CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
APPLICATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND
ATOMIC ABSORPTION SPECTROPHOTOMETER IN FOOD CHEMISTRY
LABORATORY
A graduate project submitted in partial fulfillment of the requirements for the degree of Master of Science in Family and Consumer Sciences
by
Diem Nguyen
August 2015
The graduate project of Diem Nguyen is approved:
Elizabeth Sussman, Ph.D., RD Date
Julie Ellis, M.S. Date
Terri Lisagor, Ed.D., Chair Date
California State University, Northridge
ii ACKNOWLEDGMENT
I would like to thank my committee members who supported my efforts in completing and writing this graduate project.
To my chair, Dr. Terri Lisagor, who has given assistance and direction on writing this project. Thank you for your patience, invaluable feedback and unconditional support on this project.
To Professor Julie Ellis, who has always supported me on getting materials needed to accomplish this project, thank you for taking time to get to read and evaluate this project.
To Dr. Elizabeth Sussman, who has given ideas to help develop experiments, thank you for taking the time to read and evaluate this project.
To Daria Baciu, project partner, thank you for helping with HPLC trouble shooting and fixing the AAS.
To Dr. Claudia Fajardo-Lira, previous committee chair, thank you for giving me a chance to work on this project and constantly helping me to get the materials that were needed to develop these experiments.
And thank you to Dr. Simon Garrett, analytical chemistry instructor at California
State University, Northridge, who allowed me to sit in his lectures, as well as visit his laboratory to experience the methods of HPLC and AAS. Dr. Garrett provided detailed instructions on chemicals and mechanical of running these analytical instruments, also took his precious time to discuss the possible solutions to solve the instruments error.
Thank you for your patience and valuable guidance throughout this project.
iii TABLE OF CONTENTS
Signature Page ii Acknowledgment iii List of Tables v Abstract vi
CHAPTER I – INTRODUCTION 1 Statement of the Problem 2 Purpose 2 Definitions 2 Assumptions 3 Limitations 3
CHAPTER II – REVIEW OF LITERATURE 5 The Grow of Food Science in the Food Industry 5 Application of Food Science in the Classroom 8 Application of Analytical Chemistry in Food Industry 12 Caffeine, Iron and Calcium in Food Products 16 Conclusion 21
CHAPTER III – METHODOLOGY 23
CHAPTER IV – RESULTS 26 Determination of Caffeine in Energy Drink by HPLC 27 Iron Determination in Fresh Spinach and Breakfast Cereal using GF-AAS 28 Detection of Calcium in Kale and Vitamin Tablet 29
CHAPTER V – DISCUSSION 31 Summary of Findings 31 Discussion of Findings and Modifications 31 Discussion of the Expert Evaluation 36 Discussion of the Target Population Evaluation 36 Implications 36 Conclusion 37
REFERENCES 38
APPENDIX A- The Three Developed Experiments 44 B- Experimental Data Analysis and Calculation 71 C- Analytical Math Support 80 D- Instruction on Running Experiments & Supplemental material for GF 83 E- MSDS sheets 101
iv LIST OF TABLES
Table 1 - GF-AAS Instrumental Setup Parameters 27
Table 2 - HPLC Instrumental Setup Parameters 27
Table 3 - Caffeine Standard Solutions, Unknown and Calibration Curve Equation 28
Table 4 - Fe Standard Addition Solutions, Unknown and Calibration Curve Equations 29
Table 5 – Amount of Fe in Samples 29
Table 6 - Ca Standard Addition Solutions, Unknown and Calibration Curve Equations 29
Table 7 – Amount of Ca in Samples 30
v ABSTRACT
APPLICATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND
ATOMIC ABSORPTION SPECTROPHOTOMETER IN FOOD CHEMISTRY
LABORATORY
by
Diem Nguyen
Master of Science in
Family and Consumer Science
The purpose of this graduate project was to develop three laboratory experiments based on the most current food analysis instruments, the High Performance Liquid
Chromatography (HPLC) and the Atomic Absorption Spectrophotometry (AAS). The three experiments, including determination of caffeine in energy drinks using HPLC, iron determination in fresh spinach and breakfast cereals using GF-AAS, and detection of calcium in kale and calcium supplements would be implemented in Advanced Food
Science laboratory courses at California State University, Northridge (CSUN) (i.e. FCS
401L and FCS 501L). The utility of these experiments would enhance student learning and critical thinking in food analysis. In addition, hands-on experience using HPLC and
AAS will allow students to better understand the background principles and apply these theories, which they had previously learned in the lecture courses into practical operation.
