1. i Spectral analysis of Commercially Available Honey using Low cost, Portable Vis-NIR Spectroscopy _________________________________________ An Undergraduate Thesis Proposal Presented to the Faculty of College of Mathematics and Natural Sciences Caraga State University 8600 Ampayon, Butuan City _________________________________________ In Partial Fulfillment Of the Requirements for the Degree Bachelor of Science in Physics _________________________________________ SARAH MAE EPAN MAY 2024

2. ii Table of Contents CHAPTER 1 INTRODUCTION .................................................................................... 1 Background of the Study ................................................................................... 2 1.1.1 Importance of Honey Purity: Health, Economic, and Regulatory Perspectives ................................................................................................................ 2 1.1.2 Investigating Honey's Role ......................................................................... 3 1.1.3 Honeybee Biodiversity and Indigenous Practices .................................... 4 1.1.4 Honey Adulteration .................................................................................... 4 1.1.5 Traditional Methods on testing the Honey’s Purity ................................ 5 1.1.6 Spectroscopic techniques ............................................................................ 5 1.1.7 Analysis 6 Vis-NIR Spectroscopy: A Rapid and Non-Destructive Tool for Honey Objectives of the Study ...................................................................................... 6 Significance of the Study.................................................................................... 7 Scope and Limitation ......................................................................................... 7 Theoretical Background .................................................................................... 8 1.5.1 Vis-NIR Spectroscopy Non-Destructive Quality Control ....................... 8 1.5.2 Vis-NIR Spectroscopy ................................................................................. 8 1.5.3 Applications ................................................................................................................ 9 Theoretical Foundations of Vis-NIR Spectroscopy for Analytical Review of related literature ............................................................................. 11 CHAPTER 2 METHODOLOGY ................................................................................. 15 Optical Components ......................................................................................... 15 2.1.1 Light Source: Incandescent Lamp ..................................................................................... 17 2.1.2 Diffraction gratings ............................................................................................................ 18 2.1.3 Optical Slits and Light Collimation .................................................................................... 18 2.1.4 VIS-NIR Detector ............................................................................................................... 19 2.1.5 OceanView: Software ......................................................................................................... 20 Sample Testing.................................................................................................. 21 Honey Adulteration Sample Preparation ...................................................... 21 CHAPTER 3 Design and Implementation of cost-effective Spectroscopy ................... 23

3. iii Abstract 23 Introduction ...................................................................................................... 23 Methods ............................................................................................................. 24 3.2.1 Design of the spectrometer was carried out with the SketchUp ........... 24 3.2.2 Test the Effectiveness of the used detector ............................................. 24 3.2.3 Detect the effectiveness of the sample through food dye ....................... 25 Results and Discussion ..................................................................................... 25 3.3.1 Vis-NIR Spectrometer Calibration Performance .................................. 25 3.3.2 Helium-Neon (He-Ne) laser ...................................................................... 26 3.3.3 Argon .......................................................................................................... 28 3.3.4 Hydrogen ................................................................................................... 29 3.3.5 Helium ........................................................................................................ 30 3.3.6 Food dye ..................................................................................................... 31 Conclusion ......................................................................................................... 32 CHAPTER 4 33 4.1 Introduction ........................................................................................................... 33 4.2 Methods .................................................................................................................. 34 4.3 Results and Discussion .......................................................................................... 35 4.3 Limitation and recommendation ......................................................................... 37 4.5 Conclusion .............................................................................................................. 37 CHAPTER 5 39 CONCLUSION ................................................................................................................. 39 REFERENCE 40

4. iv List of Figures Figure 1 Schematic diagram of spectroscopy ..................................................................... 9 Figure 2 The electromagnetic spectrum .............................................................................. 9 Figure 3 Vis-NIR spectra of (a) Pure SBH and adulterants and adulterated SBH by different percentages of water (b), apple cider (c), and fructose syrup (d) ..................................... 11 Figure 4 The original UV–Vis spectra of authentic honey, pure adulterants, and adulterated samples with sugars and colorants in the highest concentration in the range of 190–900 nm ........................................................................................................................................... 12 Figure 5 Intensities for honey samples depending on the type of adulterant present ....... 13 Figure 6 Transmittance of Pure honey and Adulterated honeys ....................................... 14 Figure 7 Cost effective Spectrometer with DC Power Supply ......................................... 15 Figure 8 The Vis-NIR Spectroscopy set-up ...................................................................... 16 Figure 9 Emission Spectrum of the used Incandescent light ............................................ 17 Figure 10 Diffraction Grating ........................................................................................... 18 Figure 11 USB650-VIS/NIR and P400-Vis-NIR Fiber cord ............................................ 19 Figure 12 OceanView software for Spectral Analysis ...................................................... 20 Figure 13 Transmittance ................................................................................................... 22 Figure 14 Diagram of Vis-NIR Spectroscopy .................................................................. 24 Figure 15 The Vis-NIR spectra, ranging from 350 nm to 1000nm .................................. 26 Figure 16 Vis-NIR Spectra of Helium Neon laser, Red laser and Green laser ................. 27 Figure 17 Emission spectra of Argon ............................................................................... 28 Figure 18 Emission spectra of Hydrogen.......................................................................... 29 Figure 19 Emssion spectra of Helium ............................................................................... 30 Figure 20 Obtained wavelength peaks of food dyes using an incandescent light source. 31

5. v Figure 21 Obtained wavelength peaks of pure and adulterated honey using the Vs-NIR spectroscopy ...................................................................................................................... 36

6. vi Abstract This study investigated the construction and application of a Vis-NIR spectrometer for material analysis. An incandescent lamp successfully calibrated the spectrometer, demonstrating its accuracy through consistent measurements of laser transmittance and alignment with known gas emission spectra. Spectral analysis of various dyes revealed peaks generally matching literature values, highlighting the spectrometer's potential for material characterization. However, discrepancies observed emphasize the influence of factors like dye composition and experimental setup. The Vis-NIR spectrometer distinguished pure honey from branded products based on their spectral signatures. Pure honey exhibited peaks closely matching literature values, while branded honeys displayed differing profiles, suggesting potential adulteration due to additives, processing, or floral sources. Notably, branded honeys showed lower peak intensities, indicating a reduced ability to transmit light. These findings suggest the promise of Vis-NIR spectroscopy for honey quality control. There are restrictions, though. The limited wavelength range and variable light intensity of incandescent lamps require additional research using more advanced light sources and comprehensive spectral analysis in order to conclusively identify adulterants.

7. 1 CHAPTER 1 INTRODUCTION Honey has been a valued natural product for centuries, appreciated for both its delightful taste and potential health properties. The most common type is flower honey, meticulously crafted by honeybees (Apis mellifera L.) who collect nectar from a variety of flowers. This floral source gives honey its unique flavors and variations, making it a regionally dependent treat. Traditionally, honey has been used for a range of health purposes, including strengthening the immune system, soothing coughs and sore throats, and promoting wound healing. Recent research suggests even wider benefits, with honey exhibiting anti- inflammatory, antioxidant, and even anti-cancer properties. These therapeutic effects can be delivered in two ways: through oral consumption or topical application. Studies have shown promise for treating various conditions when taken orally, such as ulcers, insomnia, and even liver problems. Topical application might benefit skin conditions like eczema and burns. Honey's unique composition is key to its properties. Primarily composed of sugars and water, it also contains a wealth of other compounds like amino acids and vitamins. The exact makeup depends on factors like the flower source, harvest season, and storage methods. This intricate chemistry contributes to the distinct flavors and potential health benefits of honey, making it a popular choice for consumers worldwide honey [1]-[2]-[3]. Honey is also a widely consumed food, and is particularly susceptible to adulteration. One major threat to honey's purity is the addition of cheaper, commercially available sugar syrups that mimic its chemical composition. Even with quality standards in place, contamination or adulteration can occur during processing, compromising the honey's integrity. Adulteration refers to the addition of any foreign substance to pure honey. This practice can have negative health consequences, potentially leading to issues like diabetes, weight gain, and high blood pressure due to increased

