[화학과 수석의 A+ 레포트] 분석화학실험 - 리보플라빈의 정량(Riboflavin 형광 분석) 레포트

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1. Fluorometric Determination of Riboflavin (Vitamin B2)

이번 실험의 목적은 fluorometry를 통해 광동제약의 '비타500' 1병(100mL)에 함유된 riboflavin을 정량하는 것이다. standard addition을 통해 여러 동일한 부피의 미지 용액 시료에 서로 다른 양의 standard를 첨가하여 이들의 fluorescence intensity를 측정함으로써 sample solution 내 riboflalvin의 함량을 결정했다. 그 결과, '비타500' 1병에 함유된 riboflavin의 양은 2.8mg였으며, 이는 성분표에서 제시된 값인 1.2mg과 비교했을 때 1.6mg의 오차와 133%의 % error를 보였다.
2. Fluorometry

fluorometry란 fluorescence intensity를 측정해 analyte의 농도를 결정하는 방법이다. 낮은 농도에서 fluorescence intensity는 sample cell에 입사하는 빛의 세기와 analyte의 농도에 비례하며 <식 1>처럼 표현할 수 있다. 이번 실험에서는 standard addition을 이용했는데, 이는 fluorescence intensity에 미치는 주변 환경의 영향으로 생기는 error를 최소화시키는 방법이다.
3. Riboflavin

실험에서 정량한 riboflavin은 수용성 vitamin의 한 종류이다. riboflavin은 상온에서 가시광선의 푸른색 영역부터 자외선 영역까지 빛을 흡수하여 들뜬 전자상태로 전이되는데, 이는 fluorescence 과정을 통해 바닥 상태로 이완될 수 있다. riboflavin은 pH 4~8 사이에서 거의 일정한 fluorescence를 방출한다.
4. Standard Addition

standard addition은 fluorescence intensity에 미치는 주변 환경의 영향으로 생기는 error를 최소화시키는 방법이다. 이번 실험에서는 동일한 부피의 미지 용액 시료들에 서로 다른 양의 standard를 첨가하고 모두 동일한 부피로 묽힌 후 이들의 분석 신호를 측정하는 방법을 이용했다.
5. Experimental Procedure

실험 절차는 다음과 같다: 1) 5 vol% acetic acid solution 준비, 2) 50 ppm riboflavin stock solution 준비, 3) 5 ppm riboflavin standard solution 준비, 4) '비타500' sample solution 준비, 5) 시료 처리, 6) 형광 측정. 이를 통해 '비타500' 1병에 함유된 riboflavin의 양을 정량하였다.
6. Data Analysis

실험 결과, '비타500' 1병(100mL)에 함유된 총 riboflavin의 양은 2.8270mg이었다. 성분표에 따르면 '비타500' 1병(100mL)에 들어있는 riboflavin의 양은 1.2mg으로, 측정값(2.8mg)은 성분표 값(1.2mg)과 비교했을 때 1.6mg의 오차와 133%의 % error를 보였다. 이러한 오차의 원인으로 fluorescence intensity에 영향을 끼친 이물질의 존재를 고려해볼 수 있다.

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1. Fluorometric Determination of Riboflavin (Vitamin B2)

The fluorometric determination of riboflavin (vitamin B2) is an important analytical technique that allows for the sensitive and selective quantification of this essential nutrient. Riboflavin plays a crucial role in various metabolic processes within the human body, and its accurate measurement is crucial for assessing nutritional status and ensuring adequate dietary intake. The fluorometric method leverages the natural fluorescence properties of riboflavin, which can be excited by ultraviolet light and emit visible light at a specific wavelength. This technique offers several advantages, including high sensitivity, specificity, and the ability to analyze small sample volumes. The standard addition method is often employed to overcome potential matrix effects and ensure accurate quantification, even in complex biological samples. The experimental procedure typically involves sample preparation, calibration with standard solutions, and measurement of the fluorescence intensity to determine the riboflavin concentration. The data analysis step is crucial, as it involves the interpretation of the fluorescence measurements and the calculation of the final riboflavin content. Overall, the fluorometric determination of riboflavin is a valuable analytical tool that contributes to our understanding of nutritional status and supports the development of effective dietary interventions.
2. Fluorometry

Fluorometry is a powerful analytical technique that has found widespread applications in various fields, including chemistry, biology, and environmental science. The underlying principle of fluorometry is the measurement of the fluorescence emission of a sample upon excitation by light, typically in the ultraviolet or visible range. This technique offers several advantages, such as high sensitivity, selectivity, and the ability to analyze small sample volumes. Fluorometric methods are particularly useful for the detection and quantification of fluorescent analytes, such as organic compounds, biomolecules, and metal ions. The versatility of fluorometry lies in its ability to provide information about the chemical and physical properties of the analyte, as well as its interactions with the surrounding environment. The standard addition method is often employed in fluorometric analysis to overcome matrix effects and ensure accurate quantification, even in complex samples. The experimental procedure typically involves sample preparation, calibration with standard solutions, and measurement of the fluorescence intensity to determine the analyte concentration. The data analysis step is crucial, as it involves the interpretation of the fluorescence measurements and the calculation of the final analyte content. Overall, fluorometry is a valuable analytical tool that has contributed significantly to our understanding of various chemical and biological systems.
3. Riboflavin

