with a Linear Model 555 single-channel recorder. Reagents. Solid silica supporting material, Partisil-10, was purchased from Whatman (Clifton, NJ). Amino bonded phase, Rosil-NH2,was purchased from Alltech Associates (Deerfield,IL). 3-[ (2-Aminoethyl)amino]propyltrimethoxysilanewas obtained from Silar Lab. (Scotia, NY). All other chemicals were reagent grade and obtained from various sources. Elemental analysis was performed by Galbraith Lab., Inc. (Knoxville, TN). Preparation of Diamine Bonded Phase. The diamine bonded phase was prepared according to the method used by Chow and Grushka with minor modifications (6). Partisil-10 was pretreated by drying under vacuum at 150 "C for 6 h and then cooling under vacuum to room temperature. To a 60-mL solution of 10% (v/v) 3-[(2-aminoethyl)amino]propyltrimethoxysilane in dry touluene was added 10 g of pretreated Partisil-10. The mixture was refluxed under nitrogen for 6 h with stirring, it was then filtered and washed successively with 2propanol, acetone, methanol, and acetone. The diamine bonded phase was then dried under vacuum at 100 "C for 2 h. Elemental analysis: C, 7.75; H, 2.21; N, 2.81. A previous study showed that for each derivatizing molecule, two of the three methoxy groups reacted with silanol groups on Partisil-10 (7). By use of the method described by Unger et al., surface coverage was calculated to be 3.02 pmol/m2 (8). According to the manufacturer's guide, the surface silanol concentration was ca. 8 wmol/m2. Thus, the surface coverage of diamine was over 75%. Column Packing. The columns were packed by using an up-flow method with a Micromeritics Model 705 stirred-slurry column packer. Carbon tetrachloride was used as the solvent for column packing. Before being packed, the bonded phases were dispersed in the solvent and treated by an ultrasonic vibrator for 1 min so that a well-mixed suspension was formed (9). The procedures for column packing were described in a previous publication (IO). Chromatographic Procedures. Before the separation experiments, the columns were washed with 1% 2-propanol in n-heptane which is the lowest concentration of the polar modifier used. A flow rate of 1mL/min was used for the washing process. After washing 12 h, the column was allowed to stand for another 12 h. The process was repeated three to four times. After preequilibrium was achieved, a flow rate of 2 mL/min was used. The temperature-dependent measurements of capacity factor (k') were performed starting with a lower polar modifier concentration and then progressing to higher polar modifier concentrations (temperature ranges from ambient to 56 "C). Additional washing with a flow rate of 2 mL/min for 8 h was performed when the concentration of polar modifier was changed. The 2-propanol was purchased from Fisher and used without further purification. The trace water content in such solvent did not present a problem in all studies. The separation of a mixture of various substituted anilines was pretested. Depending on the separation condition, solute mixtures were prepared so that overlapping peaks did not occur and good 0 1985 Amerlcan Chemical Society
996
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
Table I Elution Sequences of Several Disubstituted Benzenes in Three BPCs”
polar modifier
%
2-propanol in n-heptane
5 10 25
stationary phase
sequence (increaaing k)
silica amino diamine silica amino diamine amino diamine
PhN02, 0-C1, o-N, o-T, m-T, m-C1, A, p-T, p-C1, m-N, p-N PhN02, 0 4 1 , o-T, m-T, p-T, A, m-C1, p-C1, o-N, m-N, p-N PhN02, 0-C1, m-C1, p-C1, o-N, m-N, p-N 0-C1, o-N, o-T, m-T, m-C1, A, p-T, p-C1, m-N, p-N 0-C1, p-T, m-T, p-T, A, rn-C1, p-C1, o-N, m-N, p-N 0-C1, rn-C1, p-C1, o-N, m-N, p-N o-T, A, rn-C1, o-N, p-C1, m-N, p-N 0-C1, m-C1, p-C1, o-N, m-N, p-N
a 0-,m-, and p - represent ortho, meta and para isomers; A represents aniline; T represents toluidine; C1 represents chloroaniline; N reDresents nitroaniline: PhNO, reDresents nitrobenzene.
Table 11. Capacity Factors and Dipole Moments of Several Solutes of Interest
dipole moment” k (I)b k (IIY
p-T
m-T
A
o-T
041
m-C1
p-C1
o-N
PhN02
m-N
p-N
1.31 1.62 3.75
1.44 1.54 3.00
1.53 1.91 3.28
1.58 1.06 1.94
1.84 0.91 0.62
2.91 3.04 3.19
3.0 3.56 5.05
4.06 4.96 1.85
4.22 0.31
4.91 12.02 7.19
6.32 47.10 14.46
“Values selected from “Hand Book of Chemistry and Physics”, 61 ed.; CRC Press: Boca Ratan, FL, 1980. bThe capacity factors are obtained from amino BPC using 3% 2-propanollheptane solution as eluent. ‘The capacity factors are obtained from silica column using 3% 2-propanollheptane solution as eluent. resolutions (R)were obtained (Le., R > 1.0). In each separation, solute mixtures were prepared using the eluent. The concentration of each solute was ca. 1-2 mg/mL. An amount of 1pL of the solute mixture was injeded into the system. A back pressure less than 1000 psi was usually observed throughout the experiment. All data points were collected by averaging more than three reproducible separations and treated with normal statistical methods. p-Xylene or benzene was used to determine tofor each column.