8. 2 glucose and blood lipid levels [1]. Common adulterants include readily available syrups like high-fructose corn syrup and corn syrup The growing global population and consumer demand for high-quality food highlight the importance of the food industry. However, low-quality or adulterated food can pose serious health risks. Food adulteration, which involves adding inferior ingredients or removing essential components, further amplifies this threat. When adulterated products contain cheaper, lower-grade elements that compromise consumer health, they are deemed unsafe. Consequently, ensuring the quality and safety of honey has become a top priority for international regulatory bodies [4]-[2]. Scientists are increasingly using spectroscopy to quickly and easily check honey purity without harming the sample. To address this issue, various methods, both traditional and modern, have been developed to distinguish pure honey from adulterated products. This technique is gaining popularity because it's safe for the environment and doesn't destroy the honey. As people find new ways to tamper with products, methods to verify authenticity need to improve as well. Newer spectroscopy techniques, like Raman spectroscopy, are being developed to address this. Combining near-infrared (NIR) spectroscopy with other methods offers a simple, fast, and safe way to detect adulteration in honey. It's important to note that honey can be thin, thick, or crystallized, but this method still works because it relies on water content, which most natural products have. This approach, using NIR spectroscopy, is being actively researched to create better methods for detecting adulterated honey. Background of the Study 1.1.1Importance of Honey Purity: Health, Economic, and Regulatory Perspectives The purity of honey holds paramount significance from multiple perspectives encompassing health, economic, and regulatory domains. From a health standpoint, consumers rely on honey as a natural alternative to refined sugars and artificial sweeteners, often seeking

9. 3 its purported health benefits, including antioxidant properties and potential antimicrobial effects [5]. However, the presence of impurities, such as added sugars or contaminants, compromises these perceived health advantages and may even pose risks to vulnerable populations, such as infants or individuals with allergies [6]. Economically, honey is a valuable commodity both domestically and internationally, with its quality directly impacting market value. Consumers are willing to pay premium prices for pure, high-quality honey, making authenticity and transparency crucial for maintaining consumer trust and fostering fair trade practices within the honey industry. Furthermore, from a regulatory perspective, ensuring honey purity is essential for compliance with food safety standards and regulations. Governments and regulatory bodies worldwide establish guidelines to safeguard consumer health and prevent deceptive practices, necessitating reliable methods for honey purity assessment and enforcement of quality control measures throughout the supply chain [7]. Therefore, addressing the complexities of honey purity assessment through innovative techniques, such as low-cost, portable spectroscopic analysis, is not only a scientific endeavor but also a critical imperative with far-reaching implications for public health, market integrity, and regulatory compliance. 1.1.2Investigating Honey's Role For centuries, honey reigned supreme as a sweetener. Beyond its delicious taste, it was also prized for its medicinal properties. People used honey to make mead, a fermented drink, and even added it to wine and other alcoholic beverages. In Egypt, honey played a unique role in the embalming process. Across Asia, including India, honey served as a natural preservative for fruits and a key ingredient in cakes, candies, and various other foods [8]. Honey is a complex mixture, boasting over 180 different compounds. This includes water, sugars, amino acids, proteins, enzymes, minerals, vitamins, and even unique plant chemicals. The exact makeup of each honey varies depending on several factors. The type of flowers the bees collect

10. 4 nectar from plays a big role, along with the geographic location, climate, and even the specific bee species involved. Processing techniques and storage methods can also influence the final product. This intricate composition is what contributes to the distinct flavors and colors found in different honey varieties [9]. 1.1.3Honeybee Biodiversity and Indigenous Practices In the Philippines, honey is sourced from a variety of bee species, namely: 1) Apis dorsata, recognized as wild honeybees or "pukyutan," 2) Apis cerana, also known as "anig," 3) Tetragonula biroi, commonly referred to as stingless bees or "Lukot," and 4) Apis mellifera, the European honeybees [10]. The Sierra Madre Mountain range acts as a protective barrier for the Cagayan Valley Region against storms, boasting rich biodiversity. Beyond its ecological significance, the Sierra Madre plays a crucial role in the lives of the Cagayanos, serving as their primary source of livelihood, including the gathering of wild honey. Honey holds revered status as "liquid gold" among the locals in northern Cagayan due to its nutritional value and medicinal properties, often utilized as an alternative treatment for various ailments. Despite the laborious process of honey gathering, indigenous communities such as the Aeta families residing in the Sierra Madre mountains engage in this activity as a means of earning income [11]. 1.1.4Honey Adulteration The honey trade is a major player on the global stage, offering both economic advantages and nutritional value [12]. However, ensuring the quality and purity of this product requires tackling some key challenges. Food safety standards play a crucial role in safeguarding the honey trade. Honey adulteration, where cheaper substances are added to dilute honey, it is a growing concern. Researchers from the Department of Science and Technology-Philippine Nuclear Research Institute (DOST-PNRI) have made a concerning discovery regarding the purity of honey products available in various retail outlets. Their study revealed that approximately 80% of honey products, whether sold in groceries, souvenir shops, or online

11. 5 platforms, do not meet the standards of pure honey [13]. According to the Philippine National Standard for Honey set by the Bureau of Agriculture and Fisheries Standards, honey sold in the market should be free from any food additives and additional substances. Any additives present must be clearly stated on the product labeling. Additionally, the labels should include the geographical origin of the honey [14]. This finding highlights a significant issue in the honey industry, as the demand for honey has led to fraudulent practices in its production. Despite the potential for honey farming to be a lucrative venture, the prevalence of adulterated products undermines consumer trust and the integrity of the market. 1.1.5Traditional Methods on testing the Honey’s Purity Traditional methods for testing the purity of honey typically involve a combination of physical, chemical, and microscopic analyses. Physicochemical parameters such as moisture content, electrical conductivity, and pH are often measured to assess honey quality. Additionally, analyses of specific chemical marker such as diastase activity, hydroxymethylfurfural (HMF) [15] content, and sugar composition provide insights into honey authenticity and adulteration. Microscopic examination, particularly pollen analysis, is another widely used method for determining the botanical origin and authenticity of honey [16]. However, these traditional techniques have several limitations including time-consuming procedures [17], the requirement for specialized equipment and trained personnel, and the inability to detect certain types of adulteration [18]. As a result, there is a growing demand for innovative and cost-effective approaches such as spectroscopic techniques, which offer rapid, non-destructive, and potentially portable alternatives for honey quality assessment [19]. 1.1.6Spectroscopic techniques In response to the fast-paced nature of the global honey market, there has been the development of rapid detection methods for honey adulteration utilizing spectroscopic techniques combined with chemometrics. These methods encompass Fourier-transform

12. 6 infrared spectroscopy (FTIR), near-infrared spectroscopy (NIR) and ultraviolet-visible spectroscopy (UV-Vis) [20]. 1.1.7Vis-NIR Spectroscopy: A Rapid and Non-Destructive Tool for Honey Analysis Vis-NIR (Visible and Near-Infrared) spectroscopy has emerged as a promising technique for honey analysis due to its rapid, non-destructive nature. This spectroscopic method analyzes the interaction of light in the visible (400-780 nm) and near-infrared (780- 2500 nm) regions with honey components [21]-[22]. Different molecules absorb specific wavelengths of light, creating a unique spectral fingerprint for the sample. By analyzing these spectral patterns, researchers gain valuable insights into the chemical composition of honey. This technique offers significant advantages over traditional methods, which can be time- consuming, destructive, or require specialized expertise. Vis-NIR spectroscopy provides a rapid and objective means to assess honey quality, detect adulteration, and potentially classify honey based on its floral origin [22]. Despite advancements in honey analysis techniques, ensuring honey purity and quality remains challenging. Traditional methods are often time-consuming, destructive, or require specialized expertise, leading to inefficiencies and potential inaccuracies. Vis-NIR spectroscopy offers rapid, non-destructive assessment, but its effectiveness in detecting honey purity, adulteration, and classifying floral origin needs further investigation. This research aims to explore the efficacy of Vis-NIR spectroscopy in developing a reliable method for honey analysis in the food industry. Objectives of the Study Honey adulteration is a growing concern, highlighting the need for rapid and cost- effective detection methods. This study aims to address this challenge by developing a low- cost Visible-Near Infrared (Vis-NIR) spectrometer for pure honey authentication;