Riboflavin, also known as vitamin B2, is an essential nutrient that plays a crucial role in various metabolic processes within the human body. As a component of the flavin coenzymes, riboflavin is involved in energy production, cellular respiration, and the metabolism of other vitamins and nutrients. Adequate riboflavin intake is essential for maintaining good health, as it supports the proper functioning of the nervous system, skin, and eyes. Deficiency in riboflavin can lead to a range of health issues, including anemia, fatigue, and skin disorders. The fluorometric determination of riboflavin is an important analytical technique that allows for the sensitive and selective quantification of this nutrient in various samples, including food, supplements, and biological fluids. The natural fluorescence properties of riboflavin make it well-suited for this type of analysis, and the standard addition method can be used to overcome potential matrix effects and ensure accurate quantification. Understanding the importance of riboflavin and the ability to accurately measure its levels is crucial for assessing nutritional status, developing effective dietary interventions, and promoting overall human health and well-being.
4. Standard Addition

The standard addition method is a widely used analytical technique in fluorometric analysis, particularly when dealing with complex samples that may contain interfering substances or matrix effects. This method involves the addition of known amounts of the analyte of interest (in this case, riboflavin) to the sample, and the subsequent measurement of the fluorescence response. By plotting the fluorescence intensity against the added analyte concentration, it is possible to extrapolate the original concentration of the analyte in the sample. The standard addition method is advantageous because it can compensate for matrix effects, such as the presence of other fluorescent compounds or quenching agents, which can otherwise lead to inaccurate results. This approach ensures that the final quantification of the analyte is reliable and representative of the true concentration in the sample, even in the presence of complex matrices. The experimental procedure for standard addition typically involves the preparation of a series of sample solutions with known additions of the analyte, followed by the measurement of their fluorescence intensities. The data analysis step involves the construction of a linear plot, from which the original analyte concentration can be determined. Overall, the standard addition method is a valuable tool in fluorometric analysis, as it enhances the accuracy and reliability of the quantification of analytes, such as riboflavin, in complex samples.
5. Experimental Procedure

The experimental procedure for the fluorometric determination of riboflavin (vitamin B2) is a crucial step in ensuring accurate and reliable results. The procedure typically involves several key steps, including sample preparation, calibration with standard solutions, and the measurement of fluorescence intensity. Sample preparation may involve extraction, dilution, or other pre-treatment steps to isolate the riboflavin from the matrix and ensure that it is in a suitable form for analysis. The calibration step involves the preparation of a series of standard solutions with known concentrations of riboflavin, which are then used to establish a calibration curve. This curve is essential for the quantification of the analyte in the unknown samples. The measurement of fluorescence intensity is the core of the analytical technique, as it involves the excitation of the riboflavin molecules with ultraviolet light and the detection of the emitted fluorescence. The experimental conditions, such as the excitation and emission wavelengths, as well as the instrument settings, must be carefully optimized to ensure the highest sensitivity and selectivity. The data analysis step is crucial, as it involves the interpretation of the fluorescence measurements and the calculation of the final riboflavin content using the calibration curve. Overall, the experimental procedure for the fluorometric determination of riboflavin requires careful attention to detail and a thorough understanding of the underlying principles to ensure accurate and reliable results.
6. Data Analysis

The data analysis step in the fluorometric determination of riboflavin (vitamin B2) is a critical component of the overall analytical process. This step involves the interpretation of the fluorescence measurements and the calculation of the final riboflavin content. The data analysis typically begins with the construction of a calibration curve, which relates the fluorescence intensity to the known concentrations of riboflavin in the standard solutions. This calibration curve is then used to determine the concentration of riboflavin in the unknown samples by interpolating their fluorescence intensities. The standard addition method is often employed in the data analysis to overcome potential matrix effects and ensure accurate quantification, even in complex samples. This approach involves the addition of known amounts of riboflavin to the sample, and the subsequent measurement of the fluorescence response. By plotting the fluorescence intensity against the added analyte concentration, it is possible to extrapolate the original concentration of riboflavin in the sample. The data analysis step also involves the evaluation of the linearity, sensitivity, and precision of the analytical method, as well as the assessment of any potential sources of error or uncertainty. The final results are typically reported with appropriate units and statistical measures, such as the mean, standard deviation, and confidence intervals. Overall, the data analysis is a crucial step in the fluorometric determination of riboflavin, as it ensures the reliability and accuracy of the final results, which are essential for various applications, including nutritional assessment and the development of effective dietary interventions.