Public Lab Research note


DIY injection device for chemical reaction kinetics.

by JSummers | December 31, 2018 21:28 | 137 views | 4 comments | #18031 | 137 views | 4 comments | #18031 31 Dec 21:28

Read more: publiclab.org/n/18031


****work by Jack S. Summers, Taylor Stack, Hailey Medder, Kristen Eggles.

Introduction: The study of chemical reaction rates provides fundamental information about reaction mechanisms that can be determined in no other way. Knowledge of enzyme reaction mechanisms provides greater understanding of biological processes and can be used to devise rational approaches to drug design. Despite their importance, the measurement of reaction rates is seldom taught at the undergraduate level. We believe this is due to the high cost of instruments used in this field. One of the most widely used methods for studying reaction kinetics is stopped flow spectroscopy. Stopped flow methods involve the rapid mixing of two reaction solutions and the introduction of the resulting solution into a spectrophotometer cell for analysis. The spectrometer must be capable of acquiring data rapidly and must be triggered soon after the solutions are mixed. In this research note, we describe a minimal injection device consisting of a dual syringe pump, servo-driven injection valve, motor driving and acquisition triggering electronics and software. The device can be built with minimal tools and experience. We also fabricated a flow cell that withstands the pressures generated in the experiment. We wrote code to interface the device with a relatively low cost, commercially available uv-visible spectrometer and another program to help analyze the data. As proof of concept, we characterized the rate of the reaction of crystal violet (CV) with hydroxide in aqueous solution by monitoring bleaching of the CV visible absorbance with time. Our results were consistent with the known chemistry of this system.

Hardware: The following description of the hardware is seriously abreviated. For more information, ask below. The hardware for the current prototype is based on the 250 mm C-beam linear actuator sold at OpenBuilds.com. The kit was assemble according to the online instructions. Additional hardware was fabricated from plate and angle aluminum. We chose plastic 3 mL syringes that are available with luer locks at a low cost.

Teflon tubing 1/16" od with ¼" 24 fittings were purchased from Idex-hs.com. The mixing chamber is a standard T-fitting with ¼" 24 fittings. The valve is a Hamilton Company HVXL 6-6 manual valve. This is a six port valve with the flow path shown in the Figure below. The ports were connected such that inputs from the two syringes were 180 degrees apart and the two cell connections were next to each other (ie, the syringes are attached to the top and bottom ports in the Figure and the mixing chamber is connected to the two ports on the right). The remaining two ports (those on the left) are attached to the reservoirs for the two reagents. In this way, ports for the syringes can be switched between the cell and the reservoirs by rotating the valve 60 degrees.image description

Spectrometer: The CCS100 Spectrometer from Thor Laboratories is capable of measuring between 350 and 700 nm light and costs a little over $2000 USD. The uv-vis light source was a BDS130 from BWTek. This setup also requires two fiber optic cables to connect the cell to the light source and spectrometer.

Electronic Hardware: The electronic hardware for the spectrometer builds on the Texas Instruments Tiva Launchpad development board. The Launchpad interfaces with the spectrometer and mechanical hardware through a printed circuit board that we developed to run our auto-titrator. The auto-titrator board has connectivity for a Pololu stepper motor driver board and a servo controlled valve (which requires a voltage converter). It also has connectivity for a pH probe with is not used. While it might seem straightforward to leave the handle on the valve and not use a servo, we were unable to irreducibly align the ports by hand, making servo control a much better option. Spectral acquisition is triggered by +5 volt pulses, via a solid state relay on a separate small circuit board.

Microcontroller firmware: Firmware for the microcontroller was written using the program Energia. Energia is available for free download from this site: www.energia.nu. The code is available from our github site, here.
Graphic User Interface: The Thor spectrometer required use of a C library, preventing us from writing a user interface in our usual Java based program, Processing. Instead, we wrote a program in C# using Visual Studio. That program is available here. We were unable to find a freeware set of tools to make graphics for this user interface and decided to write a second program using the program Processing to do the analysis. Processing is available for free download from www.processing .org. The spectral analysis software is available here.


4 Comments

The set up seems very similar to one we used for flow injection analysis (F IA).

Interesting article. It looks like you have one artic!e left. Are you headed toward chromatography or some other application?

Thanks.

Is this a question? Click here to post it to the Questions page.

Reply to this comment...


Hi Ag8n, Thanks for your interest. No, we are not headed toward chromatography although the flow cell & spectrometer are pretty well set up for that application (and the valve could be re-purposed as an injector). Rather, our plan is to develop experiments for teaching undergraduates about the mechanics of measuring chemical reaction rates. This coming spring semester, we plan to develop a set of enzyme kinetics experiments for our biochemistry lab course. Specifically, we will look at the effects of pH on the enzymatic hydrolysis of p-nitrophenylacetate by chymotrypsin. This reaction is discussed fairly extensively in Lehninger's chapter on enzyme kinetics, so I anticipate this will be a reasonable system to tackle. Best, Jack

Reply to this comment...


Can you give me the reference ( e.g.Lehninger)? I'd like to look it up.

Regards

Is this a question? Click here to post it to the Questions page.

Reply to this comment...


Lehninger, Principles of Biochemistry, Fourth Edition, David L. Nelson and Michael, M. Cox. WHFreeman & Co, NY. Chapter 6, Sections 6.3 (Enzyme kinetics as an approach to understanding mechanism) and 6.4 (Examples of Enzymatic reactions), pages 202-225. The bit about chymotrypsin starts on page 213. Best, Jack

Reply to this comment...


Login to comment.

Public Lab is open for anyone and will always be free. By signing up you'll join a diverse group of community researchers and tap into a lot of grassroots expertise.

Sign up