Capacitor discharge temperature-jump apparatus with nanosecond

The temperature-jump technique for the study of fast reactions in solution. J E Crooks. Journal of Physics E: Scientific Instruments 1983 16 (12), 114...
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Capacitor Discharge Temperature-Jump Apparatus with Nanosecond Capabilities Rlchard M. Reich' American Instrument Company, Silver Spring, Maryland 209 10

John R. Sutter" Chemistry Department, Howard University, Washington, D.C. 20059

The complexity of previously reported temperature-jump spectrophotometers varies significantly depending on the method of heating and the minimum time resolution. The most commonly used method of heating is Joule heating produced by the discharge of an energy storage capacitor through a solution containing an inert electrolyte. If the concentration of the electrolyte is high, the cell resistance will be low, resulting in a rapid transfer of energy. Capacitor discharge systems have been generally employed to produce heating in the 1-20 ps range (1-4). Such systems may use charging voltages as high as 100 kV to produce a temperature rise of 2-10 "C. The time resolution of systems using Joule heating has been extended below 80 ns (5) by replacing the energy storage capacitor with a 5-m length of coaxial cable. The impedance of the cell was matched to the characteristic impedance of the cable. The coaxial cable is charged to 100 kV and switched through the cell with a high pressure spark gap. Switching of the spark gap was initiated by releasing the pressure. This instrument was able to increase the temperature of a 40-pL sample 10 "C. The fastest reported temperature-jump Spectrophotometer utilizes a high powered Nd3+-glasspulse laser to produce a heating time of about 18 ns (6). The output frequency of the laser is shifted through the stimulated Raman effect in hydrogen gas to produce a 15-11s half-width pulse at 1.89 pm. The absorption of energy at 1.89 pm by aqueous solutions is very high, resulting in a rapid temperature rise. Of all the reported heating techniques, Joule heating through the discharge of an energy storage capacitor is the most widely used. The other techniques are either prohibitively expensive, difficult to construct, or pose a safety hazard to the operator. The basic limitations of the capacitor discharge type system have been a minimum heating time limitation of 1-5 ps and the necessity for the solution to have a specific resistance characteristic. The reasoning for the minimum time limitation of 1-5 ps has not been made explicitly clear in the literature. The factors which have been discussed as possibly contributing to the limitation are shock waves produced by the rapid heating, and increased electromagnetic interference at wider signal bandwidths. The minimum cell resistance is also limited by the stray inductance in the discharge circuit. The cell resistance must be maintained large enough to prevent oscillatory discharge.

THE CELL A cell has been developed that has a 1-cm optical path length and a 200-pL heated volume. The main cell body is constructed from Kel-F which has excellent electrical insulating properties, is chemically inert, and is easily machinable. The cell which was constructed can be seen in Figure 1. The cell windows were made from 3-mm diameter Pyrex rod glued into the cell body. Because of the difficulty of fastening the Pyrex windows into the Kel-F cell body, special preparation of the window material was necessary. Before a 3-mm diameter Pyrex rod was cut to the proper length, the outside surface was 'Present address, Teledyne Philbrick, Dedham, Mass. 02026.

roughened with sandpaper to provide more surface area for gluing. The rod was then cut to length and the end surfaces were optically polished. An etching material, Fluorocarbon Etchant No. 40, which is suitable for preparing Teflon and Kel-F surfaces for bonding, was obtained through Technical Fluorocarbons Engineering, Inc. (100 Gilbane Street, Warwick, R.I. 02886). After the surfaces of the cell which will be in contact with the window were etched, the windows were glued in place with epoxy. The geometricalconfiguration of the cell has the following characteristics: 1, optical path length, 1 cm; Q, cell heating volume, 200 pL minimum; sample volume, less than 250 pL; d, inter-electrode spacing, 0.4 cm; w,cell width (internal), 0.5 cm; ratio d/A = 0.8. The electrode material was stainless steel. PHOTODETECTION SYSTEM The measurement of a relaxation process due to a temperature-jump perturbation requires a photodetection system of both high sensitivity and wide signal bandwidth. A typical relaxation process will cause a change in signal which is less than 1 % of its preperturbed value. The photodetection system must be capable of measuring relaxation processes occurring in less than a few hundred ns. The photodetection system consists of an RCA931A photomultiplier tube followed by an operational amplifier circuit which converts the photocurrent to a proportional voltage and acts as a buffer between the photomultiplier tube load resistor and a storage oscilloscope. Assuming that shot noise produced by the photocathode is the predominant noise source and that the gain per stage is in the 3 to 5 range, the signal to noise ratio of the photodetection system can be described by (7)