13. 7 1.To design and construct a low-cost, wood-based Vis-NIR spectrometer 2.Test the effectiveness of the improvised spectrometer in the Physics Laboratory of Caraga State University. 3.To detect and develop a method for differentiating between pure and adulterated honey samples using the spectral data acquired from the constructed low-cost Vis-NIR spectrometer Significance of the Study Ensuring honey purity is crucial for both consumers seeking genuine health benefits and producers striving to maintain brand trust. This study explores the potential of a cost- effective, Visible-Near Infrared (Vis-NIR) spectrometer as a tool for detecting adulteration in Honey with water and apple cider vinegar. By quantifying spectral changes caused by adulteration at various levels, the research aims to establish a reliable and rapid method for identifying compromised honey samples. This approach offers significant advantages. The affordability of the Vis-NIR spectrometer makes it a more accessible solution compared to expensive traditional methods. Additionally, the non-destructive nature of the technique allows for testing without harming the honey sample itself. Ultimately, developing a practical and cost-effective honey quality control tool benefits both consumers by guaranteeing product authenticity and producers by safeguarding their brand reputation. Scope and Limitation This study utilizes a low-cost Vis-NIR spectrometer for the detection of adulteration in honey. The chosen spectrometer operates within the visible and near-infrared range, specifically from 400nm to 1000nm. It's important to acknowledge that this range might not capture all spectral signatures associated with potential adulterants. While this limitation exists, the chosen wavelength range offers a balance between cost-effectiveness and capturing key

14. 8 spectral information relevant to honey analysis. Additionally, this research will be conducted in the Physics Laboratory of Caraga State University, located in Butuan City, ensuring controlled experimental conditions and access to necessary equipment and expertise. Theoretical Background 1.5.1Vis-NIR Spectroscopy Non-Destructive Quality Control Honey adulteration, where cheaper substances are added to dilute honey, it is a growing concern. Spectroscopic techniques have emerged as promising tools for rapid and non- destructive honey quality control. The theoretical foundation lies in the unique light absorption properties of different molecules [23]. Each substance possesses a characteristic spectral fingerprint, and adulteration disrupts this fingerprint by introducing new absorption peaks or altering existing ones. Visible-Near Infrared (Vis-NIR) spectroscopy is particularly suitable for honey analysis due to its sensitivity to water content and sugar composition, both of which are commonly targeted for adulteration [24]. The electromagnetic spectrum encompasses a vast range of wavelengths, only a portion of which is visible to the human eye. This segment, known simply as visible light, spans wavelengths from roughly 380 to 700 nanometers [25]. Beyond the red end of the visible spectrum lies the near-infrared (NIR) region. NIR radiation boasts wavelengths slightly longer than visible light, ranging from 700 nanometers to 2500 nanometers. This distinction between visible and NIR light proves crucial for various analytical techniques, with vis-NIR spectroscopy leveraging the unique properties of both regions for material analysis [26]-[27]. 1.5.2Vis-NIR Spectroscopy The vis-NIR portion of the electromagnetic spectrum offers a valuable means of distinguishing between different molecules based on how they absorb light. Each molecule has its own arrangement of atoms and bonds, leading to distinct light absorption properties [25]- [28]. When light interacts with a molecule's structure, specific wavelengths containing energy

15. 9 are absorbed, corresponding to molecular vibrations or electron excitations. Because molecules vary in structure and energy levels, they absorb light at different wavelengths. Vis-NIR spectroscopy, by analyzing these absorbed wavelengths, effectively generates a molecular "fingerprint." This unique signature reveals how a molecule interacts with light, aiding in its identification and potential quantification in a given sample [21]. Figure 1 Schematic diagram of spectroscopy 1.5.3Theoretical Foundations of Vis-NIR Spectroscopy for Analytical Applications Vis-NIR spectroscopy, also known as visible and near-infrared spectroscopy, relies on the interaction of electromagnetic radiation [29] with matter to provide information about the molecular composition of a sample. This spectroscopic technique covers a broad spectral range from the visible (VIS) to the near-infrared (NIR) regions, typically spanning wavelengths from approximately 400 to 2500 nanometers [27]-[29]. The theoretical background of Vis-NIR spectroscopy is grounded in the principles of molecular absorption and reflection. Figure 2 The electromagnetic spectrum

16. 10 In Vis-NIR spectroscopy, molecules within the sample absorb and scatter light at specific wavelengths, resulting in characteristic absorption bands or spectral features. These absorption bands are associated with molecular vibrations, such as stretching, bending, and combination modes of chemical bonds [27]. In the visible region, electronic transitions of chromophores contribute to absorption features, while in the NIR region, overtone and combination bands of fundamental vibrations are observed. The Beer-Lambert law, a fundamental principle in spectroscopy, describes the relationship between the absorbance of light by a sample and its concentration [30]. According to this law, absorbance (A) is directly proportional to the path length (l) of the sample, the concentration (C) of the absorbing species, and the molar absorptivity (ε) of the substance at a given wavelength: ? = ? ? ? In Vis-NIR spectroscopy, the Beer-Lambert law is applied to quantify the concentration of chemical components in the sample based on their absorbance spectra. Overall, Vis-NIR spectroscopy offers several advantages for analytical applications, including rapid data acquisition, non-destructive sampling, and the ability to analyze samples in various physical states (e.g., solids, liquids, and gases) [24]-[3]. Its theoretical foundation in molecular spectroscopy enables the quantitative determination of multiple chemical constituents in a wide range of materials, making it a valuable tool for quality control, process monitoring, and research in various industries, including agriculture, food, pharmaceuticals, and environmental science.

17. 11 Review of related literature The study entitled "Identification of Stingless Bee Honey Adulteration Using Visible- Near Infrared Spectroscopy Combined with Aquaphotomics," authored by Muna E. Raypah et al., investigated adulteration in Stingless Bee Honey (SBH) through a combination of visible (Vis) and near-infrared (NIR) spectroscopy, focusing on the short-wavelength NIR region (800-1100 nm). SBH samples were adulterated with water, vinegar, or syrup across varying levels (10-90%). While visible spectroscopy only detected adulteration above 50% levels, NIR spectroscopy identified adulteration for water and vinegar, attributed to changes in water structure. This novel method utilizing both visible and near-infrared light spectroscopy successfully detected SBH adulteration, even at levels as low as 10%, with high accuracy. The technique, emphasizing a specific near-infrared wavelength range, exhibits potential for development into portable tools for convenient quality control of SBH. [1]. Figure 3 Vis-NIR spectra of (a) Pure SBH and adulterants and adulterated SBH by different percentages of water (b), apple cider (c), and fructose syrup (d)

18. 12 Another study titled "Application of UV–Vis spectroscopy for the detection of adulteration in Mediterranean honeys," authored by Dafni Dimakopoulou-Papazoglou et al., highlights the effectiveness of coupling UV–vis technique with Principal Component Analysis (PCA) in discerning pure honey from those adulterated with glucose syrup. Through PCA analysis, the study revealed that a significant 98% of the total variance within the model could be elucidated by wavelengths ranging from 280 to 300 nanometers (nm). Further validation of this method using Linear Discriminant Analysis (LDA) achieved an impressive variance of 99.8%, emphasizing the reliability of this approach in identifying honey adulteration. The outlined protocol involves simple sample preparation steps, eliminating the need for hazardous chemicals, and incorporates rapid scanning and straightforward data processing, making it suitable for honey analysis and quality assessment. Phenolic compounds, such as flavonoids and phenolic acids, play a crucial role in honey's antioxidant properties and contribute to its distinctive color, flavor, and biological functions. These compounds typically absorb ultraviolet (UV) light within the range of approximately 280 to 300 nanometers (nm), with flavonoids serving as markers for detection and quantification, thereby providing valuable insights into honey's composition and quality. The integration of UV–vis technique with the developed chemometrics protocol shows promise in identifying honey fraud efficiently and cost-effectively, thereby upholding the quality standards of this essential bee product within the beekeeping industry [31]. Figure 4 The original UV–Vis spectra of authentic honey, pure adulterants, and adulterated samples with sugars and colorants in the highest concentration in the range of 190–900 nm

