In the Laboratory edited by
Cost-Effective Teacher
Harold H. Harris
A Simple, Inexpensive Water-Jacketed Cuvette for the Spectronic 20
University of Missouri—St. Louis St. Louis, MO 63121
Jonathan E. Thompson*† and Jason Ting Department of Chemistry, Troy State University, Troy, AL 36082; *
[email protected] Due to the instrument’s reliability and low cost, the Spectronic 20 has become the standard in spectrophotometers for the undergraduate laboratory curriculum since it was first introduced nearly 50 years ago (1). Spectronic 20s are most commonly employed for the spectrophotometric determination of various species in solution using the Beer–Lambert law (2), but also are employed to determine equilibrium constants (3) or to measure rates of reactions (2). Although the descriptions of square (4) and flow-through cuvettes can be found in the literature (5, 6), we have not found a published report of a water-jacketed cuvette for the Spectronic 20. Such a cuvette would prove useful in the undergraduate laboratory as it would provide a simple modification of the Spectronic 20 instrument that would allow students to analyze the effect of temperature on reaction rates spectrophotometrically. Additionally, it is well known that absorption of light by a sample placed in a spectrophotometer can raise the temperature of the solution in the cuvette and that an increase in temperature can alter equilibrium constants and reaction rate constants; the jacketed cuvette would provide a simple means to thermostat a sample for more accurate measurements. In this article, we describe the construction of a simple, inexpensive, water-jacketed cuvette for the Spectronic 20. We then utilize the cuvette to determine the activation energy (Ea) for the reaction between crystal violet and sodium hydroxide (Scheme I). The concentration of crystal violet in solution is determined at fixed time intervals over the course of this reaction with a Spectronic 20 using the Beer–Lambert law at 530 nm. The reaction between crystal violet and hydroxide ion is a simple bimolecular process that is first order with respect to both crystal violet (CV) and hydroxide ion.
The overall reaction rate constant, k, can then be determined by dividing the observed rate constant, k1, by the concentration of hydroxide ion in the reaction solution. This experiment is repeated at several temperatures and an Arrhenius plot is prepared according to the following equation: − Ea + ln A (3) RT Since ln k has a linear relationship with the inverse of the Kelvin temperature, a plot of ln k versus 1兾T will be a straight line whose slope is ᎑Ea兾R (where R is 8.3145 J兾mol K), so Ea can be determined from a graph of k and T data sets. ln k =
Materials and Methods
Chemicals A crystal violet (hexamethyltriaminotriphenylcarbinol chloride) solution was prepared as 1.985 × 10᎑5 M in water. Sodium hydroxide was prepared as a 0.1037 M solution and diluted for use in the experiment. Equipment The water-jacketed cuvette we have developed is illustrated in Figure 1. For analysis, the sample was placed inside of a 5.5-mm i.d., 6.35-mm o.d. glass tube fashioned from a disposable 9-in. Pasteur pipet (Fisher, Cat. No. 13-678-20D). The pipet was scored and cracked 5 in. from the tapered end and the broken end was closed by heating in a Bunsen burner flame. The sample tube was then inserted into the inner (∼4.75 mm) hole of a #6 septum, which provided a seal. Prior
N(CH3)2
Rate = k [CV ][OHⴚ] = k1 [CV ] where k1 = k [OHⴚ] (1)
In this experiment, the quantity of hydroxide ion is in large excess (> 500 fold concentration) relative to the crystal violet, so that the concentration of hydroxide remains essentially constant throughout the experiment and the rate of reaction depends only on the concentration of crystal violet in solution. Thus, a plot of the natural log of crystal violet concentration versus time should yield a straight line with the slope equal to ᎑k1, according to the integrated rate equation for a first-order reaction: ln [CV ]t = −k1t + ln [CV ]0
ⴙ
C
(H3C)2N
N(CH3)2
violet
N(CH3)2
OHⴚ C
(2)
OH
N(CH3)2
(H3C)2N † Current address: Department of Chemistry, University of Nebraska–Kearney, Kearney, NE 68849-1150.
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colorless
Scheme I. Reaction between crystal violet and sodium hydroxide.
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to insertion of the sample cuvette, two 20-gauge stainless steel needles were inserted through the septum. One needle was 3.8 cm in length while the other reached nearly to the bottom of the cuvette at 12.1 cm in length. The purpose of the varying needle lengths was to minimize temperature gradients that might exist within the cuvette if heating or cooling water was added to and transferred from the water jacket in the same general area. The apparatus was then placed within a standard 1-cm round cuvette commonly used with Spectronic 20s with the septum providing a seal both for the inner sample tube as described previously and the 1-cm cuvette, which serves as the water jacket. The apparatus could then be placed into the Spectronic 20 for analysis. Black felt was placed over the tubing and cuvette to eliminate room light from being detected by the phototube. Flow was provided to the jacket through 1 m of 3-mm i.d., 7-mm o.d. Tygon tubing connected to the cuvette apparatus via Luer-lock connections. Flow from the cell was directed through 1.5 m of identical tubing and collected in a waste beaker on the floor of the lab. Gravity flow was sufficient to induce a flow of 21.4 mL兾min through the cuvette apparatus when the height difference between tubing inlet and outlet was 1.35 m. The 21.4 mL兾min flow rate we report is the average flow rate from several measurements conducted in a serial fashion. Our data suggest the water flow was relatively constant throughout the experiment as the observed flow rate never deviated by more than 1 mL兾min from the average value in our trials. As the water jacket holds 5.45
mL of water when operational, this suggests the water in the jacket is replaced in ∼15 s. To initiate flow, the system was primed with a syringe at the tubing outlet. In some trials, small air bubbles remained in the cuvette after the flow began. As the bubbles scattered light and reduced the transmittance of the sample, they were eliminated by tapping the wall of the 1-cm cuvette before the analysis began. The temperature of the reaction system was varied by either flowing water from an ice water bath or warm water bath into the jacket. In our trials, water was warmed with a hot plate; however a Bunsen burner can also be used to provide an elevated temperature if a hotplate is not available. Typically, a 1-L beaker served as the water reservoir. At a flow rate of ∼20 mL兾min this volume will provide flow for 40– 50 min, which is adequate time to collect meaningful kinetics data for this reaction.
Figure 1. Drawing of the water-jacketed cuvette and flow system for use with the Spectronic 20; (A) the Luer lock connections for the needles, (B) the inner tube that contains the sample, and (C) the rubber septum used to seal both the inner and outer tubes. The lower beaker is resting on the floor.
Hazards
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Experimental Procedure The Spectronic 20 was first zeroed and then 100% transmittance was set by assembling the cuvette with deionized water in the sample chamber. The cuvette was inserted into the Spectronic 20 so that the needles did not block the light path. After calibration of the transmittance scale, a Beer–Lambert law plot was constructed by successive dilutions of the stock crystal violet solution. For the determination of activation energy, 1.0 mL of the 1.985 x 10᎑5 M crystal violet solution was transferred into the sample cuvette with a syringe, the cuvette–water jacket was assembled, and flow was initiated through the system. The syringe was then washed and 1.0 mL of the NaOH solution drawn into the syringe. For trials at 7.5, 21, and 45 ⬚C, the concentration of NaOH added to the cuvette was 0.050 M. The NaOH solution was diluted to 0.010 M for the trial at 68.8 ⬚C to slow the reaction to the point where a suitable number of data points could be collected over the course of the reaction. The syringe containing the NaOH was brought to the appropriate temperature in an ice or hot water bath. The syringe needle was then pushed through the rubber septum and the 1.0 mL of NaOH was quickly added to the crystal violet. Transmittance readings were recorded every 10 s (every 5 s for the trial at 69 ⬚C) after this point until the reaction was complete. The first 30 s of data were not used in the data workup since the solutions were mixing in the cuvette during this time. Upon completion of the reaction, the septum was quickly removed from the cuvette and the temperature of the water measured with a digital temperature probe. Transmittances were then converted to absorbance measurements and the concentration of the crystal violet versus time was determined using the equation of the line in our Beer– Lambert law plot. The exact time the reaction should be monitored will vary with conditions of temperature and concentration of reactants. Under our conditions of concentration and temperature, the reaction was monitored between 80 to 1200 s (1.3–20 min).
It is important to utilize care handling the disposable pipet after heating it in the burner flame as the glass can remain hot for long periods of time. Additionally, if the hot
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In the Laboratory
Results and Discussion Initial concerns in this project focused on the possibility that inconsistencies in the assembly or disassembly of the cuvette would significantly alter the light path through the cuvette from run to run, thus not allowing reliable transmittance measurements. However, it was found that the cuvette can be assembled or disassembled multiple times without influencing the transmittance measurement to a large extent. For instance, if the cuvette assembly is removed from the spectrophotometer, disassembled, and then reassembled containing the same concentration of crystal violet, the average relative standard deviation of the measurements was found to be about 0.95%. The Beer–Lambert law plot obtained with our cuvette is illustrated in Figure 2. As observed, the plot is linear over the range of concentrations of crystal violet used in this experiment. From the slope of the best fit line (m = 2.37 ⫻ 104) and the molar absorptivity of crystal violet at 530 nm (ε = 43,650), the path length of the sample cuvette was calculated to be 5.43 mm, which agrees well with the measured value of 5.5 mm. This result and the general linearity of the plot suggest the cuvette we have developed is suitable for making quantitative measurements with the Spectronic 20. The kinetics experiments conducted at 7.5, 21, 45, and 69 ⬚C yielded reaction rate constants (k) of 0.034, 0.118, 0.721, and 4.05 M᎑1 s᎑1 respectively from the slope of the straight line formed from a plot of the ln [CV] versus time (s). The Arrhenius plot prepared from the rate constant (k) data is illustrated in Figure 3. As observed, the slope of the best fit line was ᎑7421.5, yielding an activation energy of 62 kJ兾mol for the reaction of crystal violet with the hydroxide ion. A previous study (7) reports the activation energy for the crystal violet reaction to be 64.8 kJ兾mol so the value for activation energy obtained by our method is consistent with other experimental results. Summary A simple, low-cost, water-jacketed cuvette designed for use with the Spectronic 20 has been described. This cuvette has been utilized to determine the activation energy of the reaction between crystal violet and hydroxide ion spectrophotometrically. The cuvette may find use in kinetics studies as well as in equilibrium constant determinations commonly encountered in the undergraduate laboratory curriculum.
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0.5
Absorbance
0.4
0.3
R 2 = 0.9987
0.2
0.1
0 0
0.5
1.0
1.5
2.0
2.5
Concentration / (10ⴚ5 mol/L) Figure 2. Beer–Lambert law plot obtained for crystal violet solutions with the water-jacketed cuvette and a Spectronic 20 at 530 nm.
4 3 4
y = −7421.5x + 23.077 R 2 = 0.9995
1
lnk
glass is immediately placed against a cold object (i.e., table etc.) it can shatter. Care should be utilized when sealing both the inner and outer glass tube with the septum as both tubes are fragile. Elevation of a beaker containing hot water and a hot plate poses a significant hazard. Care should be taken when working with this apparatus. To provide a safer working area, a variety of pumps could be used to induce flow through the water jacket. Since inclusion of a pump added significant expense to the apparatus as a whole and suitable pumps may not necessarily be present in many teaching laboratories, this step was not attempted in our work. Crystal violet is toxic if ingested and sodium hydroxide is caustic. Complete hazard information can be obtained by consulting the MSDS.
0 -1 -2 -3 -4 2.5
3.0
1 T
3.5
4.0
(10ⴚ3 Kⴚ1)
Figure 3. Arrhenius plot prepared from the experimental rate constant (k) data for the reaction between crystal violet and sodium hydroxide.
Literature Cited 1. Jarnutowski, R.; Ferraro, J. R.; Lankin, D. C. Spectroscopy 1992, 7, 24. 2. Dartmouth College, Chemistry 6 Lab Manual Home Page. http://www.dartmouth.edu/~chemlab/ (accessed Apr 2004). 3. Bishop, C. B.; Bishop, M. B.; Whitten, K. W.; Gailey, K. D. Experiments in General Chemistry, 2nd ed.; Harcourt Brace College Publishers: Orlando, FL, 1992; p 291. 4. Aronson, J. N. J. Chem. Educ. 1975, 52, 800. 5. Jezorek, J. R.; Faltynski, K. H.; Finch, J. W. J. Chem. Educ. 1986, 63, 354–357. 6. Shepp, J. M.; Messerly, J. P.; Bomstein, J. Anal. Biochem. 1964, 8, 122–124. 7. Langvad, T. Acta Chemica Scandinavica 1950, 4, 300–306.
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