RESULTS AND DISCUSSION Elution Sequence. The elution sequences of several aniline derivatives in three stationary phases with various eluents are listed in Table I. According to Kiselev’s classification (3),both chloroanilines and nitroanilines belong to the class which consists of compounds with two polar functional groups and intramolecular hydrogen bonding can occur for ortho isomers. Toluidines belong to the class which consists of compounds having one polar functional group and one nonpolar group. Because of the formation of intramolecular hydrogen bonding, o-chloroaniline always eluts faster than o-toluidine. This is consistent with what has been reported previously (3). Silica Column. The retention of toluidines, chloroanilines, and nitroanilines in silica column has been studied by Kiselev et al. (3). Dipole moments were used to interpret the retention order of the chloroanilines and nitroanilines. In addition, steric effects of the methyl group were brought up to explain the elution order of toluidines. However, the fact that they did not compare the capacity factors of all those substituted anilines using the same eluent precludes them from obtaining a complete view of the retention behaviors of those solutes in the silica column. As shown in Table I, the three ortho isomers always elute f i t , followed by meta isomers except for m-nitroaniline, which are then followed by para isomers. This clearly shows that the retention order can not be simply interpreted by consideration of only dipole interactions (Table 11). It is very likely that several effects due to the presence of two functional groups as well as the strength of hydrogen bonding between the solute and silica surface are operative when retention times are concerned. Amino and Diamine BPCs. In amino or diamine bonded phase columns (BPC), the retention time tends to follow an
order of ortho Imeta Ipara isomer. In contrast to Kiselev’s rationalization, this order is not really related to the dipole moments of the solutes (Table 11). It is also seen that all three nitroaniline isomers elute after those of chloroanilines which follow toluidines, except for o-chloroaniline. The retention time of o-nitroaniline is very greatly affected by the concentration of the polar modifier in the eluent in all three columns as compared to other solutes. This is probably due to a favorable solvation of o-nitroaniline in polar or hydrogen bonding solvents. Differences in selectivities between amino and diamine BPCs are also found by carefully examining the elution sequences of the solutes. For example, the elution order is reversed for o-nitroaniline and p-chloroaniline using an amino BPC as compared to a diamine BPC. The retention times are always longer for solutes in a diamine column although its surface coverage is similar to the amino column, Le., ca. 3 pmol/m2. Furthermore, the effect of 2-propanol on the elution of o-nitroaniline in amino BPC is more pronounced than its effect in diamine BPC. These observations imply that bifunctional interactions may occur in the diamine column. Similar observations have been reported by Chang and T u in the separations of dihydroxybenzene isomers (11). Relationship between Concentrationof Polar Modifier and Capacity Factor. Considering only solute-stationary phase and solute-mobile phase interactions, Soczewinski derived an equation to account for the effects of solvent (12) l o g k ’ = l o gKaxXae [~]-logX,
(1)
where k’is the capacity factor, X,is the mole fraction of the strong solvent in a binary mobile phase, X,, is the mole fraction of the strong solvent on the adsorbed phase, and K, and K , are the adsorption constants of the solute and solvent, respectively. Snyder derived an equation concerning the effect of solvent in a binary mobile phase (13)
AS
log k’, = log k; - - log
x,
nb
where A, is the molecular area of the solute, nb is the molecular area of the strong solvent, k’, is the capacity factor for a solute
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY
Table 111. Capacity Factors of Several Solutes of Interest in Silica Column at 30.5 the Eluent
0-T m-T P-T A 0-c1 m-C1 p-c1 0-N m-N P-N
1%
2%
3%
2.71 4.10 5.12 4.41 0.89 4.47 6.99 3.20 12.56 29.26
2.17 3.81 3.91 3.57 0.89 3.60 5.40 3.16 12.05
1.94 3.00 3.75 3.28 0.62 3.19 5.05 1.85 7.19 14.46
O C
Using 2-Propanol/Heptane Solution as
capacity factors at the following % 2-propanol 4% 5% 6% 7% 1.72 2.65 3.25 2.86 0.58 2.78 4.40 1.62 6.03 9.81
1.52 2.31 2.83 2.52 0.50 2.43 3.82 1.41 4.99 9.60
1.50 2.31 2.84 2.53 0.48 2.35 3.76
1.36 2.04 2.46 2.25 0.46 2.11 3.34 1.09 3.93 6.89
1.22
4.56 8.24
1985 999
8%
9%
10%
1.28
1.20 1.76 2.08 1.93 0.40 1.80 2.83 0.91 3.12 5.16
1.63 1.90 1.79 0.38 1.64 2.57 0.80 2.84 4.48
1.94 2.25 2.10 0.43 1.94 3.05 0.95 3.50 5.88
1.11
Table IV. Capacity Factors of Several Solutes of Interest in an Amino BPC at 30.5 "C Using Z-Propanol/n -Heptane Solution as the Eluent
0-T m-T P-T A 0x1 m-C1 p-c1 0-N m-N P-N PhNOz
1%
1.5%
2%
1.73 2.16 2.34 2.65 1.23 4.53 5.33 8.17 22.03
1.34 1.96 2.10 2.41 1.13 4.09 4.80 7.15 18.76
1.19 1.73 1.83 2.13 1.01 3.53 4.12 6.02 15.15
0.36
0.35
capacity factors at the following % 2-propanol 3% 4% 5% 7.5% 10% 15% 1.06 1.54 1.62 1.91 0.91 3.04 3.56 4.96 12.02 47.10 0.31
0.32
0.93 1.34 1.42 1.67 0.82 2.60 3.03 4.09 9.62 35.83 0.30
0.85 1.20 1.26 1.50 0.75 2.29 2.65 3.50 7.96 28.13
0.71 1.00
1.04 1.25 0.62 1.80 2.08 2.54 5.52 17.04
0.62 0.89 0.90 1.09 0.54 1.50 1.72 1.98 4.21 11.75
0.46 0.65 0.63 0.78 0.41 1.04 1.18 1.30 2.54 6.20
20%
25%
30%
0.39
0.34
0.29
0.64
0.55
0.48
0.80 0.91 0.93 1.80 3.79
0.66 0.75 0.71 1.35 2.58
0.56 0.62 0.56 1.11
1.84
e 0
e
-
2.0
e A
1.8-
e
u
a
.