19. 13 Another also study utilizes techniques such as solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS), infrared (IR) spectroscopy, and Raman spectroscopy to authenticate the botanical origin and geographical source of honey and to detect any adulteration. Specifically, it includes average visible-near infrared (Vis-NIR) spectra for pure multi-floral honey and four different sweeteners used for adulteration: rice syrup (RS), invert sugar (IS), brown cane sugar (BS), and fructose syrup (FS). In the Vis-NIR spectra, different regions labeled 1, 2, and 3 show varying intensities for honey samples depending on the type of adulterant present. This indicates that the Vis-NIR spectra of honey change in distinct ways when different types of sweeteners are added to them. These spectral differences serve as markers for detecting adulteration, helping to ensure the authenticity and quality of honey products [32]. Figure 5 Intensities for honey samples depending on the type of adulterant present The study titled "Adulterated Stingless Bee Honey Identification Using VIS-NIR Spectroscopy Technique" conducted in 2020 focused on identifying adulteration in stingless bee honey using visible-near infrared (VIS-NIR) spectroscopy. Specifically, the wavelength of 787.677 nm was selected for comparison among treatments for soluble solids content (SSC) and refractive index

20. 14 (RI) due to its high transmittance value and superior signal-to-noise ratio, enhancing measurement precision. The results revealed significant differences in the transmittance rates of stingless bee honey across five different treatments, with pure honey exhibiting the highest transmittance compared to adulterated samples. Notably, the addition of sucrose solution to stingless bee honey, likely due to the adulteration process altering the honey's moisture content and viscosity, consequently affecting transmittance. This phenomenon aligns with the principles of the Lambert-Beer law, commonly known as Beer's law, where changes in honey concentration impact VIS-NIR properties. Figure 6 Transmittance of Pure honey and Adulterated honeys

21. 15 CHAPTER 2 METHODOLOGY Spectroscopy studies how light interacts with matter and is used in many areas, from testing fruit ripeness to space exploration and determining atomic structures. Near InfraRed (NIR) spectroscopy, which covers the 780-2526 nm range, is used for analyzing milk, bio constituents, and in printing and production to find accurate colors [29]-[28]. However, spectrometers for this range can be quite expensive. Additionally, there are studies that use Vis-NIR spectroscopy to detect the purity of honey [24]. In this study, the researchers will build a cost-effective Vis-NIR spectrometer. This will cover from 350nm -1000 nm . Optical Components A dispersive spectrometer separates light into its constituent wavelengths for spectroscopic analysis. The initial stage involves collimation, where a slit or optical fiber restricts and defines the incident light beam. This enhances spectral resolution by controlling the divergence and quantity of light entering the system. Subsequently, collimating optics, often lenses, are employed to ensure the light rays propagate in a parallel manner. This minimizes spectral dispersion, which is the broadening of wavelengths due to their interaction with the dispersive element [23]. Finally, a prism or diffraction grating acts as the dispersive element, causing wavelength-dependent deflection of the collimated light. The spatially separated wavelengths are then focused onto a detector by an imaging lens. The resulting spectrum, where the position on the detector corresponds to the specific wavelength, allows for the qualitative and quantitative analysis of the original light source [33]-[10].

22. 16 Figure 8 The Vis-NIR Spectroscopy set-up

23. 17 2.1.1 Light Source: Incandescent Lamp The incandescent lamp, also known as a bulb or light, serves as a reliable light source for detection purposes. Operating on the principle of incandescence, these electric lights emit light by heating a filament [34]-[35]. Available in various sizes, voltages, and wattages, incandescent lamps typically operate within a voltage range of 1.5V to 300V. The mechanism involves passing current through a thin metal filament, typically made of tungsten due to its high melting point. Once heated, the filament emits light, reaching temperatures of up to 4,500 degrees Fahrenheit. To prevent overheating and oxidation of the filament, the lamp is enclosed in a glass casing, ensuring efficient light generation. Due to its effectiveness in producing light, the incandescent lamp finds applications across a wide range of fields [34]-[36] The figure below illustrates the spectrum of an incandescent lamp, displaying its wavelength range from 350 nm to 1000 nm. Figure 9 Emission Spectrum of the used Incandescent light

24. 18 2.1.2 Diffraction gratings In the visible and near-infrared (VIS-NIR) range, diffraction gratings offer a cost- effective alternative to prisms for constructing a spectroscopy setup. These gratings act by dispersing light based on wavelength. As light strikes the grating, its component wavelengths diffract at slightly different angles. This creates a characteristic pattern where the position of each diffracted wavelength on the detector corresponds to its specific value. By employing a collimating lens after the entrance slit, the setup ensures parallel light rays interact with the grating, maximizing the separation between wavelengths and enhancing spectral resolution [37]. Figure 10 Diffraction Grating 2.1.3 Optical Slits and Light Collimation The spectrometer's entrance slit, which controls light intake, was made by positioning tiny, circular holes close together. In this low-cost approach, a series of small, circular holes were used to create a functioning entrance slit for the spectrometer. This method offered several advantages. First, it was economical and utilized readily available materials. Second, by carefully adjusting the spacing between the holes, a thin and uniform slit was achieved. This is crucial because the slit width directly affects the resolution of the spectrometer. A wider slit allows more light in, resulting in a brighter spectrum, but at the expense of resolution. Different wavelengths begin to overlap, making it difficult to distinguish between them. Conversely, a narrower slit improves resolution by providing sharper separation between wavelengths.

25. 19 However, this also leads to a dimmer spectrum due to the reduced light intake. The final slit width was optimized by observing the diffracted light from a standard incandescent lamp. A clear set of emission lines from the lamp indicated a well-defined slit that balanced sufficient light intensity with good spectral resolution [38]. 2.1.4 VIS-NIR Detector To achieve a cost-effective design for the constructed VIS-NIR spectrometer, a commercially available P400-Vis-NIR Fiber cord was selected as the detector. The material used for detecting honey purity via cost-effective Vis-NIR spectroscopy, it has an important feature: it allows light in the critical ranges of 400-700 nm (visible) and 700-2100 nm (NIR) to pass through, vital for accurate analysis. By using low OH fibers, it reduces interference from hydroxyl groups, ensuring precise transmission for analysis and ideal for fieldwork. Additionally, it comes in various core diameters and lengths, with custom options available, ensuring it can meet specific experiment needs. Precision SMA 905 connectors make handling easy, securing stable connections, crucial for reliable measurements. Designed to withstand different environments, from labs to industrial settings, it ensures it can be used repeatedly without losing performance. [39]. Figure 11 USB650-VIS/NIR and P400-Vis-NIR Fiber cord

26. 20 2.1.5 OceanView: Software OceanView 2.0 spectroscopy software will be used in this method for it provides real- time control and processing of measurements made with Ocean Insight spectrometers and light sources. Advanced features like boxcar averaging and auto integration time allow users to receive the answers and insights they are looking for quickly. It allows the researcher to design its own measurement procedures using a “visual schematic” view that you drag-and-drop spectrometers, transform functions and display nodes to automate the unique post-processing workflow [40]-[41]. Figure 12 OceanView software for Spectral Analysis

27. 21 Sample Testing Mix the solution Pure Honey Distilled Water Di-Water Honey Put the sample into the Vis-NIR Spectroscopy Data Analysis 3.5ml sample in Cuvette Honey Adulteration Sample Preparation The pure honey sample was taken from San Isidro, Santiago, Agusan del Norte, Philippines. In this study, the researcher used two types of adulterants, brand A identified as ambrosia and brand B as bidlisiw, both of which are known to be adulterated honey. These were purchased from Puregold, Langihan, Butuan City, Philippines. The visible and near-infrared (Vis-NIR) absorbance spectroscopy measurement of the samples was conducted using the cost-effective spectrometer with a spectral range between 350 and 1000 nm [24]-[39]. The measurement of transmittance for the sample is determined in relation to an empty cuvette. This comparison serves as a baseline for assessing the extent to which light passes through the sample. The transmittance is calculated using an equation that takes account into the intensity of light transmitted through the sample compared to the intensity of light transmitted through the empty cuvette. This calculation helps quantify the percentage of light that is able to pass through the sample, providing valuable information about its optical properties. By referencing the transmittance relative to the empty cuvette,

28. 22 researchers can effectively evaluate the characteristics and composition of the sample under analysis [42]-[43]. Figure 13 Transmittance Where T is Transmittance, defined as a ratio of the intensity of incident light (I0) to the amount of intensity passes through the object (I). The equation quantifies the ratio of light intensity passing through the sample relative to the intensity passing through the empty cuvette. The resulting transmittance value is usually expressed as a percentage [42].

