ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1 9 7 8
minimum detectable quantity, defined as the concentration of sample t h a t produces a signal equal to twice the baseline noise, is about 3 x lo-" g/mL for choline or acetylcholine. While this detector is significantly less sensitive for these compounds than GC (10-13), no lengthy sample pretreatment is necessary. Figure 5 shows an application of the membrane detector to the determination of choline in a soy meal extract. The large, off-scale peak is Li', from LiOH, used to raise the p H of the extract. The second peak is choline, determined to be 2.6 mg/mL of extract. Details of this analysis, including extraction conditions and comparisons with other methods will be presented elsewhere.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
(IO) (11) (12)
M. E. Auerbach, Ind. Eng. Chem., Anal. Ed., 15, 492-493 (1943). S. Eksborg and 6.A. Persson, Acta Pharm. Suec., 8, 605-608 (1971). St. J. H. Bhkeiey and V. J. Zatka, Anal. Chim. Acta, 74, 139-146 (1975). S. 0. Jansson, R. Modin, and G. Schill, Talanta, 21, 905-918 (1974). R. Modin and S. Back, Acta. Pharm. Suec.. 8, 585-590 (1971). B. A. Persson, Acta Pharm. Suec., 8, 217-226 (1971). C. Radecka, K. Genest, and D. W. Hughes, Arzneim.-Fwsch, 21, 548-550 (1971). W. F. H. McLean and K. Jewers, J . Chromatogr., 74, 297-302 (1972). S. E. Brooker and K. J. Harkiss, J . Chromatogr., 89, 96-98 (1974). P. I.A. Szilagyi, D. E. Schmidt, and J. P. Green, Anal. Chem., 40, 2009-2013 (1968). D. J. Jenden, R. A. Booth, and M. Roch, Anal. Chem., 44, 1879-1881 (1972). D. E. Schmidt and R. C. Speth, Anal. Biochem., 67, 353-357 (1975).
1333
(13) J. L. W. Pohlmann and S. L. k h a n , J. Chromatcgr., 131, 297-301 (1977). (14) 1. Hanin and R. F. Skinner, Anal. Biochem., 66, 568-583 (1975). (15) C. G. Hammar, I.Hanin. B. Holmstedt. R J. Kitz. D. J. Jenden, and B. Karien, Nature (London). 220, 915-917 (1968). (16) S. Eksborg and G. Schiil, Anal. Chem., 45, 2092-2100 (1973). (17) J. S. Hayes, M. A. Alizade, and K. Brendel, Anal. Chim. Acta, 80, 361-367 (19751. (16) D. Speed and M. Richardson. J . Chrorrtatogr , 35, 497-505 (1968). (19) F. Chastellain and P. Hirsbrunner, Fresenius' 2. Anal. Chem., 278, 207-208 (1976). (20) W. K. Gnrley, C. D. Haas, and S. Bakermarl, Anal. Biochem., 19, 197-200 (19671. (21) T. W.'Gilberi and R. A. Dobbs, Anal. Chem., 45, 1390-1393 (1973). (22) Larry C. Hansen, Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio, 1973. (23) M. D. Arguello and J. S. Fritz, Anal. Chem., 49, 1595-1598 (1977). (24) W. K. W. Chen, R. B. Mesrobian, D. S. Baihntine, D. J. Metz, and A. Glines, J . Poiym, Sci., 23, 903-913 (1957) (25) Richard A. Dobbs, Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio, 1973. (26) D. J. Jenden and L. B. Campbell, in "Methods of Biochemical Analysis, Supplemental Volume", D. Glick, Ed., Interscience, New York, N.Y. 1971. (27) I. A. Fowliss and R. P. W. Scott, J . Chromatogr., 11, 1 (1963).
RECEIVED for review December 19, 1977. Accepted May 8, 1978. J.G.D. gratefully acknowledges the University of Cincinnati Research Council for support from a Summer Fellowship. M.S.D. gratefully acknowledges the University of Cincinnati for support through a Twitchell Fellowship. Partial support of this work in the form of a Frederick Gardner Cottrell Grant from the Research Corporation is also gratefully acknowledged.
Evaluation of a Computer-Controlled Stopped-Flow System for Fundamental Kinetic Studies Glen E. Mieling and Harry L. Pardue" Department of Chemistry, Purdue University, West Lafayetfe, Indiana
This paper describes the application of a computer-controlled stopped-flow system for a kinetic study of the Fe(II1)thiocyanate reaction. Kinetic equations are developed for a proposed mechanism involving two parallel pathways, and results of some 456 experiments run under computer control are Interpreted and processed to provide expliclt values for forward and reverse rate constants for the proposed mechanism as well as activation and thermodynamic parameters. Of the 456 experiments run, three were rejected as being inconsistent with other data and only nine were lost completely because of an inadequate supply of stock reagent. The imprecision was about 0.03 s-' for rate constants in the range of 1.8 to 5 s-'.
The stopped-flow mixing method represents one of the most useful techniques for kinetic studies involving reactions with half-lives in the range from a few milliseconds to a few seconds. Because the method generates data a t a relatively rapid rate, some investigators have used small laboratory computers on-line with stopped-flow instrumentation (1-3). While these innovations have greatly improved the convenience and reliability of stopped-flow studies, most of them do not take full advantage of the capabilities of the computers because they are used only for data acquisition and processing. Coupled with automatic reagent and sample handling equipment, a
47907
small computer can also control and execute most of the operations required to carry out major phases of stopped-flow kinetic studies. An earlier report from this laboratory described a unique sampling/mixing system that permitted control of the sampling and mixing steps via electrical switches ( 4 ) . A more recent report described a reagent preparation system that could mix up to five solutions and one diluent in a wide range of proportions and deliver many such solutions in sequence to the sampling/mixing system ( 5 ) . The total integrated system is controlled by a computer system so that after stock reagents are supplied and the conditions for several experiments are entered into the computer via Teletype, then all steps required to perform the desired experiments and process the data are carried out automatically under computer control and without operator intervention. Although data were presented to illustrate some features of the quantitative performance of the system, no data were presented to evaluate performance for a detailed kinetic study and that is the subject of this paper. In this work, the Fe(II1)-SCN- reaction is used as a model system to further evaluate the performance of the computer controlled stopped-flow instrument. Although the thermodynamic and spectral properties of' the monothiocyanato complex of Fe(II1) in acid solution have been well characterized (6-8), there are significant discrepancies among kinetic rate constants reported for the system (9-23). Therefore, in
0003-2700/78/0350-1333$01.00/0 0 1978 American Chemical Society
1334
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
addition to evaluating t h e automated stopped-flow system, this study also has provided useful information related to the model reaction.
GENERAL CONSIDERATIONS T h e rationale for experiments performed in this study is best described in terms of a proposed mechanism and rate equation for the reaction. Earlier work provided evidence for a reaction between Fe3+(,,) and SCN- (14). Later work that demonstrated increases in reaction velocity with p H (9) suggests t h e probable involvement of a hydroxy complex of Fe(II1). An overall reaction scheme that accounts for these observations is kl
Fe3+(,q)+ SCN- -=
1
Khl
Fe(OH)Zt + SCN-
FeSCN"
-1 k2
Kh2
Fe(0H)SCN"
k-2
+
(1)
+ H'
H'
where Khl and Kh2are equilibrium constants for hydrolysis steps that are assumed to be very fast in comparison with the other processes. Starting with a rate equation in terms of forward and reverse rate constants and concentrations of all species in Equation 1, and applying conservation of mass equations, it can be shown that this reaction scheme leads to a rate equation of t h e form -
d[SCN-] dt
= (hl
+ kzKhl/[H+])[Fe3+][SCN-] (k1+ h-2Kh2/[H+])[FeSCN2+] ( 2 )
If the Fe3+ concentration is maintained at least 20 times larger t h a n the SCN-. concentration (CFe(III)7 20 [SCN-I), then reactions 1 can be treated as two competing first-order reactions, a n d t h e integrated form of the rate equation under this set of conditions is
In
(
[&I
[FeSCN2+], [FeSCN2+],
-
>-
[FeSCN2+],
+ h2K,,l/[H+l)[Fe3+l + (h-I + k-2Kh2/[H+l)lt
(34
where [FeSCN"], represents the concentration of this species when t h e reaction is at equilibrium. In the discussion t h a t follows i t will simplify notation to refer to t h e quantities in parentheses on t h e right side of Equation 3a as the forward a n d reverse constants ( k fE k l + k2Khl/ [H+] and k , E k - l + k-ZKh2 / [ H+I ). It has been observed t h a t t h e absorbance a t 460 nm is proportional t o [FeSCN,'] = 4.6 X lo3 L/mol cm). Therefore, Equation 3a can be rewritten in the simplified form
In ( A ,
-
A,) =
-
(hf[Fe3+]
+ h,)t + In AA
(3b)
where AA is t h e total change in absorbance from t = 0 to equilibrium (AA = A, - Ao). It is apparent from Equation 3b that plots of In (A, - A,) vs. t should be linear if t h e previously proposed mechanism a n d simplifications are correct. Also, t h e slope should have t h e form of an apparent first-order rate constant
ha = hf[Fe3+]
+ k,
(4)
If values of k , are evaluated at different values of Fe3+ concentration for fixed values of [H+],then plots of k , vs. F@+ for each [H+] concentration should be linear with slope equal t o k f and intercept equal to k,. Noting t h e quantities represented by h f and k,, it follows t h a t a plot of kf vs. l / [ H + ] should be linear with slope equal to k2Khland intercept equal
to h l , and a plot of k , vs. l / [ H + ] should be linear with slope equal to k-,Kh2 and intercept equal to kl. Previously determined values of Khl and Kh2can be used to compute explicit values for k 2 and k-, (7, 15). Clearly, all of the rate constants included in reactions 1 can be determined by the procedures outlined above. However, in order to avoid large uncertainties t h a t can arise from the data processing procedure alone, especially those involving extrapolated intercepts, it is important that all determinations be based upon several data points over a relatively wide range of the independent variable in each case. One set of experiments designed to evaluate these rate constants at 25 "C involved triplicate runs at seven different Fe3+ concentrations a t each of eight perchloric acid concentrations (see Table I) corresponding to a total of 168 experiments. A second set of experiments designed to evaluate the temperature dependence of the rate constants and associated information involved duplicate runs on each of 48 solutions a t each of three temperatures corresponding to 288 experiments. These types of studies that involve large numbers of experiments run under well defined conditions are ideally suited to automation and serve as excellent examples for evaluation of the computer controlled stopped-flow system.
EXPERIMENTAL Apparatus. Details of the stopped-flow system including computer hardware and software concepts have been presented earlier ( 5 ) and are not discussed here. Reagents. All solutions were prepared in distilled water that had been passed through a mixed cation-anion exchange resin bed. Iron(ZZZ) Perchlorate. A 0.077 mol/L iron(II1)perchlorate stock solution in 0.200 mol/L HC10, was prepared from reagent grade Fe(C104)3(G. Frederick Smith Chemical Co., Columbus, Ohio) and standardized potentiometrically at pH 2 with 0.010 mol/L standard EDTA containing no NTA (J. T. Baker Chemical Co., Phillipsburg, N.J. 08865). Perchloric Acid. A 2.354 mol/L perchloric acid stock solution was prepared from reagent ACS grade HCIO,, 70% (Fisher Scientific Co., Fair Lawn, N.J. 07410) and standardized against weighed amounts of Baker Analyzed Reagent grade anhydrous Na2C03(J. T. Baker Chemical Co., Phillipsburg, N.J. 08865) using methyl orange as indicator. Sodium Perchlorate. A 1.413 mol/L sodium perchlorate stock solution was prepared by diluting 2.354 mol/L perchloric acid stock solution with water. The pH of the stock NaC10, solution was adjusted to 7.0 with analytical reagent grade anhydrous Na2COJ(Mallinckrodt, Inc., St. Louis, Mo. 63147) and the solution was heated to remove dissolved carbon dioxide. mol/L sodium thiocyanate Sodium Thiocyanate. A 2.0 X stock solution in 1.000 mol/L NaC104 was prepared from dried Baker Analyzed Reagent grade NaSCN (J. T. Baker Chemical Co., Phillipsburg, N.J. 08865) and the appropriate amount of 1.413 mol/L NaClO, stock solution. Procedure. The reagent preparation system was calibrated by dispensing water into preweighed vials for different amounts of time, weighing the vials, and computing the least squares slope and intercept of the mass vs. time data. Stock solutions of iron(II1) perchlorate, perchloric acid, sodium perchlorate and water are placed in containers with delivery tubes to the reagent preparation system and are adjusted to and maintained at 25 "C in a water bath. Desired volumes of these three solutions and water are dispensed into 30-mL vials on a turntable and each solution is mixed with a magnetic stirring bar placed in each vial at the start of a series of experiments. The turntable delivers each different reagent to the sampler/mixer feed line. A stock solution of sodium thiocyanate maintained at 25 "C is connected to the sample side of the sampler/mixer via a delivery tube. For the temperature study, solutions prepared with the reagent preparation system were adjusted to the desired temperature prior to being run on the stopped-flow. When all reagents are in place, all instrumental and photometric adjustments are complete, and all pertinent information related to the desired experiments is entered into the computer, then the
ANALYTICAL CHEMISTRY, VOL. 50, NO.
9,AUGUST 1978 -
1335
Table I. Effects of [ H’]and [ Fe3+]upon Apparent First-Order Rate Constant at 25 “ C 0.0050
CHC1O,,
CFe(ClO,),, ( m o w ) 0.0100 0.0125 0.0150 Apparent first-order rate constants (s-’)
0.0075
mol/L
a
0.200 0.225 0.250 0.275 0.300 0.325 0.350 0.375 Standard
0.03= 2.79 i 0.03 3.23 i. 0.06 3.73 i 0.03 2.58 i 0.03 3.11 i 0.04 3.49 ? 0.01 0.01 2.58 i 0.03 2.94 i 0.04 3.30 i 0.02 0.03 2.42 i 0.03 2.85 t 0.03 3.20 i 0.04 0.02 2.35 i. 0.03 2.74 t 0.02 3.07 0.04 0.02 2.67 i 0.02 2.99 t 0.03 0.02 2.27 i 0.01 0.02 2.23 i 0.02 2.62 i 0.02 2.90 I0.05 2.51 i 0.003 2.81 i 0.02 1.81 i 0.01 2.18 i 0.02 Point not used. Stock deviation of three replicate runs. 2.29 i 2.17 i 2.07 i 2.01 i 1.91 i 1.89 f 1.85 i
4.03 i 0.03 3.84 i 0.02 3.71 i 0.07 3.54 i 0.02 3.49 i 0.04 3.38 t 0.01 3.27 + 0.05 3.19 i 0.01
--
0.0175 4.47 i 4.28 i 4.09 i [3.90 i 3.87 i 3.71 i 3.70 i 3.56 i
0.03 0.02 0.03 0.O1lb 0.07 0.02 0.02 0.01
0.0200 4.99 4.76 4.52
i
0.05
t
0.02 0.01
i
C
4.16 4.08
i
i
0.02 0.04
C
c
reagent depleted.
Table 11. Regression Data for Apparent First-Order Rate Constant vs. Fe3+Concentration std error CHClO,, slope, intercept, of estimate, mol/L k f , Limo1.s k,, scl S-’ 0.200 0.225 0.250 0.275 0.300 0.325 0.350 0.375
A ‘f --
2-
175 i 170i 1592 154 i
3 2 2 1 150* 2 145 i 1 144 i. 2 138+1
1.46 t 0.04 1.34 ? 0.03 1.32 c 0.03 1.27 t 0.03 1.20 t 0.03 1.19 i 0.02 1.14 I0.03 1.12 t 0.02
0.06 0.05 0.04 0.04 0.05 0.03 0.04 0.02
I
,
“ZZ
-
_. ’A 7_
1 -
1
1
L.-.u I
-E-,
,TL
I
- - < _.___
i.ZX
1.625
Flgure 1. Plots of apparent rate constant vs. Fe3+concentration. [H’]: (0)0.200;(0)0.255;( X ) 0.250;@e) 0.275;(6) 0.300;(+) 0.325; (0)0.350; (A)0.375. system is activated and the experiments are carried out automatically except for a manual 100% T adjustment during the first experiment.
RESULTS AND DISCUSSION I n the study at 25 O C , data were obtained for all but three of the 56 sets of conditions represented in Table I. Each experiment included a trial run during which the computer selected a data rate t h a t was appropriate for the range of reaction rates for that set of conditions. After the trial run, then three replicate runs were made on one solution prepared for each set of conditions. The 162 runs represented by the data in Table I required approximately 7 h for completion after all stock reagents and program information were set up. The only operational problem encountered during that period was related to a n insufficient supply of some of the stock solutions (see Table I). Each datum in Table I represents the average of three values of the apparent first-order rate constant based upon the slope of In ( A , - A , ) vs. t plots (See Equation 3b). The uncertainties quoted in the table represent the standard deviation (*lS) of the three results averaged in each case. Standard deviations of individual least squares slopes of the In ( A , - A,) vs. t plots from which the averages were computed ranged between 0.002 and 0.005 s-l. Therefore, most of the uncertainty included for each datum is related to differences among runs and very little of it is related to within run variations. Figure 1 shows the observed rate constants a t each acid concentration plotted vs. Fe3+concentration a t 25 OC. These plots exhibit the linear relationship predicted by Equation 4. The least squares slopes and intercepts that represent Kf and K, are included in Table I1 along with standard deviations
Figure 2. Plots of k , and k , as functions of [H+]-’. (A) k , vs. [H+]-’; slope = 15.9 0.8;intercept = 97 f 3. (0)k , vs. [H+]-’; slope = 0.140 f 0.008;intercept = 0.75 f 0.03.
*
of slopes and intercepts and the standard errors of estimate. The standard errors of estimate indicate that the upper limit for the scatter in the rate constants imposed by the total system including reagent preparation, sampling and mixing, measurement, temperature control, etc. is in the range of 0.02 to 0.06 s-’. The individual values of k f and k,. in Table I1 are plotted vs. 1/[H+] in Figure 2 producing linear plots as predicted by Equation 3a. Least-squares values of the intercepts correspond to kl = 97 f 3 L/rnol.s and k-l = 0.75 f 0.03 s-l. Values of Khl = 1.65 x 1 0 -mol/L ~ (15) and Kh2 = 6.5 x mol/L (7) are used with the slopes to compute k z = (9.6 & 0.5) X lo3 L/mol-s and k..2 = ( 2 . 2 & 0.1) X 10’ s-]. These values of rate constants are summarized in Table III along with values obtained by other workers using a variety of methods. The value for k-z included from an earlier paper (11) is based on a different mechanism that uses the dissociation constant for water where we used Kh2 to compute k-z. The slope of the k , vs. l / [ H + ] from that paper is 0.19 mol/L.s
1336
ANALYTICAL CHEMISTRY, VOL. 50,
NO. 9,
AUGUST 1978
Table 111. Compilation of Rate Constants a t 2 5 C from Several Studies method (ref.) (ionic strength) stopped-flow ( 9 ) ( F = 0.4) pressure jump ( 10 ) ( p = 0.5) stopped-flow ( 11 ) (p =
0.5)
pressure jump ( 12 ) (1 = 0.2) temperature jump ( 13 ) ( p = 0.5) present work (F =
k , , Limo1.s 127 i 1 0
h , , Lirno1.s
150
i
50
(2.6
i
0.2) x 104
90
f
5
(5.1
i
0.5) x
1 3 2 i 50
(4.2
i
0.5) x
130 c 40
(1.3
i
0.2) x 104
i
5 ) x 103
97
i
3
k - l , s-'
k.2, s-'
1.6 i 0.2
(1.0I 0.1)x 1013
(1.0f 0.1)x 1 0 4
(9.6
1)
lo3 lo4
0.75
i
(2.2 i 0.1)x
0.03
1 0 3
-
Table IV. Regression Data for Apparent First-Order Rate Constant vs. Fe3+Concentration std error estiCHClO,, slope, intercept, mate, moi/L T , " C kf Limo1.s k, s - ' S20 i 0.1 106.1 c 0.4 0.826 i 0.003 0.005 0.200 25 i 0.1 172 t 4 1.37 * 0.03 0.04 30 i 0.1 271 + 4 2.71 t 0.03 0.04 20 i 0.1 99.6 i 0.2 0.799 i 0.002 0.002 0.225 25 i 0.1 165 f 2 1.27 i 0.03 0.02 30 i 0.1 254 f 4 2.00 i 0.04 0.04 20 i 0.1 97.0 i 0.4 0.759 i 0.003 0.004 0.250 25 i 0.1 158 i 1 1.23 i 0.01 0.008 30 i 0.1 238 i 2 1.94 i 0.02 0.03 20 i 0.1 94.1 i 0.4 0.735 i 0.003 0.004 0.275 25 i 0.1 151 i 3 1.20 i 0.03 0.04 30 i 0.1 227 i 2 1.86 i 0.02 0.03 20 -i- 0.1 91.1 t 0.3 0.713 i 0.003 0.01 0.300 25 i 0.1 148 f 1 1.14 i 0.01 0.01 30 i 0.1 215 i 4 1.79 t 0.04 0.06 20 i 0.1 88.8 i 0.2 0.696 i 0.003 0.01 0.325 25 i 0.1 141 c 3 1.13 i 0.03 0.04 30 i 0.1 212 i 2 1.71 * 0.02 0.04 20 i 0.1 86.8 i 0 . 3 0.679 i 0.003 0.004 0.350 25 i 0.1 142 k 2 1.07 i 0.02 0.03 30 i 0.1 205 i 2 1.68 i 0.02 0.02 20 i 0.1 84.6 i 0.2 0.668 i 0.002 0.003 0.375 25 i 0.1 133 i 3 1.10 i 0.03 0.02 30 i 0.1 204 i 1 1.60 i 0.01 0.02 compared with a value of 0.14 mol/L.s obtained in this work, mol/L to and when t h a t slope is used with Kh2 = 6.5 X compute k2,a value of 2.9 X lo3 is obtained in comparison with t h e value of 2.2 X lo3 obtained in this work. Little can be said about the other differences among the constants except t o note the large quoted uncertainties associated with some values of hl and to mention that the earlier stopped-flow data were evaluated manually from oscilloscope traces. One other comparison involving k , and k can be made. T h e ratio k l / k l = 129 i 6 should represent the equilibrium
-08 ,
c
40
I 3 35
330
3 40
(I/T0K)xlO3
Figure 3. Replots of temperature dependent rate constant data. (A) k1, ( 0 )k,, (A)k-1, (W k-2
constant for the hydroxide independent step in reaction 1. This value compares favorably with values of 125 f 2 and 132 f 1determined by potentiometric (16)and spectrophotometric (7) procedures, respectively. The equilibrium constant for the second step in reactions 1 can be estimated as k , / k _ 2 = 9.6 x 103/2.2 x l o 3 or 4.4 f 0.1mol/L. T h e second set of data involved measurements at 20, 25, and 30 "Cfor similar conditions represented for the 25 "Cdata in Table I. Individual values of rate constants a t several Fe(II1) concentrations were used to evaluate hf and k , as described earlier, and the results are presented in Table IV. Values of s h l = 10.2 kcal/mol (15) and f l h 2 = 12 kcal/mol (assuming the same 1.9 for both Khl and K h 2 and estimating AHh2from its free energy) were used with values of k f and k , in Table IV t o compute the forward and reverse rate constants for reactions 1. Results are included in Table V. The activation parameters included in Table V were computed from weighted least squares slopes and intercepts of t h e In ( k / n vs. 1 / T plots shown in Figure 3. T h e thermodynamic constants were evaluated from the differences between the forward and reverse activation parameters for the two reaction pathways in Equations 1 above. Literature values for the thermodynamic constants under similar conditions for the pH independent reaction vary from -1.5 (8)
_ I
Table V. Resolved Rate Constants at Various Temperatures T, "C k , , Limo1.s k ,,s-' 62i 1 0.481 i 0.006 20 i 0.1 0.75 i 0.03 25 i 0.1 93 i 3 120 i 0.6 1.09 i 0.05 30 i 0 . 1 Activation parameters A H , kcalimol 11.8 i 0.6 13.9 i 0.6 A s + , eu -9 i 2 -13 i 2
k,(L/mol.s) x 7.2 * 0.3 9.7 t 0.5 13.6 i 0.5 10.8 I 0.8
-3.5
z
2
t i
0.8
Thermodynamic constants A H , kcal/mol A S . eu
-2.1 i 0.8 4 + 3
4.8
20.2
2
k.*(s-') x
1.50 * 0.03 1.8 i 0.1
2.2
i
0.2
6.0 5 0.1 -23.4 t 0.4
ANALYTICAL CHEMISTRY. VOL. 50, NO. 9, AUGUST 1978
1337
LITERATURE CITED
to -1.3 (17) kcal/mol for AH and from 4 ( 8 ) to 5 (17) eu for AS, and these values are within the 95% confidence level uncertainties of values reported in Table V. Some checks for internal consistency can be made between the two d a t a sets run several months apart. The standard errors of estimate for the data in Tables I1 and IV reflect the maximum scatter in the data imposed by the total system. The standard errors at 20 "C are consistently lower than those a t 25 and 30 "C, and this may reflect better temperature control for the experiments nearer room temperature. When the rate constants in Table I are regressed against rate constants for the same conditions for the other data set a t 25 "C, the slope and intercept were 0.95 f 0.01 and 0.03 f 0.03, respectively, with a standard error of estimate of 0.04 s-' and a correlation coefficient of 0.997. T h e slope shows that t h e combined effects of chemical and instrumental variables contribute a difference of only 5% between the two data sets. We believe the results presented above represent convincing evidence t h a t the computer controlled stopped-flow system has much to offer for aspects of fundamental kinetic studies that involve large numbers of experiments under reasonably well defined conditions. Results presented elsewhere (18) described t h e performance for routine kinetic analyses.
R. J. Desa and 0.H. Gibson, Comput. Biomed. Res., 2, 494 (1969). B. G. Willis, J. A. Bittikofer, H. L. Pardue, and D. W. Margerum, Anal. Chem., 42, 1340 (1970). P. M. Beckwith and S. R. Crouch, Anal. Chem.. 44, 221 (1972). D.Sanderson, J. A. Bittikofer. and H. L. Pardue, Anal. Chem., 44, 1934 (19721. G.E. Mieling, R. W. Taylor, L. G. Hargis, J. English, and H. L. Pardue, Anal. Chem., 48, 1686 (1976). G. S. Lawrence, Trans. Faraday Soc., 52, 236 (1956). M. W. Lister and D. E. Rivington, Can. J . Chem., 33, 1572 (1955). V. E. Mironov and Yu. I. Rutkovskii, Zhur. Neorg. Khim., 10, 2670 (1965). J. F. Below. R. E. Connick. and C. P. Cotmel. . . J , A m . Chem. Soc.. 80. 2961 (1965). H. Wendt and H. Streklow, Z . Eiektrochem., 66, 228 (1962). S. Funakaski, S.Adachi, and M. Tanoka, Bull. Chem. SOC.Jpn., 46, 479 (19731. F P Cavasino and M Eigen, Ric S o , Part 2 Sez A , 4, 509 (1964) D M Goodall P W Harrison M J Hardv. and C J Kirk. J Chem Educ , 49, 675 (1972). H. E. Bent and C. L. French, J . Am. Chem. SOC., 63, 568 (1941). R. M.Milburn, J . Am. Chem. Soc., 7 9 , 537 (1957). R . Portanova et al.. Gazz. Chim. Ita/.. 98. 1290 (1968). "Critical Stability Constants, Vol. 4: Inorganic Complexes", R . M. Smith and A. E. Martell, Plenum Press, New York and London, 1976. 23, 1230 (1977). H. L. Pardue et al.. Clin. Chem. ( Winston-Salem, N.C.),
RECEIVED for review December 28, 1977. Accepted May 12, 1978. This work was supported by Grant No. CHE 75-1550 A01 from the National Science Foundation.
In-Situ Chemically-Modified Surfaces for Normal-Phase Liquid Chromatography R. K. Gilpin" and W. R. Sisco Research Division, McNea Laboratories, Camp Hi// Road, Fort Washington, Pennsylvania 79034
Another application for bonded phases is as adsorptive (Le., normal-phase mode) packings. A few workers have reported normal-phase studies on bonded materials (9-14). In this mode, even greater potential in selectivity may eventually be realized. T h e feasibility of forming bonded siloxane phases totally in-situ on porous layer materials and on completely porous small-particle silica has been demonstrated (15,16). These materials were evaluated for use in a reversed-phase mode. In this paper, three short-chain trichlorosilane modified silica gels for use as normal-phase high pressure liquid chromatographic packings have been prepared by a similar in-situ approach. Detailed investigations of efficiency and selectivity on these short-chain packings have been carried out using a series of 14 test solutes. All compound retentions have been examined on a relative basis using aniline as the reference compound.
I n this paper, the preparation of three short-chainlength trichlorosilane (n-butyl, 2-carbomethoxyethyl, and 3-cyanopropyl) modified silica gels by a totally in-situ process is reported. Detailed investigations of efficiency and selectivity of these modified materials along with unmodified silica have been carried out in a normal-phase mode using a series of 14 test solutes with water-saturated n-hexane as the mobile phase. All compound retentions have been examined on a relative basis using aniline as the reference solute. Columns have been found to vary in polarity in the order: silica, n-butyl, 2-carbomethoxyethyl, 3-cyanopropyl. I n addition, columnto-column reproducibility of these packings has been evaluated for multipreparations. Typical average deviations of relative retention for the 14 test solutes have ranged between 4 4 % .
In recent years t h e number of commercially available high-efficiency liquid chromatographic packings has grown a t a remarkable rate ( 1 , 2). This has been especially true for the chemically modified materials (3, 4 ) . In parallel growth has been the number of literature references of the application of these materials to analytical problems. In addition a number of reports have appeared describing the experimental preparation and characterization of various bonded-phase materials for liquid chromatographic application. These have been extensively reviewed in t h e literature (3-8). I n a majority of t h e reported studies and applications, bonded-phase materials have been used in the reversed-phase mode ( 4 ) ,with a combination of either water or an aqueous buffer and either methanol or acetonitrile as the mobile phase. 0003-2700/78/0350-1337$01.00/0
EXPERIMENTAL Column Preparation. The 2.4-mm i.d. by 25 cm stainless steel columns were slurry-packed with LiChrosorb SI 60 silica (av. dp -10 pm) as previously described (16). Following packing, each column was conditioned with 100 mL, of water, methanol, isopropanol, diethyl ether, 1,2-dichloroethane,and at least 500 mL of water-saturated n-hexane. After conditioning, all columns were characterized in terms of efficiencies losing nitrobenzene as the test solute and a mobile phase of water-saturated n-hexane. In addition, capacity factors for each test solute were calculated using benzene as the unretained peak. These data were obtained from duplicate injections using 6 to 8 different linear velocities covering a range from 0.11 to 1.1 cm/s (0.3 to 3.0 mL/min). Following packing and evaluation, suitable columns were bonded in-situ as described (16). In each case, 100 mL of C
1978 American Chemical Society