0
A A
0 A
.. 0 A
1.G
c
@
A
0
1.L
A
-
A
0 A
'
1.2 A
e
.
A
0
,1.0 1
D
A
0
0
.a -
0 A
0
- .2
i
I1
e
0
0 : : O
0
&
A
A
A
e A
0 8 0
oa
@
-1.G
-1.6
-1.2
-1.0 'og
e.8
e.6
-.I
xs
Figure 1. Plot of log k vs. log X a t 30.5 OC in an amino column using rn-T (A)p-T (0),A 2-propanollheptane mixture as eluent: 0-T (a), (a),0-Ci (O),m-Ci (A),P-CI (01, 0-N (ObW-N (A),p - N (*I.
eluted with the binary mobile phase, and k', is the capacity factor of the solute eluted with just the strong solvent. The equation has been applied to an amino column for some polar solutes with some success (14). On the other hand, Scott and Kucera derived an equation to account for the solute interactions in liquid-solid chromatography. One form of the equation is (15)
l/k' = A
+ BC,
(3)
'1. 2 - propanol
Figure 2. Plot of l l k vs. % of 2-propanol in heptane at 30.5 OC in an amino column. The symbols for solutes are the same as those given in Figure 1.
where A and B are constants, k' is the capacity factor, and C, is the (%, v/v) concentration of the stronger solvent in a binary mobile phase. Tables 111-V list the capacity factors which are obtained at 30.5 "C in three columns of interest. The plots of values of log k'vs. log X , and llk'vs. C, for the retention of several substituted anilines in an amino column using 2-propanol/ n-heptane mixtures as eluent are shown in Figures 1 and 2.
1000
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6,MAY 1985
Table V. Capacity Factors of Several Solutes of Interest in a Diamine BPC at 30.5 OC Using 2-Propanol/n -Heptane Solution as the Eluent
0x1 m-C1 p-C1 0-N m-N P-N
1%
2%
2.51 9.07 11.66 17.25
2.02 6.84 7.96 12.54 31.56
3%
4%
capacity factors at the following % a - ~ r o ~ a n o l 5% 6% 7.5% 10% 15%
1.63 5.08 5.58 8.57 20.29
1.29 3.60 3.86 5.46 12.04 39.05
1.14 2.98 3.13 4.27 9.14 26.90
0.90 2.17 2.27 2.86 5.82 14.23
20%
25%
30%
0.73 1.69 1.74 2.02 4.01 8.82
0.66 1.29 1.43 1.58 3.02 5.89
1.26 2.42 4.28
75 */e
4 '/*
/
/
/
1.0I
E
/
, I
,I'
5
/
/A
Q
.5-
0
i 1 3.;
,.
1.5
i.0
2.0
,,d 2.0 2.5
,
2.5
I ,
3.0
- A H Kcol /mole
2D
.o
2.5
3.0
15
Flgure 3. Plot of log krrm vs. -AH in a sillca column using 2propanoVheptane mixture as eluent. The symbols for solutes are the same as those glven in Figure 1.
0 >'
,
L
o
;
K 20 25 30 H, Kcal/ mole Flgure 5. Plot of log krhm vs. -AH in a diamine column using 2propanoVheptane as eluent. The symbols for solutes are the same as those given in Figure 1. 25
30
-A
misinterpret the data which may involve statistical errors. Due to the nature of data reduction, it is always possible to obtain spurious linear entropy-enthalpy plots. In order to discern between the true compensation and the false, the method described by Krug et al. is used (19,20), which involves the following equation:
In ki = (In A - AH/RT,,)
1
I
5.2
3.0
-
3.5
25
30.
3.5
H, Kcai / m o l e
Flgure 4. Plot of log kTh, vs. -AH in an amino column using 2propanoVheptane as eluents. The symbols for solutes are the same as those given In Flgure 1.