29. 23 CHAPTER 3 Design and Implementation of cost-effective Spectroscopy Abstract Optical spectroscopy serves as a versatile tool across various fields, facilitating the analysis of emitted or absorbed light to unveil the chemical and physical properties of substances [22]. However, the high cost of commercial spectrometers often limits their accessibility. This study explores the potential of low-cost DIY spectrometers in optical spectroscopy, focusing on methods to construct and calibrate these devices. By experimentally determining precise optical component distances, a functional setup is established [38]. Utilizing an incandescent lamp as a calibration source for a Vis-NIR spectrometer, spectral data spanning from 400 nm to 1000 nm is captured [36], offering insights into material optical properties across a wide spectral range. Notably, calibration processes align the spectrometer with known laser wavelengths, demonstrating accuracy. Subsequent analyses of the emission spectra of argon and hydrogen reveal significant peaks consistent with literature, affirming the detector's efficacy. Additionally, investigations into spectral data variations across different dyes indicate discrepancies from literature [44]-[45]-[44] values but highlight the consistent performance of the detector. Overall, this study showcases the feasibility and reliability of low- cost DIY spectrometers in various applications, advancing accessibility and understanding in optical spectroscopy. Introduction Optical spectroscopy is a powerful tool used across science, medicine, and engineering. It analyzes the light a substance emits or absorbs, revealing its chemical makeup and properties [33]. While commercial spectrometers offer exceptional resolution and reliability, their high cost often keeps them out of reach for educators and hobbyists. In essence, optical

30. 24 spectroscopy, even in its basic forms, offers a fascinating window into the chemical and physical nature of the world around us. A variety of low-cost DIY spectrometers designs exist in literature. Methods Initially, the procedure involves conducting an experiment to determine the precise distances between the optical components. Subsequently, the researcher establishes that the correct distance from the light source to the lens measures 10.6 cm, while the distance from the lens to the sample is found to be 6.8 cm. The lens utilized in this experiment is a biconvex lens with a focal length of 8.7 cm. 10.6 cm 6.8 cm Figure 14 Diagram of Vis-NIR Spectroscopy 3.2.1Design of the spectrometer was carried out with the SketchUp The design of the spectrometer was carried out with the SketchUp: online 3D design software. 3.2.2 Test the Effectiveness of the used detector Prepare the Light source Place the light source to the detector analyze the data

31. 25 3.2.3Detect the effectiveness of the sample through food dye put 0a specific amount of food dye to a 100ml of distilled water in a beaker and mix them together Prepare the samples put 3.5 ml solution to the cuvette apply the sample to the Spectrometer Analyze the Data Results and Discussion 3.3.1Vis-NIR Spectrometer Calibration Performance An incandescent lamp was used as a calibration source for a vis-NIR spectrometer. The method involves capturing the spectrum of the incandescent lamp.The incandescent lamp emits light across a broad range of wavelengths in the visible and near-infrared (vis-NIR) region. The results obtained from the VIS-NIR spectra, spanning from 350 nm to 1000nm [46], provide valuable insights into the optical properties of the material under investigation across a wide spectral range.

32. 26 This range encompasses both the visible and near-infrared regions, allowing for a comprehensive analysis of how the material interacts with light across different wavelengths. One interesting finding is the confirmation, supported by literature, that incandescent lamps emit light within this wavelength range [47] Figure 15 The Vis-NIR spectra, ranging from 350 nm to 1000nm 3.3.2Helium-Neon (He-Ne) laser The calibration process successfully aligned the spectrometer with the known wavelengths of the lasers. The measured transmittance of the Helium-Neon (He-Ne) laser was around 630 nm, which is consistent with the literature value of 632.8 nm [48]-[49]. The measured transmittance of the red laser was 650 nm, which is in line with the literature value of 650 nm [50]-[51]. Similarly, the measured transmittance of the green laser was 515 nm, which matches the literature value of 515 nm [45]. These close matches between the measured and literature values demonstrate the accuracy of the calibration process. In essence, the

33. 27 calibration ensures that the spectrometer provides accurate measurements of wavelengths within the vis-NIR range. Figure 16 Vis-NIR Spectra of Helium Neon laser, Red laser and Green laser

34. 28 3.3.3Argon The emission spectra of argon were obtained, revealing prominent peaks at wavelengths of 750 nm, 810 nm, 840 nm, and around 910 nm, with the lowest peaks observed around 400 nm and 700 nm. These findings align with existing literature on the optical emission spectrum of argon. Specifically, significant peaks in the spectrum correspond closely to established wavelengths for argon, including 696.54 nm, 706.72 nm, 750.38 nm, 763.51 nm, 800.61 nm, 810.36 nm, and 912.29 nm [52]. The consistency between the results and the literature supports the efficacy of the detector used in this study. Figure 17 Emission spectra of Argon

35. 29 3.3.4Hydrogen In the analysis of the hydrogen emission spectrum, significant peaks were identified at wavelengths of approximately 650 nm and 490 nm, with lesser peaks observed at around 415 nm and 590 nm. These results were compared with the documented optical emission spectrum of argon, which showed major peaks at 656.2 nm, 486.1 nm, 434.0 nm, and 410.1 nm [53]- [54]. Remarkably, the observed hydrogen peaks closely corresponded to those reported in the literature, indicating the reliability of our findings. The presence of most hydrogen peaks in the obtained spectrum suggests that the detector functioned effectively in capturing these emissions. This alignment between experimental results and theoretical expectations underscores the accuracy and validity of the study, contributing to the broader understanding of hydrogen emission properties. Figure 18 Emission spectra of Hydrogen

36. 30 3.3.5Helium In the analysis of the hydrogen emission spectrum involved identifying prominent peaks at specific wavelengths, such as approximately 590 nm, 680 nm, 700 nm, and 710 nm, with the lowest observed at 500 nm and 400 nm. This data was then compared with the documented optical emission spectrum of helium, which is known to exhibit distinct lines in the visible spectrum at wavelengths like 388.8 nm, 447.1 nm, 471.3 nm, and so on [49]. Remarkably, the helium peaks observed closely matched those reported for helium in the literature, indicating the reliability of our findings. This correspondence validates the accuracy of our experimental setup and methodology. Additionally, the consistent presence of most hydrogen peaks in our obtained spectrum suggests that the detector effectively captured these emissions. This alignment between our experimental results and the theoretical expectations based on the helium spectrum underscores the validity of our study's conclusions. Consequently, the research contributes to a deeper understanding of hydrogen emission properties, enhancing the broader scientific knowledge in this field [55] . Figure 19 Emssion spectra of Helium

37. 31 3.3.6Food dye In the results and discussion section of the thesis, the spectral analysis data of various dyes were meticulously compared with existing literature values. For the green dye, the observed peaks ranged from approximately 500 nm to 550 nm, whereas literature values indicated peaks at 520 nm and 565 nm [56] in UV-VIS-NIR results. This discrepancy suggests potential differences in dye composition or experimental conditions. Similarly, for the blue dye, observed peaks at 450 nm and 550 nm differed from the literature-reported peaks at 445 nm and 520 nm [44], which may be attributed to variations in dye concentration or purity. The orange dye exhibited peaks at 500 nm and 700 nm, contrasting with literature values of 565 nm and 590 nm, possibly due to different dye formulations or measurement techniques. Lastly, the red dye showed peaks at 600 nm to 700 nm, while literature indicated peaks at 625 nm to 740 nm [56]. These differences highlight the potential influences of experimental setup, dye batch variations, and environmental factors on spectral data [57]. Despite these variations, the consistent identification of spectral peaks within close ranges to those documented in the literature underscores the reliability and accuracy of the constructed Vis-NIR spectrometer used in this study. Figure 20 Obtained wavelength peaks of food dyes using an incandescent light source.

