Chapter 4: Results and Discussion
4.4 Transesterification Reaction by Microfluidic Platform
As mentioned before, the study is focus on the microfluidic reactors for production the biodiesel and detection the transesterification reaction. The study uses microfluidic system by pumping 0.6 mm Teflon tubes through the channel and reactor chamber with lipase immobilized silicon surface or solar cell which is shown in figure 3-20. Teflon tubing is used to resistant the organic solvents, and has internal diameter appropriate for making connections to the feedstock, peanut oil and methanol, for the flow lines and detection.
We pumping the sample through relatively a tube and a channel and flowing in a large surface is to mix the sample well. Due to tendency to easily fabricate as well as low cost mixing approaches, the mixing is typically performed by diffusion, requiring a microchannel and reactor chamber. That means that the surface of the channel versus the volume of sample is large. In this situation, the substrate solution is constantly pumped through the microreactor tube. First, the feedstock solution is pumped through a Teflon tube with I.D. 0.6 mm, and then flow through a needle with I.D. is 0.25 mm, and the solution then flow through L-channel into a semicircle channe l with I.D approximately 130μm. As the image, the intersection of the injection channel reveals straight edges with 90° angles in figure 4-12. The channel created in the PDMS replica has a nearly semicircle geometry with about 130 μm width and the height of semicircle is 55 μm. The side view of PDMS channel is shown in figure 3-16(b).
Figure 4-12 Schematic of microfluidic platform with the L-channel intersection.
The primary objective of the present work is to develop a simple fluidic system to enable for activity of immobilized lipase enzymes catalyzing transesterification in the microreactor. The substrate solution is pumped through the system with a syringe pump and the cut-off volume (pumping forward and reverse) for syringe pump is 0.06 ml at flow rate of 0.67 ml/min. A secondary objective is to explore the detection methods by UV–Vis spectrophotometer. Under this detection, the following are analyzing the characteristics by NMR and monitoring the biocatalytic process with photo-electronic detection. All experiments processed in microfluidic reactor and the conversion is much higher compared with other lipase biocatalysis. [73] The reactions are very slow, with requiring from 4 to 40 hours, or more. The experiment in the microfluidic system has higher conversion, duo to the optimal texturisation process for increasing the surface and activity of lipase-immobilized. The microfluidic platform for increasing the surface-to-volume ratio, and improve the conversion yield of transesterification reaction.
4.4-1 Ultraviolet-Visible Spectrophotometer Analysis
Biodiesel has been widely researched using many different types of vegetable oils. However, interest of this study is focused on peanut oil, due to lower coat and its different change of transmission during transesterification reaction. In order to faster and completely catalysis reaction, we first use the alkali-catalysis, KOH, just for comparison of the triglyceride (oil) and near 100 % of biodiesel. Figure 4-13 indicates the transmission in three different types of vegetables; (a) shows the transmission of soybean oil and (b) the transmission of soybean oil. In these two types of oil, there is not apparent change of transmission before and after transesterification; (c) shows the transmission of peanut oil which has obvious changes between the triglyceride (oil) and biodiesel in transmission, especially at the 400 nm wavelength with the difference of 29.26 %. In figure 4-13, the transmission change largest is using peanut oil for production the biodiesel which is using alkali-catalysis for easy observation.
(a)
Figure 4-13 Comparison of the transmission with different vegetable oils by alkali-catalysis. (a), (b) and (c) are the transmission with oils and biodiesel by
(b)
(c)
According to this phenomenon, we can detect the transeaterification reaction with lipase-catalysis by using peanut oil and evaluating the transmission of UV-Vis spectrophotometer for the following analysis. As mentioned, the detection system is focus on microfluidic reactor with textured silicon surface. Once optimized with immobilization of Candida rugosa lipase on the textured silicon substrate, the microfluidic reactor is applied to detect transesterification reaction of pumping through a channel and measuring the transmission at the wavelength from 700 nm to 300 nm. Since the transmission increases with the transesterification reaction as shown in figure 4-13, we can detect optical responses with time. Figure 4-14 shows the detection of transmission with different incubation time and the reaction is using lipase-catalysis.
Figure 4-14 Measurement of the transmission spectrum by lipase catalyst with time.
The most different in the transmission curve from the all wavelengths is under 400 nm and the difference of 29.26 % which are 23.12 % and 52.87 % of peanut oil and biodiesel, respectively shown in figure 4-13 (c). Compared to the figure 4-13 (c) and 4-14, the transesterification reaction reacted with time, the trend of transmission curve from the oil moved toward the curve of biodiesel. That means the triglyceride has been catalyzed by lipase to form biodiesel and detected by the change of transmission. However, the transmission curves of the first two hours are below peanut oil transmission curve as shown in figure 4-15. The transmissions of first two hours are 13.5 % and 19.53 % and the triglyceride of peanut oil is 23.12 %. It is because the reactants are still under emulsifying and mixing. Then the transmission trends to increase with longer catalysis time.
Figure 4-15 The relationship of transmission and catalysis time at 400nm. The
This experiment proves the feasible and easy way for observation the transmission by UV-Vis spectrophotometer of driven biocatalytic system using immobilized lipase in a microreactor. We can determine the occurrence of transesterification reaction by the analysis of transmission changes. However, this method only can provide the phenomenon of transesterification reaction; it can not confirm the conversion yield. For more precise analyses the transesterification reaction, we use another methods which are discussed in next two sections.
4.4-2 Transesterification From NMR Analysis
Different from UV-Vis transmission, the Nuclear magnetic resonance (NMR) spectroscopy is given to exploit the magnetic properties of certain nuclei and identify the carbon-hydrogen framework of the compound during the transesterification reaction. In this section, we used the 1H-NMR with respect to hydrogen, since the protons chemical shift of CH3 groups between triglyceride and esters shown in figure 4-16. [27][28][74]
Figure 4-16 Schematic diagrams of the chemical shift of CH3 groups. (a) Chemical shifts of proton in triglyceride, and (b) chemical shifts of proton in eaters (biodiesel).
The alkyl groups of methanol and alkoxy groups of triglyceride will react and exchange for production biodiesel during the transesterification shown in figure 2-1.
The product of eaters (biodiesel) with two ends of CH3 groups, shown in figure 4-16 (b), is made from the feedstock of one molar triglyceride with CH3 groups and 3 molar methanols. The groups of R1, R2 and R3 does not react the responses in the whole reaction, and the chemical shift of CH3 groups with R1, R2 and R3 is about 0.9 ppm and another site of esters with CH3 groups is about 3.6 ppm. Since the two end sites of esters are with the same molar of CH3
Depending on the local chemical environment, different protons in a molecule resonate at slightly different frequencies. Hence, both frequency shift and the frequency of the fundamental resonant are directly proportional to the strength of the
groups, the peak areas is equal to the H molar in H-NMR. Therefore, the conversion can be defined as the 3.6 ppm of peak areas divide by 0.9 ppm of peak areas shown in figure 4-17.
The frequency shifts are extremely small in comparison to the NMR frequency.
All of the 1 500
NMR spectrometer with description 5mm, 7” length tubes, and the solvent is d-Chloroform (CDCl
H-NMR experiments are performed on a Varian Unityinova
3). The typical frequency shift might be 500 Hz, and the chemical shift is generally expressed in parts per million (ppm). That means the percentage yield (weight conversion) was defined as (ppm of biodiesel ÷ ppm of initial peanut oil) × 100% and the percentage yield is estimated using peak area integrated by NMR spectrum. The 1H-NMR spectrum is discussed with two-dimensional spectroscopy.
Figure 4-17 Comparison of 1H-NMR spectrum before and after transesterification by Alkali-catalyzed for previously observation the chemical shift position of triglyceride and esters. (a) Before the reaction there is no peak in the chemical shift of 3.6 ppm which means there is not production of biodiesel, and (b) after the reaction the integrated of peak areas in chemical shift 3.6 ppm is 15.28 and 0.9 ppm is 16.51. The reaction conversion is calculated with 15.28/16.51 × 100 % = 92.55 %.
We have known that the 1H-NMR spectrometers can be used to detect the transesterification reaction. In this study, the reaction is discussed on the microfluidic reactors and the microfluidic reactor with textured substrate is pumped continuous.
After detection of transmission in the microfluidic reactor, the reaction solution is collected each hour for preparing 1H-NMR sample (UV/Vis-NMR analysis system) shown in figure 4-18. The results of CH3 groups with each hour in 1H-NMR spectrum is shown in figure 4-19.
Figure 4-18 Schematic of UV/Vis-NMR analysis system.
Figure 4-19 The 1H-NMR spectrums exhibit the chemical shift of CH3 groups with triglyceride and eaters for each hour. The peak of triglyceride and eaters are 0.9 ppm and 3.6 ppm respectively. The conversion is defined as the integrated of peak areas in 3.6 ppm divide by peak areas in 0.9 ppm.
Figure 4-20 Effect of lipase catalyses the transesterification reaction for the conversion increasing with time.
The figure 4-18 shows the conversion increase with transesterifacation reaction progress, and that can be calculated. In this experiment, we propose a new approach for detection the transesterification reaction. With UV-Vis spectroscopy is a feasible and easy way for quickly detecting the reaction. Following are the analysis by 1H-NMR can be quantity and calculated the conversion of transesterification reaction. The analysis is for detection of transmission in the microfluidic reactor, and the reaction solution is collected each hour for preparing 1H-NMR sample (UV/Vis-NMR analysis system). Since the phenomenon of transmission can be expressed concretely quantity by calculated H-NMR. The relationship of them is shown in figure 4-21.
Figure 4-21 The relationship of transmission and conversion with the transeaterifacation reaction by lipase-catalyzed.
For the detection of transesterification reaction, the transmission in the first two hours is 13.5 % and 19.53 % which is under the transmission of triglyceride (oil), 23.12 %. We think reactants are still under emulsifying and mixing and the reaction is not really under biocatalysis. These results can be proved by the NMR which confirms the conversion is 0 % at the first two hours. After that, the conversion of reaction increase with longer time of biocatalysis, the appearance can also observe with increasing transmission. Therefore, this method allows either commercially available immobilized lipase or transesterification reaction to be tested in a short series of experiments.
4.4-3 Electrical Properties of Commercial Cell
The lipase-catalyzed reaction is evaluated by the UV –Vis spectrophotometer at 400 nm wavelength has been discussed in chapter 4.3-1. Application of enzymatic microfluidic reactors usually allows for continuous real-time monitoring of reaction progress. Here we would like to fabricate a photodetector to monitor the biocatalysis in the microfluidic system. We use a shadow mask for coating 5 nm Cr and 10 nm Au on the silicon substrate. The metal structure is composed of two contact pads and interdigitated lines, which form the active area of the device shown in figure 4-22.
Figure 4-22 Schematic view of photodetector. (a) full layout of photodetector, the middle area is interdigitated lines, and the two squares in both side is contact pad, (b)
The device works by absorbing optical energy and converting incident photons into a time-varying electrical signal. When the active area of the device is illuminated, carriers in the semiconductor absorption layer (also known as electron-hole pairs) are generated by incident photons having energy greater than the band gap energy (Eg).
The carriers are transported to the metal contact pads, and current is detected in the external circuit under the application of an external bias voltage. However, the current of device is unstable and low on/off current is difficult to separate the photo-induced electrons shown in figure 4-23.
Figure 4-23 I-V characteristic of photodetector with interdigitated lines.
In figure 4-23, the device has low on/off current and not sensitive to the optical energy source. In this study, we focus on the phenomenon of difference transmission with the biocatalysis of transesterification reaction; therefore the sensitivity of light source is an important role for the detected device. The photodetector we used is
commercial solar cell for the same characteristic with random pyramids of textured surface by TMAH. [37]
The system utilizes a photo-electric transported work principles of real-time monitoring transesterifiaction reaction with lipase-catalyzed. Under the larger difference of transmission is at 400 nm, the lipase is immobilized on the solar cell and exposed to the 400 nm. This detection method is shown in figure 4-24.
Figure 4-24 Schematic of UV/Vis-photodetector analysis system.
The solar cell is composed of the microfluidic reactor which is exposed to 400 nm light source and the output transducer is measured the photo-electrical signal.
When the light is passing through the PDMS mold, the catalysis is evaluated in the
solar cell is amorphous silicon of SC 5030 with 50 mm in width, 29 mm in length, 2 mm in thickness, and operation at 26 mA in current and 1.8V in voltage.
Figure 4-25 The relationship of log current and voltage with solar cells which are stressed the voltage from -8V to 8V. The solar cell has better sensitivity under external negative bias operation in -5V.
Under the under the application of an external bias voltage with -5V, the photo current of the solar cell was decrease from 50 μA to 5 μA, due to that lipase attachment of solar cell. With biocatalysis time increasing, the current increases from 20.50 μA to 33.11 μA shown in figure 4-26.
Figure 4-26 The I-t curve of lipase-immobilized on the solar cell and exposed to 400nm at bias of -5V.
After pumping the feedstock into the microfluidic system continuous, the photo signals decrease further attributed to the less transparent characteristic caused by samples mixing at the first two hours. This observation also is revealed in UV-Vis transmission spectrum at the first two hour. After mixing, the photo current signals increase gradually with time. The photo current here represents the amount of 400 nm light passed through product of transesterification reaction. Therefore, the gradually increasing photo current indicates the increasing catalysis reaction, the production of transesterification. This method allows photodetector to real-time monitoring amount of biodiesel by the electric signal. From the I-t curve, we can found the microfluidic platform has the sensitivity to detect the transesterification by lipase-catalysis. The
4.4-4 Analysis of Transesterification Reaction
One of our studies is directed towards using the photodetector for simultaneous real-time monitoring, which have the potential for using differential UV-Vis measurements and for commercial cell assays.
The understanding of the relationship between sensor parameters and performance would allow biosensors to more optimally meet the connection and consequence of those detection results. The analysis of solar cell with the lipase catalysis in the microfluidic reactor would be affected by transmission and photo-electrical current. However, we should consider the transmittance of PDMS, due to the incident light of 400 nm pass through the PDMS mold into the solar cell.
Besides, the efficiency of solar cell and input power of 400 nm amethyst LED also are important factors in detecting solar cell.
The transmittance of PDMS mold is nearly 90.55 %, which is measured by a stable lamp. The power of light source is 70 mW and the efficiency of solar cell is 3.23 %. Here, we can calculate the electrical power accumulations of solar cell covered by microfluidic reactor, since the devices is illuminated and generated electrical signal by incident photons.
In equation 8, the calculation power indicates that it has the relationship between the transmission and solar cell. In this experiment, the relationship of the transmission and photo-current signal of solar cell is shown in figure 4-27.
Figure 4-27 The relationship of transmission and current of solar cll with the transeaterifacation reaction by lipase-catalyzed.
As mentioned before, the activity of the lipases after surface immobilization can be evaluated by 400 nm wavelength. This method detected the different of transmission by the UV –Vis spectrophotometer, at the same time we detected by the 1H-NMR and collected the electrical signal of solar cell for 6 hours. As the results of reaction catalysis, the detection of current increases with the transmission changes with time and enhance the conversion of reaction. Combine the figure 4-21 and 4-27, the connectionbetween the three detecting method is studied and shown in the figure 4-28.
Figure 4-28 The relationship of the transesterification reaction compared with the UV/Vis-NMR (▲) and UV/Vis-photodetector (●) analysis system. The symbols with different colors represent different hours. The colours from black to purple represent sequential reaction time from 1 to 6 hours.
There is no obvious change of conversion at first two hours by NMR measurement, due to there is no reaction during solution emulsifying and mixing.
Interestingly, the UV-Vis transmission is below the transmission of triglyceride and the photo current signal shows less change characteristic during the two hours of transesterification reaction. After that, the reaction catalysis with time and the result can be detected from the value of transmission. For the real-time monitoring, we used the solar cell for converting the light into the photo-electrical signal. In this experiment, we have the conclusion that the transesterification reaction has the characterization to the transmission and we have the conversion of the transmission
with time. Therefore, we can detect the reaction for real-time monitoring and, at the same time, know the convertion of the biocatalysis because of combining the UV/Vis-photodetector and UV/Vis-NMR analysis system.