Instrumentation
pulsed-Flow spectroscopic Grover D. Owens Dale W. Margerum Department of Chemistry Purdue University West Lafayette, Ind. 47907
Specification of chemical mecha nisms requires knowledge of the ele mentary steps in the overall chemical reaction. A variety of techniques has been developed over the past few dec ades which allows the study of fast re actions in solution (1-6). T h e most widely used techniques are stoppedflow methods and relaxation methods. T h e relaxation techniques have al lowed the characterization of extreme ly rapid reactions but are limited to reversible reactions. Flow methods do not have this limitation. Although continuous flow methods were the first to be developed, they have been overwhelmingly replaced in practice by stopped-flow methods because of the smaller reactant volume and con venience of use. Nevertheless, contin uous flow techniques are capable of measuring faster reactions than stopped-flow, and there is still a great need for such measurements with irre versible systems. Pulsed-flow tech niques refer to continuous flow meth ods in which the duration of fluid de livery is short and therefore smaller reagent volumes are needed. Hartridge and Roughton (7) intro duced the first continuous flow instru ment in their classical investigation of some of the reactions of hemoglobin. Hydrostatic or gas pressure was used to drive solutions into a mixer and then down an observation tube as shown in Figure la. T h e progress of the reaction was monitored optically 0003-2700/79/0351-091 A$01.00/0 © 1979 American Chemical Society
at various positions downstream from the mixer. T h e Hartridge-Roughton instrument could measure half-lives as short as 3 ms, but from 3 to 7.5 L of each reactant were needed per deter mination and visual methods of detec tion were used. Photoelectric methods were introduced in 1936 by Roughton and Millikan (8) along with design changes to permit smaller volumes of reagents as shown in Table I. Rapid mixing techniques combined with op tical observation of concentration changes have been widely applied and improved in the decades t h a t have fol lowed this pioneering work. Some highlights of this developmental pro cess follow and are summarized in Table I. In 1940 Chance (9) suggested that the volume of reagents required for each determination could be reduced considerably by using a range of flow velocities for a given sample (the ac celerated flow method). T h e initial in strument used a manual push to dis charge two 1-mL tuberculin syringes into the mixing chamber and observa tion tube. T h e mode of observation was the same as the Hartridge-Rough ton instrument (Figure la). T h e sepa ration between the mixer and the point of observation was usually 7 mm. Flow velocities up to 20 m/s and half-lives as small as 0.5 ms were origi nally cited for this instrument. In 1951 Chance (10) described developments in the electronic circuitry for the same
a
) Solution A
\
Observation
Observation Tube
Solution Β b) Solution A
\
Observation
Observation Tube Solution Β c)
Solution A t
Observation
π
I Observation Tub
t Solution Β Figure 1. Modes of observation of flow mixing systems, (a) Continuous flow and stopped-flow, (b) stopped-flow, (c) CFMIO and pulsed-flow
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980 · 91 A
TABLE 1.
Characteristics of Several Flow Methods
Method (author-date introduced)
Configuration as per Fig. 1
Continuous flow (Hartridge, Roughton-1923) (Roughton, Millikan-1936)
Volume of Flow reagent Optical velocities expended pathattained in per length, observation mm determination tube, m / s
a a a
6.3 4.5 1
Accelerated flow (Chance-1940)
a
1
Stopped-flow (Gibson, Milnes-1964)
b
20
CFMIO (Gerischer-1965)
c
20
Stopped-flow (Berger-1968)
a
CFMIO (Gerischer, Holzwarth-1971) Pulsed-flow (Owens, Margerum) a
7.5 L 250 mL 25 mL
4 4 4
< 1 mL
>10
0.2
Minimum halflife, ms
3 10 1 0.5
3
3
500
2-6
1
3
1
up to 30
c
20
500
2-6
0.02
c
20
4
2-9
0.04/0.1 a
0.5
Calibration range for second-order/pseudo- first-order.
instrument and stated that it was possible to obtain flow velocities in exesss of 10 m/s. T h e latter publication did not specify the half-life values which could be determined. In a subsequent work Chance (11 ) adapted the above technique to study the kinetics of the respiratory chain. Chance coined the name "pulsed-flow" (12) for accelerated flow methods in which the duration of the fluid delivery is extremely short. Pulsed : flow techniques have been designed by Gutfreund for chemical quenching applications (13). Other than this work by Chance, the accelerated flow method does not appear to have been widely used. Crouch, Holler, Notz, and Beckwith (14) have reviewed stopped-flow methods in some detail. T h e first commercial stopped-flow (Durrum-Gibson) resulted from the design of Gibson and Milnes (15). T h e y developed syringe drive methods which afforded a rapid start and stop of solution flow. Greater optical sensitivity (up to a 2-cm pathlength) was obtained by looking down the axis of the flow tube as shown in Figure l b . T h e distance from the mixer to the entrance of the observation tube is