38. 32 Conclusion The experiment successfully calibrated a vis-NIR spectrometer using an incandescent lamp and validated its accuracy. The measured transmittance of lasers and the emission spectra of argon and hydrogen closely matched established values, demonstrating the effectiveness of the calibration and detection methods. Spectral analysis of various dyes revealed peaks within a range of those reported in literature, suggesting the spectrometer's potential for material characterization. However, observed discrepancies between measured and literature values for dyes highlight the influence of factors like dye composition and experimental conditions. Overall, the study demonstrates the construction of a reliable and accurate vis-NIR spectrometer, contributing to the understanding of material properties and potential applications in various fields.

39. 33 CHAPTER 4 Detecting Adulteration in Honey: Utilizing Spectral Data from a Low-Cost Vis-NIR Spectrometer Abstract The analysis conducted using a Vis-NIR spectrometer on pure honey and two branded honey samples (Brand A and Brand B) revealed distinctive findings. Pure honey displayed specific wavelength peaks across various ranges, while Brand A and Brand B showed differing peak patterns. A comparison with literature values for pure honey indicated both similarities and differences in the observed peak at a wavelength 787.677 nm ,736.0 nm 850nm, 950nm and 989nm. While pure honey matched some literature values closely at 730 nm to 780 nm, 800 nm to 890 nm, and around 940 nm, the branded samples displayed variations, suggesting potential compositional differences possibly due to additives, processing methods, or floral sources. The absence of certain expected peaks in the branded samples raises concerns about adulteration. This analysis effectively distinguishes pure honey from branded products based on their spectral characteristics. Further investigation and detailed analysis are necessary to fully understand these differences and confirm the presence of any adulterants, highlighting the importance of ensuring honey authenticity and purity. 4.1 Introduction Honey purity is a complex issue with significant implications for human health, economic fairness, and regulatory enforcement [58]. Consumers value honey for its perceived health benefits and are willing to pay a premium for pure, high-quality products. However, adulterated honey can compromise these benefits and pose health risks [5]. Governments

40. 34 regulate honey purity to ensure food safety and prevent deceptive practices. Traditional methods for testing honey purity have limitations, prompting the need for innovative techniques [12]. This research explores the potential of Vis-NIR spectroscopy, a rapid and non- destructive method, for reliable honey analysis in the food industry. 4.2 Methods Honey adulteration is a pressing concern, and effective methods for detection are crucial. This study explores the potential of a low-cost Vis-NIR spectrometer for identifying adulteration in honey samples. The researchers used a simple method to prepare the honey samples. First, the honey samples was prepared. Then, a measured amount of honey was carefully transferred to beakers. Next, 3.5 milliliters of honey were dispensed into cuvettes, which were then placed in the Vis-NIR spectrometer. Finally, the spectral data from the samples was collected and analyzed to identify any signs of adulteration.. Prepare the samples whch is the pure and the two adulterated honey Fill the beaker with the specified amount of honey samples. put 3.5 ml solution to the cuvette apply the sample to the Spectrometer Analyze the Data

41. 35 4.3 Results and Discussion The Vis-NIR spectrometer analysis of the pure honey and the two branded honey samples (Brand A and Brand B) yielded distinct results. Pure honey exhibited wavelength peaks at 730 nm to 780 nm, 800 nm to 890 nm, and around 940 nm. Brand A showed peaks at approximately 810 nm and 900 nm, while Brand B displayed peaks from 800 nm to 900 nm. When compared to literature values for pure honey, which report peaks at 736 nm, 787.677 nm, 850 nm, 972 nm, and 989 nm, there were some matches and some differences. The pure honey sample’s peaks partially matched the literature values, specifically at 736 nm, 787.677 nm, and 850 nm. However, the peak around 940 nm did not match the higher wavelength peaks of 972 nm and 989 nm, though it was close. The peaks in Brand A (810 nm and 900 nm) did not exactly match any specific literature values but were in the general range for pure honey. This suggests that Brand A might have different components or processing that change its spectral characteristics. Brand B’s peaks from 800 nm to 900 nm included the 850 nm peak from the literature but lacked the distinct higher wavelength peaks of 972 nm and 989 nm, indicating possible additives or processing differences. The closer match of pure honey’s peaks with literature values confirms its authenticity and purity. In contrast, the differences in Brands A and B suggest possible compositional changes due to additives, processing methods, or different floral sources. The lack of higher wavelength peaks in the branded samples suggests possible dilution or substitution. The Vis-NIR spectrometer analysis effectively differentiates pure honey from branded products based on their spectral peaks. Pure honey’s broader range of peaks closely matches the literature values, indicating authenticity. In contrast, Brands A and B show fewer peaks, suggesting differences that may affect quality and purity. Further investigation and more detailed spectral analysis are needed to pinpoint the exact nature of these differences and confirm any adulterants.

42. 36 Figure 21 Obtained wavelength peaks of pure and adulterated honey using the Vs-NIR spectroscopy

43. 37 4.3 Limitation and recommendation The study highlighted several scope and limitations regarding the use of incandescent lamps in Vis-NIR spectroscopy. The detection range of the incandescent lamp used in this study spans from 350 nm to 1000 nm, covering both the visible and near-infrared regions. However, the intensity of light emitted by the incandescent lamp can fluctuate due to variations in the power supply or aging of the filament, leading to inconsistent spectral data and affecting the reliability and accuracy of measurements. Additionally, incandescent bulbs generally have a shorter operational lifespan compared to other light sources such as LEDs or lasers. This limitation necessitates frequent replacements, increasing maintenance costs and potentially causing downtime that interrupts the continuity of the study. To improve the accuracy, reliability, and cost-effectiveness of future studies, the following recommendations are made: consider using LEDs or laser light sources, which provide more stable light intensity and have a longer operational lifespan, thereby reducing maintenance costs and minimizing downtime. Implement more robust calibration procedures and use a stable power supply to mitigate intensity fluctuations and enhance the consistency of spectral data. Finally, to broaden the scope of the study, employ light sources that cover a wider or more specific range of wavelengths beyond 350 nm to 1000 nm, which may provide more comprehensive data, especially for samples with critical spectral features outside this range. By addressing these limitations and incorporating the recommended changes, future studies can achieve more precise and consistent results in Vis-NIR spectroscopy analysis 4.5 Conclusion The Vis-NIR spectrometer analysis provided valuable insights into the composition of the tested honey samples, revealing distinct spectral characteristics for pure honey compared to branded products. Pure honey exhibited wavelength peaks consistent with literature values, indicating its authenticity and purity. However, Brands A and B displayed differing spectral

44. 38 profiles, suggesting potential differences in composition due to additives, processing methods, or floral sources which is lesser transmittance of light. The lower intensities of the peaks in the branded samples shows that the processes involve contributed to lesser ability of honey to transmit light, even at orange and red regions. This reduction of transmitted light suggest that the composition of honey has been adulterated. Further investigation, including more detailed spectral analysis, is necessary to clear the exact nature of these differences and confirm any adulterants. Overall, the study underscores the efficacy of Vis-NIR spectroscopy in distinguishing pure honey from branded products based on their spectral signatures.

45. 39 CHAPTER 5 CONCLUSION This study constructed and validated a Vis-NIR spectrometer using an incandescent lamp. The instrument demonstrated accuracy by precisely measuring laser transmittance and matching known gas emission spectra. Spectral analysis of dyes aligned generally with literature values, suggesting the spectrometer's potential for material characterization. However, discrepancies observed highlight the influence of external factors and limitations of the incandescent lamp itself. The incandescent lamp's restricted detectable wavelength range and potential for fluctuating light intensity pose limitations. Further investigation with more advanced light sources and detailed spectral analysis are necessary for definitive identification of any adulteration in the branded honey samples. Despite these limitations, the study underscores the promise of Vis-NIR spectroscopy for material analysis, particularly in honey quality control. Continued development of the technique, including employing more sophisticated light sources, will enhance its reliability and broaden its applications.