S/N =

(A)''' 6 -1 (T)l'*

2eB, where I , is the photocathode current, e is the charge on an electron, B, is the equivalentnoise bandwidth of the detection system and 6 is the gain per stage of the photomultiplier tube. The equivalent noise bandwidth is related to the signal bandwidth of the detection system, B,, by B, = 1.57 X B, for a detection system whose transfer function approximates that of an RC low pass filter. Equation 1indicates that at fixed bandwidth the signal to noise ratio can be maximized by maintainingthe photoelectron current as high as possible. The photoelectron current is directly proportional to the intensity of the monochromatic radiation incident on the photocathode. The system described was designed to operate at photocathode currents in the 0.2 to 1PA range with a maximum anode current of 0.5 mA. In order to keep the gain per stage in the three to five range under these conditions, it was necessary to utilize only the first five of the nine available dynodes. The photomultiplier tube biasing configuration is shown in Figure 2. The 6th dynode is used as the anode and dynodes 7, 8, and 9 and the original anode are connected to the same potential as dynode 5. To maintain linear operation of the photomultiplier tube, the current ID,flowing through the dynode resistors is kept ten times greater than the maximum anode current. If the anode current begins to approach the ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977

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-CELL

ZODY

-

44-

IERMINAL

Figure 1. T-Jump cell assembly

dynode current, the bias voltage of the last few dynodes will begin to change, resulting in a gain change. The first dynode will contribute most of the noise introduced by the dynode structure (8). It is important, therefore, that this dynode is operated at a high potential with respect to the photocathode. To assure that this condition is maintained a 120-V zener diode is connected between the photocathode and first dynode. If the dynode bias power supply voltage, VD, is varied to produce less than 120 V per stage on the other dynodes, the first dynode is held at 120 V. VD can be varied from 420-850 V while still maintaining the dynode current equal to or greater than 5 mA with Rl-R5 being 12-KQ, 2-W resistors. A unity gain buffer amplifier was used to isolate the 10-KQ load resistor, R7, from the coaxial interconnecting cable and oscilloscope. Without this buffer amplifier, the maximum system response time would be dependent on the length of the cable connecting the load resistor to the oscilloscope and the input capacitance of the vertical amplifier of the oscilloscope. The buffer amplifier was constructed with a high speed FET input operational amplifier as shown in Figure 2. The am-

Figure 2. Photodetection circuit 1082

ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977

plifier selected was a National Semiconductormodel LH0032C ultra fast FET operational amplifier. In a non-inverting unity gain configuration,the maximum rise time for signal changes of up to 1V is 20 ns. The input capacitance which would appear in parallel with the load resistor is typically 5 pf. The effective input capacitance can be further reduced to less than 1 pf by connecting the isolated metal case of the amplifier directly to the input. The photomultiplier tube and buffer circuit were constructed on a single vector board to minimize lead lengths and stray capacitance. The LH0032C operational amplifier has a 70-MHz unity gain bandwidth which increases its susceptibility to instabilities if the circuit is not constructed with compensation components mounted as close as possible to the amplifier pins and the power supply lines bypassed to ground. Both the +15 and -15 V power supply lines were bypassed to ground with 0.01-pF ceramic disk capacitors, C3 and C5, as close to the power supply pins of the amplifier as possible. The photodetection system is susceptible to interference from magnetic fields and radiofrequency interference. The RCA 931A photomultiplier tube, as with most photomultiplier tubes, is very sensitive to external electromagnetic interference. Magnetic fields cause defocusing of the electrons in the dynode structure resulting in loss of sensitivity. To minimize this susceptibility, the photomultiplier tube was wrapped in an adhesive backed magnetic shielding material which was then connected to cathode potential through a 20-MQresistor. The resistor is in series with the shield to prevent a safety hazard if the shield should accidently be touched. Connecting the shield to cathode potential will reduce voltage gradients across the tube envelope wall, decreasing leakage currents and noise. The complete photomultiplier tube amplifier assembly is enclosed in a steel box which will act as both an electrostatic and magnetic shield. A small hole was drilled through the box to allow light to strike the photocathode. Both the +15V and -15 V power supply leads enter the box through electromagnetic interference filters. The box is connected at a single point to the signal common. T o further reduce the system sensitivity to power line conducted interference, the f15-V power supply was also enclosed in a separate steel box which was connected a t a single point to the power line ground. The 115-V ac line was filtered though feed-through capacitors before reaching the

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ac input of the power supply.

HIGH VOLTAGE DISCHARGE SYSTEM The discharge circuit that has been developed is shown in Figure 3. The conventional spark gap switch has been replaced by a two-krytron switching circuit which will permit the energy storage capacitor to be switched over a wider range of discharge voltages by the application of a smaller trigger voltage. The discharge can be triggered with energy storage capacitor voltages ranging from less than 2 kV to up to 10 kV. The oscilloscope time base can be pre-triggered for intervals ranging from 0.1-1000 ws. The krytron is a gas-filled, cold cathode switch tube which will operate in an arc discharge mode. The krytron has an anode, cathode, trigger grid, and keep-alive electrode. A keep-alive current supplied through the keep-alive electrode, maintains a column of ionized gas in a glow mode which provides an initial source of plasma to initiate discharge. The EG&G, Inc., KN-6B Krytron is rated at maximum peak current pulse of 3000 A for a duration of 10 ks. This is ideal for temperature-jump requirements. The maximum anode hold-off voltage for a single tube is rated for 8 kV. This was considered marginal for the system requirements. Using a two-krytron configuration, the hold-off voltage range is extended above 10 kV. The circuit under best conditions should hold off 15 kV. Since the hold-off voltage of the tubes will deteriorate with usage and discharge history, the recommended operating range of the system will therefore be considered to be 10 kV or below. This will operate the switching tubes under conservative conditions and maximize their operating life. Another advantage of the krytron circuit compared to triggered spark gaps is that only 300-500 V are required on the trigger grid to fire the tubes. Triggered spark gaps of similar rating require 5-8 kV. In the circuit shown in Figure 3, the discharge is triggered by the application of a 1000-V pulse to the grid of krytron, K1, through the pulse transformer, T2. The pulse transformer is an EG&G type TR-180B which has a 112:l turns ratio. The trigger pulse on the primary side

of the transformer can therefye be less than 9 V. In the discharge system that has been developed, the discharge is initiated by pushing the momentary switch, S1, or by grounding the external trigger input. The switch, S1, is “debounced” by the cross coupled NAND gates Z1A and Z1B to prevent multiple triggering. Initiation of the discharge by either mode will cause a positive transition from logic “0” to logic “1”to occur on the output of Z1C. The output of Z1C connects directly to the inputs of monostable multivibrator, 23,and dual monostable multivibrator, 22. Both 22 and 23 are connected to trigger on a positive transition of the input. Triggering of 23 will cause a 100-ns pulse to appear on the external trigger output BNC connector 52. This output is normally at a TTL logic level “1”and will switch to logic “0” for 100 ns, providing a signal to initiate the sweep of the oscilloscope. The dual monostable multivibrator, 22, is connected to produce an output pulse after a time delay set by R6 and S4. The delay time is approximately 0.7 (R6 + R7) C (where C is the capacitor selected by S4). The output on 22 is normally at logic “0” which keeps the gate of SCRl below its turn on threshold. The delayed output pulse from 22 turns SCRl on, allowing C6 to discharge through SCRl and T2. When the current produced by the discharge of C6 decays below the holding current of SCR1, SCRl turns off and is ready for the next time. The discharge of C6 appears as a 1000-Vpulse on the trigger grid of the krytron, K2, causing it to switch into conduction. When K2 begins to conduct, the trigger pulse goes through the anode of K2 and grid resistor R11, causing K1 to switch into conduction. The energy storage capacitor, C7, will now discharge through the sample cell. After discharge, the krytrons turn off allowing C7 to recharge through R15. The charging time constant is 2 s. To maintain at least 1%repeatability of charging voltage, it is necessary to wait a t least 10 s between discharges. The high voltage power supply is a Kilovolt Corporation model KVP 10-5,O-10000 volt supply. The output high voltage is varied by changing the 60 Hz ac input voltage with the Superior Electric Co. Powerstat model 10B variable transformer, T3. The voltage ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977

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stored on C7 is continuously monitored by a 0-50 pA panel meter, M1 in series with a 200-MO resistor, R14. The scale markings on the meter were changed to read 1-10 kV. Selection of the energy storage capacitor, C7, for low series inductance and the maintenance of low stray inductance in the interconnecting wiring are critical in producing heating time constants below a few ps. For short time constants, the discharge circuit must be analyzed as a series RLC circuit where C is the capacitance of the energy storage capacitor, R is the resistance of the sample cell, and L is the sum of the internal series inductance of the energy storage capacitor and the total stray inductance of the interconnecting wiring. For minimum heating time, the circuit is operated in an overdamped condition. The requirements for overdamped response of an RLC circuit is that (9): R > 2(L/C)li2. The capacitor, C7, used in the circuit of Figure 3 was a 0.1-pf, 10 000-V dc rated unit (Del Electronics Corp., model 160-0626B),with series inductance of 25-50 nH. Assuming that the interconnecting wiring has a stray inductance of 250 nH, and using 50 nH for the series inductance of C7, and applying the above equation, the minimum cell resistance required to maintain overdamped response must be greater than 3.5 R. Under these conditions, the minimum heating time constant, RC/2, is 175 ns.

SYSTEM INTEGRATION All the components which are involved in the high voltage discharge are completely enclosed in what is effectively a single shield. This includes the discharge trigger electronics, high voltage discharge circuit, and the sample cell. The above three items are each enclosed in their own steel box. The discharge trigger logic output is connected to the high voltage discharge electronics through twinax connectors and a twinax cable. The cable shield is therefore continuous with the steel boxes of both systems. The shield is not used as a signal ground; the signal ground and trigger signal are each connected on separate wires within the shielded cable. The high voltage discharge circuit, which includes the 10000-V power supply and the energy storage capacitor, are connected to the sample cell enclosure through a 6-in. length of cylindrical galvanized steel conduit. All grounds in the system are brought to a single point where they are connected to earth and the steel enclosure. To prevent interference generated by the discharge from coupling to the ac line and conducted out of the shield, two electromagnetic interference suppression filters were installed where the ac line enters the shield. The filters were a Pi type (Erie part no. 9011-100-1005)designed to attenuate over the 10 kHz to 10 GHz frequency range. The only break in the shield integrity is a 3/16-in.diameter hole on opposite sides of the sample cell enclosure to allow passage of the analyzing beam of light from the monochromator. The cell enclosure is rigidly mounted directly to the exit aperture of the grating mochromator, Aminco part number A109-69007. Inside the cell enclosure, the cell is held in place by three thermostated aluminum plates. One plate is L shaped and in contact with two walls of the cell. The other two plates are rectangular and have their thermostating channels connected in series with the third. A refrigerated water bath is located externally and maintains the cell at the desired temperature. The three thermostated plates are spring loaded against the walls of the enclosure to firmly remain in contact with the cell walls. Access to the sample cell is provided through a removable plate on the top of the enclosure. A RFI gasketing material was installed on the surface of the cover plate which makes contact with the enclosure. The photomultiplier tube and amplifier assembly was mounted in a steel box with electromagnetic interference fiiters on the +15 and -15 V power lines. A small hole was drilled in the photometer enclosure to allow light to fall on the 1084

ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977

Trace showing heating time of apparatus. The chemical system is described in the text. Horrizontal scale 500 ns/cm. Vertical scale 0.1 V/cm amplified from 1 V/cm. Heating time constant 300

Figure 4.

ns.

photocathode. The photometer assembly shield was not connected to the shield enclosing the cell and high voltage components. The shield was connected to earth, the common terminal of the dual 15-V power supply, and the positive output terminal of the photomultiplier tube high voltage power supply. These connections were made at a single point. The high voltage power supply for the photomultiplier tube has an output voltage range of 0-1000 V at 0-20 mA (Kepco, Inc., model ABC 1000M). The signal output connector on the photometer box was an isolated BNC type. The center conductor was connected to the output of the buffer amplifier and the shield lug was connected to the common ground point. The photometer box was positioned '/2 inch from the sample cell enclosure so that the monochromatic light leaving the cell could fall directly on the photocathode. The signal output of the photometric system connects directly to the vertical amplifier of a storage oscilloscope. The lamp used was a 200-W tungsten-iodine lamp (Aminco A312-62081) powered by a Hewlett-Packard 6274B Power Supply. Figure 4 shows an oscilloscope trace for the Glycylglycine-Phenol Red buffer system. The G.G. is 0.2 M while Phenol Red is 5 x M. The pH is adjusted to 7.74 at 5 "C. The horizontal scale is 500 ns per large division. The vertical scale is 100 mV/div amplified from a total steady-state signal of 5 V, giving a 2%/div change in T. The large change in T for the system in part accounts for the low noise in the signal. Our S/N ratio is about lo00 at this wavelength, 563 nm. Since the protonation equilibrium is established extremely rapidly, the absorbance change is following the heating time of the instrument. Using a 0.05-pf capacitor and a 1 3 4 cell resistance (KN03 added), the heating time constant is 300 ns. The heated volume is 0.2 mL with an optical path of 1 cm. The initial temperature of the solution is 5 "C. In a separate experiment,a temperature jump of 3 "C was measured. Faster heating times can be obtained by adding more salt or by raising the solution temperature to decrease the solution resistance. We have used this apparatus to duplicate results previously obtained in our laboratory using a different T-Jump Apparatus, also constructed in this laboratory (I0);the agreement was quite satisfactory. After perturbation, the final absorbance will persist for about 3 s before the heated solution begins to cool exponentially back to the thermostat temperature. The cooling

time is normally around 30 s, so that one usually waits a few minutes before perturbing the system again to ensure that all has reequilibrated. Also, a solution is used, generally, only once or twice before changing. Fresh degassed solution is kept in the circulating constant temperature bath ready for use. This apparatus using typically from 8 to 10 kV perturbation voltage is capable of studying reactions whose relaxation times are longer than 300 ns. This and a 1-cm optical path greatly extend the range of reaction rate constants that may be obtained from the capacitor discharge method.

ACKNOWLEDGMENT The authors thank Paolo Priarone for the cell construction and for assistance in the system integration.

LITERATURE CITED (1) G. Gzerlinski, Rev. Sci. Insfrum., 3 3 , 1184 (1962). (2) A. D. Yu, M. D. Waissbluth, and R. A. Geieger, Rev. Sci. Insfrum., 44. 1390 (1973). (3) E. Caldin, Cbem. Br., 11, 4 (1975). (4) T. C. French and G. G. Hammes, Methods Enzymoi.. 16, 5-21 (1969). (5) G. W. Hoffman, Rev. Sci. Instrum., 42, 1643 (1971). (6) S. Ameen, Rev. Sci. Instrum., 46, 1209 (1975). (7) G. Czerlinski and A. Weiss, Appi. Opf., 4, 59 (1965). (8) “Photomuttiplier Manual”, (RCA Corp., New Jersey, 1970) Technical Series PT-61, p 62. (9) S. Fich, “Transient Analysis in Electrical Engineering”, Prentice-Hall, Englewood Cliffs, N.J., 1951, pp 77-78. (10) J. R. Sutter, M. Krishnamurthy, and P. Hambright J. Chem. Soc, @em. Commun., 13 (1975).

RECEIVED for review January 19, 1977. Accepted March 24, 1977.

Simplified Gas Chromatographic Method for the Determination of Chlorpheniramine in Serum James W. Barnhart’ and James D. Johnson’ Health and Consumer Products Department, The Dow Chemical Company, Indianapolis, Indiana 46268

An electron capture gas chromatographic method for the determination of brompheniramine in blood has been reported ( I ) . Permanganate oxidation was used to produce p bromophenyl-2-pyridyl ketone, the chromatographed species. As the authors noted, it would have equal utility for the determination of chlorpheniramine. Partition chromatography was used to remove two interfering metabolites, the monoand didemethylated compounds. Acetylation was thought to be inapplicable as a means of separating these metabolites. We have found that acetylation can be used for the removal of comparable metabolites of chlorpheniramine, resulting in a considerable saving of time.

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H C CH2CH2N(CH3)2

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K Mn04

0 H-

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EXPERIMENTAL

Instrumentation. A Packard Model 7300 gas chromatograph equipped with a tritium electron capture detector was used for the analyses. A 6 ft X 3 mm silanized glass column was packed with 3% SP-2250 on 100-120 mesh Supelcoport. Detector and injector temperatures were 225 “C and the oven temperature was 195 “C. Nitrogen flow was 70 mL/min and the detector voltage setting was 20 V. Procedure. An appropriate quantity of brompheniramine (internal standard) was added to 2.0 mL of plasma or serum in a test tube and mixed with 0.2 mL of 10 N KOH. The samples were heated in a boiling water bath for 10 min. After cooling, the samples were mixed vigorously for 1-2 min on a rotary mixer with 4 mL of diethyl ether (Fisher anhydrous). After centrifugation, the ether layer was transferred to a second tube and mixed with 1 drop of acetic anhydride (Fisher reagent grade, redistilled). After 5 min, 1.0 mL of 0.1 M pH 5.5 citrate-phosphate buffer was added and mixed vigorously for 1-2 min. After centrifugation, the ether layer was discarded. The aqueous layer was then washed twice with 3-mL portions of ether. All traces of residual ether were removed by placing the samples under a stream of nitrogen for 15 min in a 100 “C sand bath. After adding 0.15 mL of 2 N KOH and 1.5 mL of 1%KMn04,the tubes were stoppered loosely with marbles and heated in a boiling water bath Present address, 3M Company, St. Paul, Minn.

Table I. Human Serum Chlorpheniramine Levels After Single Oral Dose Time, Serum chlorpheniramine, ng/mL as the free base h 4-mg dose 8-mg dose 12-mg dose 0

0.5 1.0 1.5 2.0 4.0 8.0 12 24

0.1 0.6 2.1 3.7 4.5 5.5 4.3 3.7 2.2

f

0.0 (SD) 0.2 1.0 1.2 1.1

t

1.0

t

0.5 0.6

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t

*

* 0.7 i

0.3 i 0.6 i 3.2 t 6.0 t 7.2 i 10.4 i 8 . 3 ?: 6.7 i 4.1 ?:

0 . 4 (SD) 0.8 3.1 4.4 3.7 2.6 2.4 1.6 1.3

0.1 i 1.4 i 6.7 t 13.2 t 15.3 i 17.6 i 14.7 i 12.8 t 8 . 6 ?:

0 . 1 (SD) 1.2 4.6 6.7 4.5 5.1 3.9 4.8 4.7

for 10 min. The cooled samples were extracted with 1 mL of

isooctane (Mallinckrodt Nanograde) on a rotary mixer. After evaporation of the solvent, the residue was dissolved in 25-50 pL of isooctane containing 5% acetone. Standards were taken through the entire procedure and peak height ratios plotted vs. ng of chlorpheniramine. All reagents were reagent grade and double glass-distilled water was used throughout.

RESULTS AND DISCUSSION Since the permanganate oxidation results in the same product whether chlorpheniramine or the demethylated metabolites are the starting material, it is essential that the latter be efficiently removed. Even though a pyridyl moiety remains in the resultant acetamide, it is a very weak base and does distribute to the organic phase a t the pH employed. Ionization constants for chlorpheniramine are 9.2 and 4.0 for pKa, and pKaz,respectively (2). The ionization constants of the acetylated metabolites are unknown. Assuming they are of the same magnitude as pK, of chlorpheniramine, significant protonation would not be present at pH values >5. This was confirmed by determining the degree of interference of both metabolites under different pH conditions (Figure 1). The stated pH was the pH of the original buffer. The actual pH during the extraction was slightly lower because of the acetic anhydride added earlier. Interference was less than 1% a t pH values of 4 or above. In another experiment, quantities of both demethylated metabolites ranging from 0.25-5 pug were added to control plasma and carried through the analysis. Less than 1% of these amounts was recovered as apparent ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977

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