It clearly shows that most of the plots are not completely linear indicating more complex retention behavior (16). Similar results are observed for silica and diamine columns. Entropy-Enthalpy Compensation. Entropy-enthalpy compensation has been observed in many organic and inorganic reaction systems, and several observations in HPLC separations have also been reported (17,18). Although the phenomenum is not unusual, care must be exercised not to
-
A H / R [ ( l / T J- ( l / T h m ) ] (4)
where A and R are both constants, ki is the capacity factor at temperature T,, and T b is the harmonic mean of all temperatures studied. The term AH refers to the enthalpy change of solute transfer from the mobile phase to the stationary phase. If the plot of values of AH (which can be obtained from van't Hoff plots) vs. -RT In k'Th, is linear, a true entropyenthalpy compensation is obtained. ktTh is the capacity factor at the harmonic mean temperature. The slope of an entropy-enthalpy compensation has been used to interpret the retention mechanism in reversed-phase chromatography (17).For a set of compounds in which the entropy-enthalpy compensation exists, the slope will be the same for the same type of reaction. On the other hand, two completely different slopes for a set of compounds in two systems mean the retention mechanisms are likely to be different. Values of the entropy change and enthalpy change obtained from van't Hoff plots in the amino and the diamine BPC, and
ANALYTICAL CHEMISTRY, VOL. 57,
NO. 6, MAY 1985
1001
Table VI. Results of van't Hoff Plot in an Amino BPC Using 2-Propanol/Heptaneas the Eluentasb so1ut es
1%
-AH
0-T
-AS
corr
-AH
m-T
-AS
corr P-T
-AH
A
-AH
-AS
corr -AS
coir
-AH
0x1
-AS
corr m-C1
-AH -AS
corr p-c1
-AH -AS coir
-AH
0-N
-AS
corr
-AH
m-N
-AS
corr -AH
P-N
-AS
corr PhNOz -AH -AS
corr
1.5%
2%
3%
2.63 3.05 9.90 8.30 0.9992 0.9995 3.33 2.94 10.11 8.59 0.9983 0.9998 3.38 2.92 10.15 8.38 0.9956 0.9994 3.46 3.06 10.09 8.56 0.9978 0.9999 2.88 2.55 9.68 8.35 0.9928 0.9990 3.52 3.27 9.37 8.25 0.9973 0.9995 3.66 3.42 9.52 8.41 0.9978 0.9997 3.12 3.33 7.78 6.68 0.9959 0.9990 3.69 3.42 7.21 5.84 0.9944 0.9991 4.40 6.85 0.9995 1.32 0.80 1.86 6.33 4.72 8.39 0.9510 0.9819 0.9668 1.83 5.24 0.9983 2.11 5.42 0.9925 2.19 5.52 0.9999 2.30 5.65 0.9943 1.90 5.82 0.9924 2.50 5.21 0.9960 2.62 5.30 0.9963 2.53 4.13 0.9955 2.57 2.30 0.9960
2.28 6.92 0.9942 2.58 7.12 0.9949 2.67 7.28 0.9984 2.76 7.31 0.9955 2.16 6.84 0.9979 3.03 7.15 0.9956 3.18 7.32 0.9967 2.98 5.88 0.9963 3.25 4.87 0.9959
4% 3.08 10.29 0.9983 3.36 10.46 0.9996 3.34 10.30 0.9998 3.51 10.51 0.9996 2.71 9.32 0.9967 3.52 9.67 0.9998 3.70 9.95 1.000 3.33 8.16 1.000 3.83 8.11 1.000 4.54 7.85 1.000
5%
7.5%
10%
3.12 3.28 10.60 11.49 0.9996 0.9979 3.36 3.23 10.64 10.69 0.9999 0.9997 3.42 3.43 10.81 11.17 0.9993 0.9994 3.52 3.50 10.76 11.10 0.9998 0.9981 3.00 2.98 10.46 10.75 0.9966 1.000 3.48 3.53 10.29 9.97 1.000 1.000 3.65 3.59 10.08 10.36 0.9995 1.000 3.27 3.03 8.12 8.27 0.9989 0.9998 3.63 3.84 8.56 8.51 0.9993 0.9991 3.92 4.41 7.26 7.87 0.9994 0.9963
15%
20 %
25%
30 %
35%
3.30 3.40 3.44 3.53 3.48 12.40 13.08 13.50 14.07 12.40 0.9988 0.9983 0.9996 0.9967 0.9994 3.52 3.46 12.47 11.65 0.9998 0.9980 3.61 3.60 12.75 12.04 0.9955 1.000 3.47 3.39 3.52 3.68 11.62 11.96 12.73 12.28 0.9994 0.9976 0.9987 1.000 3.38 3.67 12.87 13.22 0.9963 0.9817 3.41 3.31 3.16 3.41 3.55 11.15 11.32 11.20 12.38 10.88 0.9997 0.9977 0.9961 0.9998 1.000 3.43 3.29 3.28 3.32 3.71 10.51 11.48 12.22 10.97 11.12 0.9999 0.9987 0.9987 0.9986 1.000 2.69 2.52 2.12 2.48 3.00 8.34 8.46 7.66 9.29 8.52 0.9973 0.9901 0.9999 0.9999 0.9986 3.11 3.23 3.04 3.13 3.69 8.78 9.39 10.18 9.08 9.29 0.9996 0.9996 0.9993 0.9986 0.9998 3.10 2.51 2.55 2.87 3.80 6.56 6.37 7.17 6.80 7.61 1.000 1.000 0.9998 1.000 0.9999
3.31 12.41 0.9981 3.44 12.64 0.9992 2.58 10.06 0.9840 3.04 10.38 0.9993 2.54 7.75 0.9998
Corr is correlated coefficient. AS is actually AS + R In (VJ V,) where V, and V , are respective volumes of the stationary phase and the mobile phase in the column. AH (kcal/mol), AS (entropy units). Table VII. Results of van%Hoff plot in A Diamine BPC Using 2-Propanol/Heptaneas the Eluenta
solutes 0-c1
-AH -AS
corr m-C1
-AH -AS
p-c1
-AH -AS
0-N
-AH -AS
m-N
-AH -AS
P-N
-AH -AS
corr corr corr corr corr a
1%
2%
4%
7.5%
10%
15%
20 %
25%
30 %
2.01 4.80 0.9938 2.74 4.63 0.9975 2.73 4.11 0.9969 2.85 3.72 0.9994
2.37 6.41 0.9996 3.17 6.63 0.9992 3.23 6.52 0.9985 3.18 5.47 0.9995 3.55 4.85 0.9997
2.66 7.80 0.9991 3.42 8.04 0.9997 3.36 7.65 0.9998 3.23 6.38 1.000 3.84 6.67 0.9999
2.60 8.05 0.9966 3.31 8.36 0.9997 3.27 8.11 0.9996 2.97 6.42 0.9999 3.75 7.42 0.9998 4.41 6.37 1.000
2.77 8.86 0.9984 3.44 9.14 0.9998 3.40 8.92 0.9999 2.95 6.82 1.000 3.70 7.80 0.9997 3.98 6.59 0.9997
2.78 9.36 0.9981 3.06 8.76 0.9990 3.19 8.90 0.9993 2.56 5.90 0.9987 3.40 7.73 0.9994 3.40 5.93 0.9997
2.45 8.64 0.9914 3.17 9.39 0.9991 2.97 8.67 0.9987 2.31 6.23 0.9988 3.21 7.79 0.9988 3.13 5.99 1.000
2.77 9.96 0.9958 3.00 9.19 0.9999 2.94 8.97 0.9984 2.04 5.84 0.9988 2.98 7.63 0.9994 2.68 5.30 0.9999
2.10 6.06 0.9994 2.91 7.83 0.9999 2.63 5.78 0.9998
See footnote of Table VI.