46. 40 REFERENCE [1] M. E. Raypah, A. F. Omar, J. Muncan, M. Zulkurnain, and A. R. Abdul Najib, “Identification of Stingless Bee Honey Adulteration Using Visible-Near Infrared Spectroscopy Combined with Aquaphotomics,” Molecules, vol. 27, no. 7, Art. no. 7, Jan. 2022, doi: 10.3390/molecules27072324. [2] R. Fakhlaei et al., “The Toxic Impact of Honey Adulteration: A Review,” Foods, vol. 9, no. 11, p. 1538, Oct. 2020, doi: 10.3390/foods9111538. [3] D. Valinger et al., “Detection of honey adulteration – The potential of UV-VIS and NIR spectroscopy coupled with multivariate analysis,” LWT, vol. 145, p. 111316, Jun. 2021, doi: 10.1016/j.lwt.2021.111316. [4] D. S. Brar et al., “A comprehensive review on unethical honey: Validation by emerging techniques,” Food Control, vol. 10.1016/j.foodcont.2022.109482. [5] S. Samarghandian, T. Farkhondeh, and F. Samini, “Honey and Health: A Review of Recent Clinical Research,” Pharmacogn. Res., vol. 9, no. 2, pp. 121–127, 2017, doi: 10.4103/0974- 8490.204647. [6] I. A. Rather, W. Y. Koh, W. K. Paek, and J. Lim, “The Sources of Chemical Contaminants in Food and Their Health Implications,” Front. Pharmacol., vol. 8, p. 830, Nov. 2017, doi: 10.3389/fphar.2017.00830. [7] J. Vapa-Tankosić, S. Ignjatijević, J. Kiurski, J. Milenković, and I. Milojević, “Analysis of Consumers’Willingness to Pay for Organic and Local Honey in Serbia,” Sustainability, vol. 12, no. 11, Art. no. 11, Jan. 2020, doi: 10.3390/su12114686. [8] “Sucrose | Definition, Characteristics, & Natural Sources | Britannica.” Accessed: Apr. 26, 2024. [Online]. Available: https://www.britannica.com/topic/honey [9] B. R. Albuquerque, S. A. Heleno, M. B. P. P. Oliveira, L. Barros, and I. C. F. R. Ferreira, “Phenolic compounds: current industrial applications, limitations and future challenges,” Food Funct., vol. 12, no. 1, pp. 14–29, Jan. 2021, doi: 10.1039/D0FO02324H. [10] J. Barbecho, “Diversity of bees and wild pollinators in the Philippines, Thailand and Viet Nam,” the Philippines. [11] “More money for their honey,” PIA. Accessed: Apr. 30, 2024. [Online]. Available: https://pia.gov.ph/features/2024/01/19/more-money-for-their-honey [12] “The Economic Evolution of Honey: From Ancient Trade to Modern Markets | Manukora.” Accessed: May https://manukora.com/blogs/honey-guide/honey-economic-journey [13] DOST, “Sweet but not pure: DOST study shows 80% of honey sold in markets contain sugar syrup.” Accessed: May https://www.dost.gov.ph/knowledge-resources/news/67-2020-news/2072-sweet-but- not-pure-dost-study-shows-80-of-honey-sold-in-markets-contain-sugar-syrup.html [14] “DOST-PNRI Honey Team gets Pagasa Award for NCR.” Accessed: May 06, 2024. [Online]. Available: https://pnri.dost.gov.ph/index.php/2-uncategorised/779-dost-pnri- honey-team-gets-pagasa-award-for-ncr [15] U. M. Shapla, Md. Solayman, N. Alam, Md. I. Khalil, and S. H. Gan, “5- Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on bees and human health,” Chem. Cent. J., vol. 12, no. 1, p. 35, Apr. 2018, doi: 10.1186/s13065- 018-0408-3. 145, p. 109482, Mar. 2023, doi: 06, 2024. [Online]. Available: 06, 2024. [Online]. Available:

47. 41 [16] “Honey authenticity: analytical techniques, state of the art and challenges - RSC Advances (RSC Publishing) DOI:10.1039/D1RA00069A.” Accessed: May 04, 2024. [Online]. Available: https://pubs.rsc.org/en/content/articlehtml/2021/ra/d1ra00069a [17] G. Zhang et al., “An innovative molecular approach towards the cost-effective entomological authentication of honey,” Npj Sci. Food, vol. 8, no. 1, p. 24, May 2024, doi: 10.1038/s41538-024-00268-9. [18] “Foods | Free Full-Text | The Toxic Impact of Honey Adulteration: A Review.” Accessed: May 04, 2024. [Online]. 8158/9/11/1538 [19] “Foods | Free Full-Text | Non-Targeted Detection and Quantification of Food Adulteration of High-Quality Stingless Bee Honey (SBH) via a Portable LED-Based Fluorescence Spectroscopy.” Accessed: May 04, 2024. [Online]. Available: https://www.mdpi.com/2304-8158/12/16/3067 [20] Y. C. Hong, “ADULTERATION DETECTION OF STINGLESS BEE HONEY USING UNTARGETED 1H-NMR METABOLOMICS IN ADDRESSING THE LIMITATION OF STABLE CARBON ISOTOPE RATIO ANALYSIS”. [21] S. A. D. M. Zahir, A. F. Omar, M. F. Jamlos, M. A. M. Azmi, and J. Muncan, “A review of visible and near-infrared (Vis-NIR) spectroscopy application in plant stress detection,” Sens. Actuators Phys., vol. 338, p. 113468, May 2022, doi: 10.1016/j.sna.2022.113468. [22] “2.2: Visible and Near Infrared Optical Spectroscopic Sensors for Biosystems Engineering,” Engineering LibreTexts. Accessed: May 03, 2024. [Online]. Available: https://eng.libretexts.org/Bookshelves/Biological_Engineering/Introduction_to_Biosyst ems_Engineering_(Holden_et_al.)/02%3A_Information_Technology_Sensors_and_Contr ol_Systems/2.02%3A_Visible_and_Near_Infrared_Optical_Spectroscopic_Sensors_for_B iosystems_Engineering [23] “10.1: Overview of Spectroscopy,” Chemistry LibreTexts. Accessed: May 06, 2024. [Online]. https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Analytical_Chemistry_2. 1_(Harvey)/10%3A_Spectroscopic_Methods/10.01%3A_Overview_of_Spectroscopy [24] M. E. Raypah, A. F. Omar, J. Muncan, M. Zulkurnain, and A. R. Abdul Najib, “Identification of Stingless Bee Honey Adulteration Using Visible-Near Infrared Spectroscopy Combined with Aquaphotomics,” Molecules, vol. 27, no. 7, Art. no. 7, Jan. 2022, doi: 10.3390/molecules27072324. [25] “Electromagnetic spectrum | Definition, Diagram, & Uses | Britannica.” Accessed: May 06, 2024. [Online]. Available: https://www.britannica.com/science/electromagnetic- spectrum [26] “Visible Light - NASA Science.” Accessed: May 03, 2024. [Online]. Available: https://science.nasa.gov/ems/09_visiblelight/ [27] veselin, “Near-Infrared (NIR) Spectroscopy - Detailed Explanation,” tec5USA. Accessed: May 03, 2024. [Online]. Available: https://www.tec5usa.com/nir- spectroscopy/ [28] “Infrared Spectroscopy,” Chemistry LibreTexts. Accessed: May 03, 2024. [Online]. Available: https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textboo k_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/ Vibrational_Spectroscopy/Infrared_Spectroscopy/Infrared_Spectroscopy [29] “Near-Infrared Spectroscopy Analysis: FAQs (Part 1),” AZoM. Accessed: May 04, 2024. [Online]. Available: https://www.azom.com/article.aspx?ArticleID=19837 Available: https://www.mdpi.com/2304- Available:

