End of Year Report

Final End of Year Report

Throughout the 2018 – 2019 school year, I worked collaboratively with my faculty mentor, Dr. Elmer Mojica, on studying the chromatographic characterization of bee pollen from different countries around the world. The original overall objective was to determine the composition of different solvent extracts of bee products, specifically from the Philippines. This was successfully completed using the five solvents of methanol, ethanol, dichloromethane, hexane, and chloroform. This led to the natural curiosity to determine the antioxidant activity and phenolic content of said solvent extracts. Both objectives were met using the HP 6890 Gas Chromatography Mass Spectroscopy and a microplate reader where the DPPH and Folin-Ciolcalteu assays were performed. Being given the opportunity by the Undergraduate Research Initiative to brainstorm, conduct, and discuss my research via presentations and blog-posts has been a critical and valuable asset to my analytical research.

Before beginning the physical research in the lab, I established three major learning outcomes that I wanted to work diligently towards throughout the academic school year. The first goal I wanted to reach was the articulation of a proper research question with an effective objective. I wanted my research to have meaning and a purpose. For this reason, I chose to work with bee pollen in relation to the health and nutrition benefits that it could possibly have on humans. For example, working with the assay gave me an insight to the amount of antioxidant levels. Antioxidants prevent the formation of free radicals, which are often cancer-causing, in the human body. Establishing an understanding of what bee pollen consists of was imperative before moving onto further research. Next, I wanted to utilize an appropriate and efficient methodology to find an answer to my research question. I did so using instrumental methods and mass spectroscopy analysis. Lastly, I aimed to present the research in a conference setting. I applied with my research abstract and was gratefully accepted to present in an Undergraduate Poster Session at the American Chemistry Society Annual Conference in Orlando, Florida.  I can successfully say that I accomplished all three of these outcomes.

An imperative part of my research was the collaboration between my faculty mentor and myself. Dr. Mojica first helped introduce me to the GC-MS instrument and the sample preparation methods involved such as extraction and filtration methods using solvents and bee pollen. Once I was able to acquire the compounds that make up the pollen, he challenged the research to go even further by teaching me how to determine the phenol content and antioxidant levels. Two assays were used with the Biotek Cytation instrument, DPPH and Folin-Ciocalteu. The DPPH assay involved dissolving the DPPH solution and pollen extracts in methanol on a microtiter plate. The Folin-Ciocalteu assay involved diluted methanol mixed with Na2CO3. The absorbance was then measured at 745 nm post-incubation. We gathered that the phenol content was highest when methanol was used for extraction and lowest when hexane was used. The antioxidant amount was highest when extracted with chloroform and lowest when extracted with methanol. Without Dr. Mojica’s guidance and assistance in the assay processes, the procedure would not have reached the scientific caliber that it did. By meeting with him on a regular basis to discuss all aspects of the research, I heightened my ability to work with others in terms of problem-solving and effective communication.

The main initiative for my research project was being able to find an answer to the question: what components make up bee pollen? I wanted the answer to this to be used in future studies for human health benefits. While my study did not focus on how bee pollen benefits humans, it focused rather on what bee pollen consists of. For this reason, further analysis can be used to interpret the benefits of these components, specifically in terms of antioxidants. Although problems were encountered during the school year, most were fixed through collaborative problem solving. For example, during a reading of bee pollen in hexane, no peaks were found. Through doing several trials, we realized that a greater amount of hexane, in microliters, must be injected into the GC-MS for a proper reading to be established. We therefore injected 10 microliters as opposed to 5 microliters. For precise readings, it was necessary that trials be taken multiple times. It was then found that the non-polar solvents contained mainly non-polar compounds, specifically long-chain hydrocarbons.

The Undergraduate Research Initiative provided me with the motivation to ask, learn, and challenge my previous bee exploration. By collaborating with Dr. Mojica, I established a solidified understanding of the GC-MS instrumentation, filtering process, and two different assay methods. Through constant communication, we were able to establish a proper methodology and ability to solve rising issues in the lab. The conducting of chromatographic research on bee pollen has served as an educational and eye-opening experience with results that can be implemented by future researchers.

Blog 3: A Conclusive Review of my Research on the Characterization of Bee Products

March is a busy month in the world of Chemical research! I am currently preparing to attend the American Chemistry Society conference in Orlando, Florida, which will take place at the end of the month, along with the Dyson School Society of Fellows conference on March 9th. Although the plane tickets are booked and the conferences are registered for, the most important part remains in the works – the display poster. I aim to have the poster completed by Spring Break, as I am currently working on analyzing the major peaks on the gas chromatography-mass spectrometry chromatogram to understand which compounds can be found in the pollen.

Presenting at the Dyson Society of Fellow’s conference.

The extraction process for the solvents being used is now complete. I decided upon using four main solvents: ethanol, methanol, methylene chloride, and chloroform. Using a precision balance, I weighed 5 grams of pollen, specifically from Los Baños in the Philippines. Allowing the pollen to sit in a test tube with the solvent for about 24 hours results in a solution ready to be filtered. I used syringe filtration to filter each of the solutions. The filtered products were then put into mini centrifuge tubes and refrigerated for another 24 hours. Post-refrigeration, the filtered solutions were ready to be injected into the GC-MS. One sample was injected per day, using different injection needles as to avoid any cross-contaminations between the solvents.

Samples of bee pollen in five various solvents.

Experiments do not always go as planned. Through this experiment, I have learned to not only accept this, but embrace it as a learning opportunity. Whenever something does not go as planned, I work collaboratively with my faculty mentor to ask the questions “why did this go wrong?” and “how can we fix it?” One recent example of this was the possibility of contamination. Rather than rinsing out the injection needle and re-using them, the possibility of cross-contamination was eradicated by using multiple injection needles.

As the end of my research project comes near, I am focusing on an in-depth exploration and analysis of the results at hand. I am also currently working on a paper about my research in hopes of submitting it for publication. Overall, this research opportunity has provided me with investigative skills, critical thinking skills, while enhancing my wet lab skills as well.

Blog Post #2: Continuation of Research on the Methods of Extracting Bee Pollen

During the entirety of the Fall semester, I worked collaboratively with Dr. Mojica for 1-2 lab sessions per week. We met before, during, and after my time in the lab to establish the goals for the lab session, answer any relevant questions, and discuss a brief overview of the results attained. Using my lab notebook, I kept track of each step taken in this process, as well as by taking photo documentation. Dr. Mojica and I also worked to establish an abstract to submit to the American Chemical Society for the 257th National Meeting, taking place in the Spring of 2019 in Orlando, Florida. I was happily accepted, alongside some of my colleagues, to be given the opportunity to present my research at the conference with some of my colleagues. Since my study focuses on various extraction methods, it has been very beneficial to work with Dr. Mojica who, through a collaborative effort, has taught me several methods of extraction for various bee-deriving substances. With a passion for the topic of analysis, the initiative to continue my research this semester was simply to find an answer to the question at hand in hopes of using the results to help better understand how bee substances can help our society.

Throughout this semester, my UGR research has taken a turn from focusing primarily on propolis to being inclusive of pollen, as well. A recurring obstacle was noticed despite of the change in focus from propolis to pollen. It appeared that cross-contamination in the GC-MS instrument remained a prevalent issue. For this reason, we came up with a few problem-solving techniques, such as running a trial solely with the solvent before running it with the pollen. This aided in cleaning out the machine of any by-products that may have been lingering from prior research. Additionally, our addition of a new machine, described below, has helped us in the extraction process in an effort to avoid cross-contamination. Each week, we have worked to make continuous progress on this project, despite of any obstacles encountered along the way.

Fortunately, we ended the semester with a gift from NSF, the National Science Foundation. The National Science Foundation, an American government agency, provides financial aid to support research and education in the field of science. Dr. Mojica was given a grant for the Accelerated Solvent Extraction (ASE) machine, which I have had the benefit to start using. The ASE machine is used for the extraction of chemicals from a solid. I first grind the bee pollen into smaller pieces and let these pieces sit in 5 mL of the given solvent overnight. Then, I manually filter this solution using syringe filters. This is followed by an extraction in the ASE machine. Below is a photo of me with our new ASE machine and my first extraction using the machine! We are continuing to answer the question of why there are outside chemicals showing up in our results from the GC-MS and hope to have this issue solved by the end of the semester.

 

My first extract of pollen and methanol using our new ASE.

Chromatographic Characterization of Bee Propolis and Pollen from Around the World

The title of my research, as stated above, is the Chromatographic Characterization of Bee Propolis and Pollen From Around the World. I have chosen to analyze and compare the components that make up bee pollen and propolis from several species with varying origins, developing an understanding for how the type of extraction methods will affect the gathered results. The purpose of this research is to distinguish between the results gathered from using different extraction methods, while understanding the importance of the extracted components. Moving forward, we would like to understand the different health benefits that propolis can provide for humans and which benefits relate to which chromatographically discovered components.

Before stepping into the wet lab and performing physical research, Dr. Elmer Mojica and I worked collaboratively to perform literary research in order to finalize my research question and methodology being used. We studied papers from several scientific journals, such as the Food Research International and the Journal of the Science of Food and Agriculture. In today’s society, the focus of what is being put inside the foods and products consumed by humans is so vast and significant. For this reason, I expect to achieve a fully developed understanding of which extraction method is best, along with which components are found in which species of bees. I can then use this to understand which components humans are consuming when they go to health food stores to purchase propolis and pollen.

The methodology being used to answer my research question varies depending on the extraction method being used. In chemistry, extraction methods are used to separate substances that they are mixed with. I used my literary research to fully understand the Soxhlet, microwave, sonication, accelerated solvent extraction, and simple extraction. I aim to compare these methods with one another. I will first be using dichloromethane and methanol to make solutions with the propolis and pollen. I can then inject these solutions into the machine known as GC-MS, or gas chromatography-mass spectrometry. This provides us with a visual representation of the spectrum. We can then use this, alongside a library, to analyze which molecules make up the propolis and pollen. Refer to Image 1.0 for footage of the first bee pollen that we have begun analyzing.

Image 1.0 – Stingless Bee Pollen from Los Baños, Leguna, Phillippines

This semester has so far been successful with literary research and we have begun injecting samples into the GC-MS. We are looking forward to analyzing the results and successfully reaching our desired achievements to answer my research question.