the silica column using 2-propanolln-heptane as eluent are listed in Tables VI-VIII, respectively. In general, the correlation coefficients are good for most of the plots except for those where polar modifier concentrations are 1%or 2%. This could be due to the effects of having more interaction modes involved in the retention process at such solvent compositions. The AH vs. log kThmplots are shown in Figures 3-5 and will be discussed below (Thm= 40 "C). (Tests have been made to show true compensation is observed for most systems.) Amino and Diamine BPCs. In amino and diamine BPCs using 2-propanolln-heptane as eluent, the slopes gradually change from a positive value a t low concentrations of 2-
propanol in n-heptane to a negative one at high concentrations of 2-propanol (Figures 4 and 5 ) . Detailed analysis of the linear free energy plots have been performed for the amino column according to the eq (17)
where kT is the capacity factor at temperature T , @ is the compensation temperature, and AGOis the Gibbs free energy of a physicochemical interaction at temperature 6. The slopes of log k3130k vs. -AH plots lead to the following @ values a t
1002
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
Table VIII. Results of van't Hoff Plot in a Silica Column Using 2-Propanol/Heptaneas the Eluentn solutes
0-T
-AH -AS
m-T
-AH -AS
corr
P-T 0-c1
corr -AH -AS corr -AH -AS
corr m-C1
-AH -AS
p-c1
-AH -AS corr -AH -AS corr -AH -AS
coir
A
0-N m-N P-N
corr -AH -AS coir -AH -AS corr
1Yo
2%
3%
4%
5%
6%
7%
8%
9%
10%
1.10 1.63 0.9551 1.63 2.56 0.9964 1.70 2.33 0.9939 0.77 2.77 0.8120 1.89 3.24 0.9973 2.09 3.02 0.9980 1.59 2.29 0.9794 1.40 2.29 0.9871 1.71 0.60 0.9940 2.43 1.30 0.9958
1.82 4.43 0.9410 1.74 3.08 0.9934 2.11 4.19 0.9455 1.36 4.72 0.9905 2.41 5.35 0.9678 2.69 5.46 0.9729 2.37 5.26 0.9726 2.03 4.36 0.9469 2.36 2.83 0.9775
2.69 7.53 0.9992 2.97 7.61 0.9992 3.07 7.51 0.9992 1.47 5.78 0.9956 3.10 7.90 0.9997 3.46 8.20 0.9910 3.11 7.87 1.000 1.66 4.27 0.9988 2.62 4.73 0.9994 2.61 3.31 0.9996
2.88 8.41 0.9989 3.29 8.91 0.9997 3.38 8.80 0.9999 2.12 8.08 0.9938 3.27 8.73 0.9997 3.52 8.66 0.9997 3.24 8.58 0.9988 1.72 4.72 0.9993 2.71 5.34 0.9988 2.56 3.53 0.9976
2.91 8.76 0.9999 3.12 8.63 0.9988 3.24 8.63 0.9992 1.56 6.50 0.9890 2.98 8.07 0.9990 3.26 8.08 0.9994 3.17 8.62 0.9993 1.53 4.37 0.9947 2.43 4.82 0.9989 2.24 2.92 0.9977
3.16 9.59 0.9987 3.27 9.14 0.9956 3.33 8.92 0.9974 1.95 7.90 0.9990 3.65 10.35 0.9990 3.45 8.77 0.9966 3.30 9.05 0.9996 1.48 4.50 0.9962 2.52 5.32 0.9988 2.18 3.00 0.9975
3.06 9.44 0.9983 3.57 10.33 0.9995 3.59 10.03 0.9996 2.24 8.89 0.9869 3.29 9.35 0.9992 3.53 9.24 0.9998 3.38 9.51 0.9991 1.67 5.32 0.9948 2.56 5.70 0.9993 2.16 3.28 0.9976
3.06 9.60 0.9994 3.05 8.72 0.9998 3.47 9.79 0.9991 2.03 8.38 0.9943 3.51 10.26 0.9998 3.47 9.19 0.9991 3.42 9.79 0.9998 1.31 4.38 0.9949 2.42 5.48 0.9996 2.08 3.31 0.9995
3.21 10.24 0.9991 3.28 9.67 0.9996 3.33 9.54 0.9994 2.43 9.80 0.9971 3.27 9.61 0.9988 3.29 8.77 0.9994 3.30 9.55 0.9999 1.74 5.93 0.9941 2.45 5.80 0.9990 1.86 2.87 0.9989
3.09 9.97 0.9988 3.25 9.74 0.9992 3.25 9.45 0.9989 2.46 10.05 0.9782 3.14 9.38 0.9997 3.28 8.92 0.9986 3.28 9.67 0.9994 1.38 5.02 0.9943 2.56 6.39 0.9988 1.81 3.00 0.9974
See footnote of Table VI. various mobile phase compositions (@"k/% 2-propanol): -930'/4%, -515'/7.5%, -119'/10%, 87'/15%, 98'/20%, and 146'/25%. The negative values of p a t lower percentage of 2-propanol indicate that a linear free energy relationship probably does not occur and multiple retention modes are operative. When the percentage of 2-propanol is greater than 15%, positive values are obtained which may imply realistic compensation temperatures. The existence of more than one slope in the same stationary phase for the same set of solutes indicates that a change of retention mechanism very likely occurs in both amino and diamine BPC's. The different behaviors of the two ortho isomers can be rationalized by the effects of intramolecular hydrogen bonding. Because of the possibility of bifunctional hydrogen bonding for the diamine column, it is more difficult to perform a detailed analysis on linear free energy relationships. However, the plots do show a similar pattern as those of amino columns indicating some similarities between the retentions of solutes in the two columns. Silica Columh. In the silica column the 10g k-AH plots show that the plot at 1% 2-propanol is different from those at 4%, 7%, and 10% (Figure 3). The latter three are similar to one another in pattern. It seems that both toluidines and chloroanilines behave similarly during the retention process because the points group together in the plots. On the other hand, it is likely that a change in retention mode may occur for nitroanilines which form a group by themselves. Linear AH-pKb Relationships. The enthalpy changes for toluidines (i.e., the compounds with small dipole moments) are much more negative than that of nitrobenzene (a highly polar molecule). Also, the retention times are always longer for toluidines. This indicates that the dipole-dipole interactions involved in the retention process cannot be a dominant factor controlling retention time. On the basis of the nature of the solutes and stationary and mobile phases, hydrogen bonding should be considered as a possible important factor
1 '/. \
\.
0
3s L
0
2.0
A
A
5,"
-a 0
0
0
0
E
Flgure 6. Plot of -AH vs. pK, value of solutes in a silica column using 2-propanoVheptane as eluent. The symbols for solutes are the same as those given in Figure 1.
in the retention process. Earlier we have shown that the plots of -iwvs. PKb values of solutes are linear at high concentrations of 2-propanol in n-heptane indicating that hydrogen bonding is probably occurring through the lone-pair electrons of the aromatic solutes (5). Plots of -AH vs. pKb for the three coluinns using 2-propanol/ n-heptane at different proportions as eluents are shown in Figures 6-8. Silica Column. In the silica column, the -AH values increase with increasing solute basicities except at 1% 2propanol (Figure 6). Because other types of interactions such
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
b
4,O%
1003
7.5 I
4.0.c Q/*"
10-A
3.0.
0
@
0
-AH
I
Flgure 9. Three posslble interaction modes in silica column using 2-propanollheptane as eluent: (a) direct interaction: (b) interaction through a layer of 2-propanol: (c) displacement.
2 .o
IO
12
10
14
12
14
P Kb
-
Flgure 7. Plot of -AH vs. pKb values of solutes in an amino column using 2-propanollheptane as eluent. The symbols for solutes are the same as those given In Figure 1.
3.0
i
I
a1
,/O4
20,o i
2.0
1
0
IO
12
el
li
10
12
14
PKb
Figure 8. Plot of -AH vs. pKb values of solutes in an diamine column using 2-propanollheptane as eluent. The symbols for the solutes are the same as those given in Figure 1.
as dipole interaction and van der Waals interaction may also be important at very low concentrations of polar modifier, the plot of -AH vs. pKb at 1%2-propanol is more scattered. However, when the concentration of 2-propanol increases, a good correlation is obtained. From the plots, it seems that the retention is through the lone-pair electrons of the solutes and no change of interaction type is observed. Amino and Diamine BPCs. Previous discussion of the experimental results makes it reasonable to consider that a gradual change of the interaction mode is likely for the re-
tention process as the concentration of 2-propanol in the mobile phase is changed. In the amino column, at 4% 2-propanol, -AH values increase with decreasing basicities for all ortho isomers (Figure 7). The same is true for meta and para isomers. However, in the 25% 2-propanol solution, the -AH values increase with increasing basicities of the solutes. Similar results are also found in the diamine column (Figure 8). These observations clearly show that there are at least two retention modes involving hydrogen bonding. The dominant one at low concentrations of 2-propanol in n-heptane is probably an interaction involving the protons of amino groups of the solutes. The other one a t high concentrations of 2propanol may be an interaction involving the lone pair electrons of the amino group of the solutes.
Retention Behaviors in Normal-Phase HPLC Separations. Several propositions have been made concerning the retention behaviors of solutes in normal-phase HPLC separations. One proposes that the solutes may directly interact with the surface of the packing material. If there are layers of solvents on the surface, two general possibilities may occur: (1)displacement of the adsorbed solvents by the solute so that the solute may interact directly with the surface; (2) the solute may interact with the surface through a layer (or layers) of adsorbed solvent. It is our belief that the exact processes should depend upon the relative stabilities of solute-stationary phase and solvent-stationary phase interactions. Thus, the interaction modes may change with a change of solutes and the eluents used in the retention process. The solutes used in this study are aromatic amines with relatively low basicities. Hydrogen bonding can occur at the lone pair electrons of solutes, the hydrogen atoms of solute amino groups, the oxygen in nitro groups of nitroanilines, and the chlorine atoms of chloroanilines. Silica Column. The effective retention sites in silica columns are believed to be the silanol groups. Therefore, several possible interaction modes involving hydrogen bonding for the solutes with the stationary phase can be proposed as follows (Figure 9): (a) direct hydrogen bonding interaction with the hydrogen of silanol group; (b) interactions with the alcohol hydrogen if the silanol is covered by a 2-propanol molecule; (c) displacement of the covering 2-propanol molecule and subsequent interactions with the hydrogen of silanol group. The increase of the values of -AH with increasing concentration of 2-propanol in the first three sets of data, i.e.,
1004
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
ai
\
C
)c
C
c1 \
Figure 10. Four possible interaction modes before the lone pair electron sites of amino BPC are occupied by 2-propanol: (a) direct interaction through lone pair electron sites of the stationary phase; (b) displacement; (c) direct interaction through amino hydrogens of the stationary phase: (d) interaction through adsorbed 2-propanol.
1,2, and 3% of 2-propanol in n-heptane, shows that a change of interaction mode is possible. From the previous discussion, it is strongly inferred that solutes interact through their lone-pair electrons. This in turn suggests that the mechanism is changed from case (a) to case (b) or (c) as 2-propanol concentration is increased in n-heptane. Since the type of solute interaction does not change, i.e., both modes operate through the lone pair electrons of the solutes, change in slope in the entropy-enthalpy compensation plot is not found. This proposition is consistent with a report by Scott and Kucera (21) that the silica column is completely covered by a layer of 2-propanol when the concentration of 2-propanol in nheptane is over 2%. The data can also be rationalized by the recently modified displacement theory (22). 2-Propanol and all anilines are considered to be localizing solvent and solutes, respectively. Thus, when anilines are not present in the column, the silica surface is covered with polar 2-propanol molecules at active adsorption sites and some nonlocalized n-heptane molecules. The relative surface population of solvent molecules is dependent on the concentration of 2-propanol in the mobile phase. A t low concentration of 2-propanol, some active sites are occupied by the nonlocalizing n-heptane molecules which can be easily displaced by aniline molecules. When the concentration of 2-propanol is high, restricted-access delocalization of 2-propanol can occur and displacement of both localized and delocalized 2-propanol molecules by aniline molecules is possible. The configurations of adsorbed anilines depicted in Figure 9 are assumed to be vertical. However, a flat configuration cannot be ruled out particularly when 2-propanol concentration is low. Limited data have shown that the interaction of the phenol OH group with silica surface apparently involves hydrogen bonding by a surface hydroxyl to the phenol oxygen atom, consistent with our findings for anilines (23). Amino and Diamine BPCs. The existence of more than one possible site for hydrogen bonding in amino and diamine BPCs is a complicating factor, and it makes the results more difficult to interpret. The retention mechanism is more complex than that in the silica column. Given that retention mechanisms are dominated by hydrogen bonding interactions it is reasonable to assume that at very low concentration of 2-propanol, retention involving
Flgure 11. Another two possible interaction modes after the second 2-propanol covering the amino hydrogens of the stationary phase: (a) interaction through the adsorbed 2-propanol; (b) displacement.
hydrogen bonding is due to the interaction between the strongly basic lone-pair electrons of the aliphatic amine of the stationary phase and the amino protons of the solutes (case a of Figure 10). The interaction with the residual silanol groups is certainly possible, particularly for toluidines. Note that nitro and chloro groups are not the major interaction functional groups as shown by the fact that the retentions of nitrobenzene and chlorobenzene are much weaker than that of aniline. As the concentration of 2-propanol is increased, the lonepair electron sites of the aliphatic amine of the stationary phase are gradually occupied by 2-propanol. (According to Hennion et al., each amino group can take up to three alcohol molecules (24)). When these positions are completely covered, three possible interaction modes can be suggested (Figure 10, cases b, c, and d): case b, solutes displace the 2-propanol and interact with the lone-pair electrons of the amino group of the stationary phase; case c, lone-pair electrons of the amino group of solutes interact with hydrogen atoms of amino groups of the stationary phase; case d, protons of the amino group of solutes interact with the lone-pair electrons of the hydroxyl oxygen of adsorbed 2-propanol, Le., interaction is through a layer of polar solvent. It has been shown that the interactions between nitroanilines and unreacted surface silanol groups are probably less important when 2-propanol concentration in n-heptane is high (25). Since hydrogen bonding between OH-:NH is generally stronger than that of NH--:NH (26),case b and case c are the less likely mechanisms of these three. Therefore, it is reasonable to assume that the interaction mode changes from direct solute-stationary phase interaction to an interaction which is through a layer of strong hydrogen-bonding solvent molecules. When a second 2-propanol molecule is adsorbed to an amino group of the stationary phase (% 2-propanol > lo%), another two interaction modes may become important: (a) the interaction between the alcohol hydrogen and the lone pair electrons of the amino group of solutes and (b) a displacement of the second 2-propanol by solute (Figure 11). However, because of the relatively weak hydrogen bonding for HN-HN, displacement is probably insignificant. Registry No. Aniline, 62-53-3; o-chloroaniline, 95-51-2; mchloroaniline, 108-42-9;p-chloroaniline, 106-47-8;o-nitroaniline, 88-74-4; m-nitroaniline, 99-09-2; p-nitroaniline, 100-01-6; otoluidine, 95-53-4; m-toluidine, 108-44-1;p-toluidine, 106-49-0.