48. 42 [30] “The Beer-Lambert Law,” Chemistry LibreTexts. Accessed: May 06, 2024. [Online]. Available: https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textboo k_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/ Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law [31] D. Dimakopoulou-Papazoglou et al., “Application of UV–Vis spectroscopy for the detection of adulteration in Mediterranean honeys,” Eur. Food Res. Technol., pp. 3043– 3053, Dec. 2023, doi: 10.1007/s00217-023-04347-1. [32] N. Sotiropoulou et al., “The Use of SPME-GC-MS IR and Raman Techniques for Botanical and Geographical Authentication and Detection of Adulteration of Honey,” Foods, vol. 10, p. 1671, Jul. 2021, doi: 10.3390/foods10071671. [33] Technovation, “DIY Low Cost Spectrometer,” Instructables. Accessed: Apr. 30, 2024. [Online]. Available: https://www.instructables.com/DIY-Low-Cost-Spectrometer/ [34] T. Agarwal, “Incandescent Lamp : Construction, Working, Types & Its Applications,” ElProCus - Electronic Projects for Engineering Students. Accessed: May 04, 2024. [Online]. Available: https://www.elprocus.com/incandescent-lamp/ [35] Masuzi, “Incandescent Light Bulb Visible Spectrum | Americanwarmoms.org.” Accessed: May 04, https://americanwarmoms.org/incandescent-light-bulb-visible-spectrum/ [36] “incandescent_spectrum_graph.jpg (657×361).” Accessed: May 04, 2024. [Online]. Available: https://i1.wp.com/www.hollandfranklin.com/wp- content/uploads/2016/11/incandescent_spectrum_graph.jpg?fit=657%2C361 [37] “4.5: Diffraction Gratings - Physics LibreTexts.” Accessed: Apr. 30, 2024. [Online]. Available: https://phys.libretexts.org/Bookshelves/University_Physics/University_Physics_(OpenSt ax)/University_Physics_III_- _Optics_and_Modern_Physics_(OpenStax)/04%3A_Diffraction/4.05%3A_Diffraction_Gr atings [38] M. Furia, “Design and Construction of a Low- cost DIY Spectroscope”. [39] “P1000-2-VIS-NIR.” Accessed: Apr. https://www.oceaninsight.com/products/fibers-and-probes/fibers/patch-cords/p1000- 2-vis-nir/ [40] “OceanView | Spectroscopy Software | Ocean Insight.” Accessed: May 06, 2024. [Online]. Available: https://www.oceaninsight.com/products/software/acquisition-and- analysis/oceanview/ [41] “Ocean View: Software - Yahoo Search Results.” Accessed: May 06, 2024. [Online]. Available: https://ph.search.yahoo.com/search?fr=mcafee&type=E210PH739G0&p=Ocean+View% 3A+Software [42] “13.1: Transmittance and Absorbance,” Chemistry LibreTexts. Accessed: May 06, 2024. [Online]. https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Instrumental_Analysis_( LibreTexts)/13%3A_Introduction_to_Ultraviolet_Visible_Absorption_Spectrometry/13.0 1%3A_Transmittance_and_Absorbance [43] “Transmittance (Formula & Transmittance to Absorbance Calculation) | Electrical4U,” https://www.electrical4u.com/. Accessed: May 06, 2024. [Online]. Available: https://www.electrical4u.com/what-is-transmittance/ 2024. [Online]. Available: 30, 2024. [Online]. Available: Available:

49. 43 [44] “uv-vis-nir blue dye - Yahoo Image Search Results.” Accessed: May 10, 2024. [Online]. Available: https://ph.images.search.yahoo.com/search/images;_ylt=AwrKGE_7jT1mTycwyA61Rwx. ;_ylu=c2VjA3NlYXJjaARzbGsDYnV0dG9u;_ylc=X1MDMjExNDczNDAwNQRfcgMyBGZyA21j YWZlZQRmcjIDcDpzLHY6aSxtOnNiLXRvcARncHJpZANHdmJFdWx3WlMuS0tRdHN0QXJYc WlBBG5fcnNsdAMwBG5fc3VnZwMwBG9yaWdpbgNwaC5pbWFnZXMuc2VhcmNoLnlha G9vLmNvbQRwb3MDMARwcXN0cgMEcHFzdHJsAzAEcXN0cmwDMTkEcXVlcnkDdXYtdml zLW5pciUyMGJsdWUlMjBkeWUEdF9zdG1wAzE3MTUzMTAxMzU-?p=uv-vis- nir+blue+dye&fr=mcafee&fr2=p%3As%2Cv%3Ai%2Cm%3Asb-top&ei=UTF- 8&x=wrt&type=E210PH739G0#id=7&iurl=https%3A%2F%2Fwww.researchgate.net%2Fp ublication%2F326223638%2Ffigure%2Ffig4%2FAS%3A645247348510720%40153085036 4596%2FUV-visible-absorption-spectrum-for-degradation-of-dyes-dye-with-NaBH-4-C- ciliaris.png&action=click [45] “Output spectrum of the green laser emission. | Download Scientific Diagram.” Accessed: May 09, https://www.researchgate.net/figure/Output-spectrum-of-the-green-laser- emission_fig5_253835766 [46] “A Layman’s Guide to Photonic Crystals – Awesci –Science Everyday.” Accessed: May 09, 2024. [Online]. Available: https://awesci.com/light-recycling-return-of-the- incandescent-bulb/ [47] dlim, “What Are the Best Light Sources For Photosynthesis?,” Vernier. Accessed: May 09, 2024. [Online]. Available: https://www.vernier.com/blog/what-are-the-best-light- sources-for-photosynthesis/ [48] “5.6: The Helium-Neon Laser,” Chemistry LibreTexts. Accessed: May 09, 2024. [Online]. https://chem.libretexts.org/Courses/Knox_College/Chem_322%3A_Physical_Chemisty_I I/05%3A_Lasers_Laser_Spectroscopy_and_Photochemistry/5.06%3A_The_Helium- Neon_Laser [49] “Helium Neon laser construction and working | 9 applications.” Accessed: May 09, 2024. [Online]. Available: https://physicswave.com/helium-neon-laser-construction-and- working/ [50] L. Tian et al., “Investigation of the Efficacy and Safety of 650 nm Low-Level Red Light for Myopia Control in Children: A Randomized Controlled Trial,” Ophthalmol. Ther., vol. 11, no. 6, pp. 2259–2270, Dec. 2022, doi: 10.1007/s40123-022-00585-w. [51] “[SOLVED] Spectrum of laser light absorbed and re-emitted by a white object ~ Physics ~ TransWikia.com.” Accessed: May https://transwikia.com/physics/spectrum-of-laser-light-absorbed-and-re-emitted-by-a- white-object/ [52] “Fig. 3. Optical emission spectrum of argon and air plasma in liquid....,” ResearchGate. Accessed: May 09, https://www.researchgate.net/figure/Optical-emission-spectrum-of-argon-and-air- plasma-in-liquid-The-inset-is-a-zoom-of-the_fig2_309736879 [53] “Emission Spectrum of Hydrogen.” Accessed: May 09, 2024. [Online]. Available: https://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/bohr.html [54] “Spectrum of Atomic Hydrogen,” Vernier. Accessed: May 09, 2024. [Online]. Available: https://www.vernier.com/experiment/phys-abm-21_spectrum-of-atomic-hydrogen/ [55] “The Emission Spectra of Various Atoms.” Accessed: May 09, 2024. [Online]. Available: https://www.astronomy.ohio-state.edu/pogge.1/TeachRes/HandSpec/atoms.html 2024. [Online]. Available: Available: 09, 2024. [Online]. Available: 2024. [Online]. Available:

50. 44 [56] “Dye Colors & Spectra | Bangs Laboratories, Inc.” Accessed: May 14, 2024. [Online]. Available: https://www.bangslabs.com/support/technical-support/spectra-information [57] “Scheme of the experimental setup for batch experiments | Download Scientific Diagram.” Accessed: May https://www.researchgate.net/figure/Scheme-of-the-experimental-setup-for-batch- experiments_fig1_337955190 [58] A. Gela, Z. A. Hora, D. Kebebe, and A. Gebresilassie, “Physico-chemical characteristics of honey produced by stingless bees (Meliponula beccarii) from West Showa zone of Oromia Region, Ethiopia,” Heliyon, vol. 7, no. 1, p. e05875, Jan. 2021, doi: 10.1016/j.heliyon.2020.e05875. 14, 2024. [Online]. Available: