Principles and Methods of External Photochemical Titrant Generation H. D. Drew’ and J. M. Fitzgerald2 Department of Chemistry, Seton Hall University, South Orange, N. J . 07079
BRICKER AND SCHONBERG (I)first demonstrated that the rate of photochemical reaction could be made to proceed reproducibly from day to day and developed a method, called photochemical titration, wherein a titrant is generated by photons much as electrons are used in coulometric titrations. Photochemical titrations reported to date all use an in situ method; that is, the sample and the titrant generator are photolyzed together using an ultraviolet source of constant intensity. A bibliography of in situ methods is available (2). The analogy between photochemical and coulometric titrant generation can be extended to external photochemical generation, which has characteristics similar to external coulometric generation developed by DeFord and coworkers (3). It is reasonable to assume that problems similar to those of in situ coulometry will be encountered with the photochemical technique. For example, using high-energy photons to irradiate a sample might cause photolysis of the sample as well as titrant generation. Internal filter effects (4) and solvents which are strong UV absorbers would prevent use of in situ photolysis. Unlike most electrochemical processes, primary photochemical reactions are often followed by “dark” reactions involving free radicals formed in the primary process. Such is the case in the system employed in the present study. The overall quantum efficiency for titrant production requires a constant efficiency of the secondary, “dark,” reaction to produce iron(I1) (5). External generation allows the photochemical conversion to proceed to completion without interference. The important parameters for external photochemical titrant generation were studied with the iron(III)-oxalate system, which has been well characterized ( 5 , 6 ) and has been used for in situ photochemical generation ( I ) . In the latter case, small irregularities were reported in stoichiometry for titration of vanadium(V) (I). The cause is a slow, thermal oxidation of oxalate by vanadium(V) (7). By the use of external generation, iron(I1) can be photogenerated in oxalate medium and then added to vanadium(V), thus minimizing the time for the vanadium-oxalate side reaction. It is also important to consider the application of the general rate equation for photochemical titrant generation to the case of external generation. The rate of formation of titrant, B, Present address, Chemistry Faculty, Southern Illinois University, Edwardsville, Ill. 62025 * Present address, Department of Chemistry, University of Houston, Houston, Texas 77004 (1) C. E. Bricker and S. S. Schonberg, ANAL.CHEM., 30,922 (1958). (2) R. J. Lukasiewicz and J. M. Fitzgerald, Anal. Letters, 1, 455 (1968). 23, (3) D. D. DeFord, J. N. Pitts, Jr., and C. Johns, ANAL.CHEM., 938 (1951). (4) W. A. Noyes, Jr., and P. A. Leighton, “The Photochemistry of Gases,” Dover Publications, Inc., New York,N. Y., 1966, p 152. (5) C. A. Parker and C. G. Hatchard, J. Phys. Chern., 63,22 (1959). 38, 897 (1966). (6) W. M. Riggs and C. E. Bricker, ANAL.CHEM., (7) H. A. Taylor, Ph.D. Thesis, Princeton University, Princeton, N. J., 1%3. 974
ANALYTICAL CHEMISTRY
Figure 1. Apparatus for external photbchemical titrant generation R--Solution reservoir; P-Pump; F-Flow meter; R.Z.-Quartz W i g (reaction zone); L-Germicidal lamp; S-Sliding shutter; T-Bubble trap; V-Teflon (DuPont) leak valve; E-Titrant exit; B-Titration vessel; M-Magnetic stirrer.
from the generator, A, by the absorption of energy, hu, is given by :
where C , and C A are the analytical concentrations of photoproduct and titrant generator, Q is the overall quantum yield, l o is the incident intensity of photochemically effective photons, and the exponential term simply represents the absorbance of the solution at the wavelength used (8). EXPERIMENTAL
Apparatus. The flow chart for the apparatus is shown in Figure 1. The photogenerator was pumped at a constant rate past the photolysis source by a bellows pump (Manostat #72-894-2). The flow rate was measured with an in-stream meter (Manostat #36-54145) and varied by a Teflon (DuPont) leak valve (Manostat #78-425-01). The emerging photolyzed solution was delivered to the titration vessel. A 57-cm low-pressure mercury discharge vycor lamp (Hanovia Model 94A-1, Engelhard Inc., Newark, N. J.) was used for photolysis, A 60-cm length of 6-mm i.d. quartz tubing was mounted parallel to the lamp and about 5 cm away. A sliding shutter, placed between the lamp and the quartz tubing, could be positioned so that only a portion of the tube was exposed to the source, The fraction of tube length used for photolysis is referred to hereafter as the reaction zone. The lamp and tubing were covered with an aluminum reflector to increase the radiation flux and also to protect the operator. The special distribution of the lamp was determined with a Beckman DK-2A spectrophotometer in the emission mode. The main wavelength from the germicidal lamp was at 2537A (Table I). The input voltage to the lamp transformer was controlled at 120 V ( I , 2). (8) J. M. Fitzgerald, R. J. Lukasiewicz, and H. D. Drew, Anal. Letters, 1, 173 (1967).
~~
The flow meter was calibrated and the stability of the system evaluated by the weight of water delivered per min. The flow rate could be varied by adjusting the leak valve and/or pump current; an inverted “T” trap was required to trap bubbles of COZformed in iron(II1)-oxalate photolysis. At flow rates around two ml/min, the mean values differed less than 0.02 ml/min from day to day. On a given day the flow rate has an rsd of less than 1 %. Titration data, discussed confirm that the flow rate can be reproduced to better than below, 1%‘ End points of the vanadium (V)-iron(I1) titrations were determined potentiometrically (Pt/SCE) and recorded; the time of titrant delivery to the inflection point was read directly from the chart paper. Reagents. Stock solutions of iron(II1)-oxalate were prepared in 1M H & 0 4 from either K3Fe(C20&.3H20 (Alpha Inorganic) or by combining standardized ferric sulfate with a 3 mole ratio of potassium oxalate (5, 7). The total amount of photoreducible iron(II1) was determined by the method of Riggs and Bricker (6). Standard solutions of reagent grade NH4V03 in 1M H&O( were prepared by weight and used as the reference to evaluate the parameters of external generation. Procedure. With a stock solution of about 4mM iron(II1)oxalate, start the pump and adjust the needle valve to give a flow rate between 1 and 2 ml/min. Allow 15 min for conditions to stabilize; discard the delivered titrant. A sample containing between 15 and 100 micromoles of vanadium is diluted with 1M sulfuric acid. The titrant stream is then diverted so that it flows into the sample, and the recorder chart drive is started at the same time. After the end point is reached, the titrant is again diverted to a drain until a new sample is ready for titration. The time, in minutes, to reach the inflection point is read directly from the chart paper. A calibration plot of time to the end point us. micromoles of vanadium can be prepared, or the data can be treated by leastsquares. The slope of the calibration curve, in micromoles/min, is equal to the micromoles of iron(I1) delivered per minute. If the flow rate is known, then the concentration of iron(I1) in micromoles/ml may be calculated. Furthermore, if the original concentration of stock iron(II1)-oxalate is known, the percent photoconversion from iron(II1) to iron(I1) can be calculated. This method of data treatment was used to study some of the fundamental parameters of external photochemical titrant generation. It should be emphasized that satisfactory working curves for routine determinations can be prepared without knowing the exact iron(II1)-oxalate concentration or flow rate. So long as these two factors are held constant, a calibration curve can be prepared by use of a standard vanadium solution. The flow rate, concentration of iron(II1)-oxalate, and fraction of quartz tubing exposed to the lamp were varied and
Range of V taken, pmoles
~
~~~
Table I. Spectral Characteristics of the Photolysis Source Molar Absorptivities and Quantum Emciencies of Iron(II1) Oxalate Quantum Molar yield, Wavelength Relative lamp absorptivity Fe(I1) (nm) intensityo, % Fe(II1) oxalateb productionc 576 578 545 435 404 366 313 312 254
0.6 0.3 7.8 6.2 1.4 0.5 1.4 0.8 100.0
..*
... ...
0.013 0.013 0.15 1.01 1.13 1.21 1.24 1.24 1.28
38.4 120 400 2.06 x 103 2.06 x 103 2.89 x 103 Relative intensities are referred to 254 nm and are not corrected for spectral response of the 1P28 photomultiplier.
Determined from the slopes of Beer’s Law Plots. Data of Parker and Hatchard (5).
know amounts of standard vanadium were titrated in order to establish slopes of calibration curves, which were used to assess the effect of each variable. The time any one portion of solution was photolyzed (residence time in the reaction zone) can be calculated from the volume of the quartz tube (17.6 cm3), fraction of tube length exposed, and the flow rate in ml/min. Effects of turbulence were not considered in these calculations. RESULTS AND DISCUSSION
The criteria of first importance for evaluating the utility of external photochemical generation include the precision, sensitivity, dynamic range, and convenience of the method. The results of several different sets of titrations, designed to test one or more of the criteria, are presented in Table 11. It can be seen that the technique works at the micromolar level, and can yield precision of better than one per cent. The major inconvenience of the method is the necessity of disposing of unwanted titrant. In order to maintain stability of flow and photochemical reaction conditions, it is necessary to keep the system in operation continuously. A short term shutdown can be obtained by closing the needle valve; the exhaustively photolyzed solution in the reaction zone should be expelled before resuming titrations. The major convenience of the method seems to be the possibility of varying the titrant delivery rate by changing either the concentration of photogenerator, or the flow rate, or length of reaction zone as required for a particular sample concentration range.
Table 11. Titration of Vanadium(V) with Externally Photogenerated Iron(I1) Slope calib. plot,” Flow rate, Photolysis time: PhotoTitrn time, rnin pmoles/min ml/min min (calc) conversion,
rsd, %”
5-20 10.80 1.28 13.60 lOld 1.15 4-20 5.126 1.28 13.60 97.54 0.71 5-14 11.09 2.79 6.22 96.2. *.. 4-13 12.35 3.20 5.42 93.58 ... 4-12 14.53 3.95 4.39 89.2O ... 5-13 2.595 1.43 2.10’ 43.0’ 0.58 Calculated by least-squares fit of time to end point us. micromoles vanadium taken. This figure is also the delivery rate of iron(I1). 50-200 15-100 50-150 50-150 50-1 50 10-30
a
Calculated time for a given increment to be pumped through the reaction zone. (Turbulence neglected.) Coefficient of variation calculated for the median concentration of range titrated. d 8.26 mM iron(II1) oxalate used as photogenerator. Per cent photoconversion calculated from slope and flow rate. e 4.13 mM iron(II1) oxalate used as photogenerator. Per cent photoconversion calculated from slope and flow rate. f 17.0% of length of quartz tube exposed to lamp (Reaction Zone). b c
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Table III. Iron(I1) Production as a Function of Reaction Zone Exposure Reaction zone," 100.0 50.0 33.9 25.0 22.0 18.7 17.0 12.5
Generation rate (pmoles/min)b
Photolysis timeC
5.714 5.649 4.508 3.731 3.123 2.786 2.532 1.902
12.36 6.18 4.19 3.09 2.72 2.31 2.10 1.54
Photoconversion,
80
1
Z d
97.1 96.0 76.6 63.4 52.5 47.3 43.0 32.3
Per cent of maximum length available for photolysis. b Calculated slope of least-squares fit of titration data. Calculated using flow rate of 1.43 ml/min. d Calculated from slope of calibration curve and flow rate, 4.13 mM iron(II1) oxalate used.
%P 6 0 -
40:/ 20
ov 0
2
4
6
8
t (min 1
0
For example, titrations summarized in the first two lines
of Table I1 were carried out under identical conditions except for the concentration of photogenerator solution used. Since the flow rate and reaction zone parameters were such that photoconversion was complete, doubling of the photogenerator concentration doubled the rate of delivery of ironGI). An upper concentration limit of about lOmM is set by the release of bubbles of COZ. For generators which do not evolve gas as a photolysis product, the upper limit is set by the concentration where the photoreaction takes place in a shallow layer (8). A second variable of interest is the flow rate; lines 2 to 5 of Table I1 provide the results of such a variation. The situation is complex because an increase in flow rate decreases the residence time of a given portion of solution in the reaction zone, and consequently the per cent photoconversion is decreased. Nevertheless, the rate of titrant delivery, in terms of micromoles/minute, is increased; this increase in delivery rate overpowers the decrease in photoconversion. The result, as seen in Table 11, is that the slope of a calibration curve increases as the flow rate is increased. Because the solution is not photolyzed exhaustively, it now becomes important that the intensity of the lamp be constant. When photoconversion is complete, the precision of the titrations depends solely on the stability of the flow rate. The precision possible under conditions where lamp stability is a factor was also examined. The last line of Table I1 shows that it is possible to carry out titrations under such conditions with good precision. Riggs and Bricker have reported that the iron(III)-oxalate photoreduction suffers from a slight reversal when exhaustive photolysis is carried out with short wavelength UV (6). Therefore, it may actually be advantageous to operate under conditions of less than 100% photoconversion. The titrations summarized in the last line of Table I1 were carried out with the sliding shutter between the mercury lamp and quartz tube positioned so that the reaction zone used was 17% of the maximum. External generation may prove useful as a method of quickly and reproducibly changing the concentration of titrant without the necessity of dilution. It is possible to select the desired titrant molarity by merely changing the length of reaction zone. The manner in which the titrant concentration can be varied with length of reaction zone was studied and the results are given in Table 111. The relation between titrant 976
0
ANALYTICAL CHEMISTRY
Figure 2. Correlation of Fe(I1) concentration with photolysis time ZP-Per cent photolysis of 4.130 mM iron(lII) oxalate photogenerator t -Calculated time photogenerator is exposed to lamp illumination, in minutes 0 -Experimental-Reaction zone data 0 -Experimental-Flow rate data A -Theoretical calculation from Equation 3 A -Point at which absorbance becomes less than 2.00 and exponential term of Equation 2 must be considered (Seetext).
normality and reaction zone length is nonlinear, which is to be expected from consideration of Equation 1. If there were a linear relation between length of the zone and the concentration of phototitrant, selection of appropriate titrant concentration would be quite simple. It can be seen from Equation 1 that the rate of formation of phototitrant B will be constant so long as the exponential term of the equation is small which is true if the absorbance is greater than 2.00. The value of the exponent depends on the concentration of photogenerator, CA. The integration of the rate equation is simple if the exponential term can be neglected and the concentration of iron (11) at any time, CF@(II), t is thus:
where t is the time of photolysis. However, the concentration of iron(II1) is decreased by photolysis until a point where the exponential term of Equation l becomes significant. The integration of the rate expression for time-dependent values of the exponent is somewhat more difficult. This problem has been treated by Kessler (9). A detailed consideration of the equation obtained is beyond the scope of the present study. However, some simplifications of the equation are useful for rough estimations of the required conditions for generation of a given titrant concentration. First, an estimate of the incident intensity of the generating lamp, lo,is easily calculated from the slope of a plot of iron(I1) concentration versus time. From Equation 2 it follows that the initial slope of the plot is equal to @ l o . Data obtained from variation of flow rate and reaction zone length are plotted in Figure 2. The initial slope of the plot is 0.86 millimole/l-min; with the value of the quantum yield at 254 nm one calculates loas 4.18 x lozophotons/l-min. (9) H. C. Kessler, Jr., J. Phys. Chem., 71,2736 (1967).
For the volume of reaction zone in this study the incident flux is therefore 7.36 x 10'8 photons/min. This is a reasonably large value for a low-pressure lamp and demonstrates the benefits of the long length and close proximity of the reaction zone and source; the aluminum reflector also increases the intensity. Figure 2 also demonstrates the good agreement between theory and experiment. By taking the value of 10calculated from the initial slope of Figure 2,using the molar absorptivity at 254 nm (Table I), and assuming that the i.d. of the tubing, 0.6 cm, is b, one calculates that the exponential term becomes significant at an iron(III)-oxalate concentration of 2.65 mM. For the concentration of generator used here, 4.18mM, deviation from linearity should occur at about 65 photoconversion. This is the case as can be seen in Figure 2. It would be possible to construct a sliding shutter with calibration marks so that a variety of titrant concentrations could be
selected with a single flow rate, thus eliminating the need for a variable pump. Some conclusions can be drawn regarding the practical value of external photochemical generation. First, it is clear that reproducible flow rates and lamp intensity can be obtained with reasonable care. Second, it is possible to obtain a sufficient intensity from a low-power lamp to generate useful titrant concentrations, and the intensity is constant over many months of continuous operation. It should be noted that in situ titrations employ medium-pressure lamps which must be turned off when not in use ( I , 2). Finally, the titrant concentration can be easily changed by changing the length of the photolytic reaction zone. RECEIVED for review December 9,1968. Accepted March 21, 1969. Taken in part from Ph.D. Thesis of H. D. Drew; presented at the 156th National ACS Meeting, September 1968,Atlantic City, N. J.
Determination of Amphetamine and Related Amines in Blood by Gas Chromatography Robert B. Bruce and William R. Maynard, Jr. A . H.Robins Co. Inc., 1211 Sherwood Avenue, Richmond,Vu. 23220 A NUMBER OF METHODS have been presented during recent years for the determination and identification of amphetamine and its derivatives in urine. These methods are based primarily on GLC (1-3) and TLC (4-7) or specific color reactions (8, 9). However, none of these procedures are applicable to the determination of these drugs in blood because of their lower limits of detection. Axelrod (IO)adapted a nonspecific colorimetric procedure to the determination of amphetamine in plasma and tissues of animals that received relatively large doses. The method described below is based on the formation of the heptafluorobutryl derivatives of four of these amines and their subsequent determination by GLC using an electroncapture detector. Halogenated amide and ester derivatives prepared from amines and alcohols usually give high sensitivity with the electron-capture detector. A number of these were prepared and from the results obtained it appeared that the heptafluorobutyramides gave the best results.
(1) A. H. Beckett and M. Rowland, J. Phurm. Phurmacol., 17, 59 (1965). (2) H. Brandenberger and E. Hellback, Helv. Chim. Acta, 50, 958 (1967). (3) C. R. Hall, V. Cordova, and F. Rieders, Pharmucologist, 7, 148 (1965). (4)B. Davidow, Psychopharmucol. Bull., 3, 30 (1966). ( 5 ) H. Eberhardt and M. Debackere, Armeim.-Forsch., 15, 929 (1965). (6) M. Debackere and A. M. Massart k e n , Arch. Intern. Pharmucodyn., 15, 459 (1965). (7) M. L. Weischer and K. Opitz, Arzneim.-Forsch., 17, 625 (1967). ( 8 ) M. S. Karawya, M. A. El-Kiey, S. K. Wahba, and A. R. Kozman, J. Pharm. Sci., 56, lo05 (1967). (9) R. D. Eastman and P. A. G. Cox, Brit. Med. J., 1965,(5439) 924. (10) J. Axelrod, J. Phurmacol. Exp. Therap., 110, 315 (1954).
EXPERIMENTAL
Reagent. Heptafluorobutyric anhydridepierce Chemical Co., Rockford, Ill. Keep refrigerated. Method. An internal standard is used in each determination. Any of the other three amines may be used as the standard for the amine being determined. The internal standard is added to the blood sample before the initial extraction. Chlorphentermine (0.5-2 y) was used as the internal standard for amphetamine, methamphetamine, and fenfluramine. Amphetamine was the internal standard when chlorphentermine was being determined. The procedure for the analysis of a blood sample is as follows: Add the internal standard to 5.0ml of blood, mix and add 1.0 ml of 2 N NaOH and 5.0 ml of water. Extract the sample with 10.0 ml, then 5.0 ml of redistilled, chromatoquality pentane. Centrifuge the sample after each extraction and pass the pentane through a layer of anhydrous Na2SO4. After passing both extracts through the NatSOa, wash the Na2S04 with an additional two ml of pentane. Evaporate the pentane to dryness under a gentle stream of nitrogen and add 100 pl of heptafluorobutyric anhydride. Allow the reaction to proceed for 30 minutes with occasional mixing. Evaporate the excess heptafluorobutyric anhydride with a gentle stream of nitrogen and cool the tube in an ice bath. Add 0.5 ml of 2 N NaOH, keeping the mixture cold, mix and extract with 4, 3, and 3 ml portions of pentane, filtering each portion through anhydrous Na2S04 after each extraction. Wash the sodium sulfate with an additional 2 ml of pentane. Allow the sample to stand overnight at this point. Evaporate the pentane under a gentle stream of nitrogen. Dissolve the residue in 0.20 ml of pentane and inject 2 to 4 p1 into the gas chromatograph. A Barber-Colman gas chromatograph with a saNi detector was used in this study. The column was stainless steel, 6 feet long by l / d r in o.d., with 5% OV-1 on Gas-Chrom Q as the stationary phase. The temperature of the oven was 110 "C, injection port 250 "C, detector 250 "C, and the nitrogen flow rate 30 ml/minute. The retention times found under VOL. 41, NO. 7, JUNE 1969
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