Anal. Chem. 1985, 57, 1005-1009
LITERATURE CITED (1) Snyder, L. R.; Kirkland, J. J. “Introduction to Modern Liquid Chromatography”, 2nd ed.; Wiley: New York, 1979; Chapter 9. (2) Mourey, T. H.; Smith, G. A.; Snyder, L. R. Anal. Chem. 1984, 56, 1773-1777. (3) Klselev, A. V.; Aratskova, A. A.; Gvozdovitch, T. N.; Yashin, Ya. I. J. Chromatogr. 1980, 195, 205-210. (4) Yashin, Ya. I. J. Chromatogr. 1982, 251, 269-279. (5) Chang, C. A.; Tu, C.-F.; Huang, C.4. J. Chromatogr. Sci. 1984, 2 2 , 32 1-326. (6) Chow, F. K.; Grushka, E. J. Chromatogr. 1979, 185, 361-373. (7) Waddell, T. 0.; Leyden, D. E.; Debello, M. T. J. Am. Chem. Soc. 1981, 103, 5303-5307. (8) Unger, K. K.; Becker, N.; Roumeliotis, P. J. Chromatogr. 1978, 125, 115-127. (9) Bristow, P. A.; Brittaln, P. N.; Riley, C. M.; Williamson, B. F. J . Chromatogr. 1977. 131, 57-64. (10) “Guide to Mlcromerltics Model 705 Stirred-Slurry Column Packer”. (11) Chang, C. A.; Tu, C.-F. Anal. Chem. 1982, 5 4 , 1179-1182. (12) Soczewinski, E. Anal. Chem. 1989, 41, 179-182. (13) Snyder, L. R. ”Principles of Adsorption Chromatography”; Marcel Dekker: New York, 1968, Chapter 8. (14) Snyder, L. R.; Schunk. T. C. Anal. Chem. 1982, 5 4 , 1764-1772. (15) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1975, 112, 425-442. (16) Hussain, A.; Hurtubise, R. J.; Silver, H. F. J . Chromatogr. 1982, 252, 21-32. (17) Melander, W.; Cambell, D. E.; Horvath, C. J. Chromatogr. 1978, 158, 215-225.
1005
(18) Chang, C. A. Anal. Chem. 1983, 55, 971-974. (19) Krug, R. R.; Hunter, W. 0.; Grieger, R. A. J. Phys. Chem. 1976, 8 0 , 2335-2341. (20) Krug, R. R.; Hunter, W. G.; Grieger, R. A. J. Phys. Chem. 1978, 80, 2341-2351, (21) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1978, 149, 93-110. (22) Snyder, L. R. High-Perform. Liq. Chromatogr. 1983, 3 , 157-223. (23) Snyder, L. R. “Principles of Adsorption Chromatography”; Marcel Dekker: New York, 1968; p 307. (24) Hennion, M. C.; Picard, C.; Cambeilas, C.; Caude, M.; Rosset, R. J. Chromatogr. 1981, 210, 211-228. (25) Chang, C. A.; Huang, C.-S.; Tu, C.-F. Anal. Chem. 1983, 5 5 , 1390-1395. (26) Pimental, G. C.; McCiellan, A. L. “The Hydrogen Bond”; W. H. Freeman: New York, 1960; p 289.
RECEIVED for review October 11,1984. Accepted January 22, 1985. Acknowledgment is made to the Robert A. Welch Foundation of Houston, TX, and the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research. Support from National Institutes of Health-Minority Biomedical Research Support Program is also acknowledged. This paper was presented in part at the 1984 Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy.
Flow Injection Determination of Penicillins Using Immobilized Penicillinase in a Single Bead String Reactor R. Gnanasekaran’ and Horacio A. Mottola* Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
The enzyme penlclllinase [EC 3.5.2.81 lmmobillzed on glass beads has been made part of a single bead string reactor used In a flow Injectton sample processing system for the determination of penicllllns. Immoblllzation on a glass matrix, after ammonlum blfluorlde etching, uslng 95% ethanol, a short, rigid silane such as (amlnophenyl)trlmethoxysllane, and glutaraldehyde coupling provide preparations with relatlvely high activity, retalnlng 97% of the inltial activity after 10 months of dally usage. The single bead string reactor as part of a flow Injectton analysis system was used for the determination of penlcllllns In pharmaceutlcal tablets, Injectables, and fermentation broths by means of pH monltorlng. Penlcllllns G and V can be selectlvely determined In the 0.05-0.50 mM range and at a frequency of 150 injections per hour.
Several methods are available for the determination of penicillins and reviews of them can be found in the literature (2-4). None of these reviews, however, covers enzymatic methods employing immobilized enzymes. Enzymatic determinations of penicillins are based on monitoring directly or indirectly the amount of penicilloic acid resulting from the catalyzed (by penicillinase) hydrolysis of penicillins. The term penicillinase denotes p-lactamase preparations that catalyze the hydrolysis of the amide bond in the p-lactam ring of penicillins
I
H20. penicillinase c
penicillin
Analysis of samples in which the content of penicillins is of interest is a daily occurrence in pharmaceutical industries and some clinical laboratories. This exemplifies the need for analyzing a large number of samples of similar nature for the same chemical species. The use of unsegmented, continuous-flow procedures (flow injection analysis) allows, in such cases, the processing of a large number of samples per unit time. The coupling of immobilized enzyme reactors with flow injection analysis affords selective determinations and helps decrease the cost per determination. Different aspects of such reactors have been discussed recently (1). Present address: Department of Chemistry, Mansfield University, Mansfield, PA 16933.
0
i;\ F-CH3 /CH3
/I
R-C-NH-$H-CH
I
HOOC
l
l
HN-CH-COOH
(1)
penicilloic acid
Determinations of penicillins using immobilized penicillinase include those of Nihon et al. (5)making use of an enzyme electrode prepared by entrapping the enzyme in a liquid layer around a pH electrode with the help of a semipermeable membrane, Enfors (6) who designed an autoclavable enzyme electrode for the determination of penicillins in fermentation broths, Cullen et al. (7) who immobilized the enzyme by adsorption on a fritted glass disk, Rusling et al. (8) who used
0003-2700/85/0357-1005$01.50/00 1985 American Chemical Society