Furthermore, getting actual experience with food analysis instruments will help to increase the potential of obtaining a position in the dynamic job market.
vi CHAPTER I
INTRODUCTION
Consumers demand food products that are of high quality, nutritious, and inexpensive. But consumers are also concerned about the safety of their foods. In addition, government agencies have pressured food industry with rules and regulations to protect consumers. In response to these demands, the food industry has employed different methods and technologies to produce food products that are wholesome and safe to eat. Food analysis is one of the processes that has been applied in food manufacturing as part of quality management before, during, and after production. In order to maintain the highest quality for our food products, those involved in food product development must be skilled in quality assurance as part of their professional development and training. Analytical chemistry is one of the important tools used as part of food product analysis.
Over the years, analytical equipment has often been used by food chemists for food analysis. High Performance Liquid Chromatography (HPLC) and Atomic
Absorption Spectrophotometry (AAS) are two important analytical methods used to analyze food samples. These have been known as sophisticated methods that provide highly accurate measurement of food’s characteristics. As a food scientist, it is critical to have the basic skills to perform the highest level of food analysis. Thus, it is important for professional development for food science students have hands-on training in some advanced analytical methods and instrumentation in order to increase the potential of obtaining a position in the dynamic job market. HPLC and AAS training should be an important part of academic training for food science students.
1 Statement of the Problem
HPLC and AAS are part of the equipment housed in the food chemistry lab at
California State University, Northridge (CSUN). Since HPLC and AAS have many applications in food chemistry and have been used to analyze food components such as vitamins, mineral, pigments, food additives, and organic compounds, it is important for students to receive the most current analytical training. Being adept in using these analytical instruments would add to the skills of our students, those who will be vying for jobs working in food science field. For the last several years, CSUN Food Science students have not had adequate experience in using these important pieces of equipment.
Purpose
The purpose of this project is to develop three laboratory experiments specifically based on the use of the most current food analysis instruments, the High Performance
Liquid Chromatography (HPLC) and the Atomic Absorption Spectrophotometry (AAS).
These experiments will be implemented in Advanced Food Chemistry courses for undergraduate (FCS 401L) and graduate (FCS 501L) Food Science students at CSUN.
Definitions
1. Analyte is a substance or component that is being identified or measure in an
analytical procedure.
2. Atomic Absorption Spectrophotometry (AAS) is an analytical method used to
determine the concentration of an analyte in a sample.
3. Graphite Furnace AAS (GF-AAS) is a type of Atomic Spectrometry used, which
has a graphite-coated furnace to increase the sensitivity of the instrument.
4. High Performance Liquid Chromatography (HPLC) is an analytical method used to
2 separate liquid mixtures in order to analyze the different components of the sample.
This method is one of the main formats of chromatography. The separation is simply
based on the solubility of the compounds.
Assumptions
These laboratory experiments were developed based upon the following assumptions:
Students enrolled in FCS 401L and FCS 501L will know how to read and understand
the lab manual and material safety data sheets (MSDS).
Students enrolled in FCS 401L and FCS 501L will have a solid understanding of
working in the food chemistry laboratory.
Students enrolled in FCS 401L and FCS 501L will have successfully completed all of
the prerequisite science classes, including Introductory Chemistry, Organic
Chemistry, and/ or Analytical Chemistry.
Each section of the laboratory manual is informative and has adequate scientific
information and is easy for students to understand.
Laboratory instructors must understand the principles of the lab instruments and
know how to perform them.
The reagents met the methods qualification and purity.
Limitations
These laboratory experiments have following limitations:
Lab instruments need to be calibrated and maintained regularly.
The laboratory chemicals and materials should be available to support students
3 working on a research or project.
Poor quality instruments should be updated or replaced.
These experiments are expensive to run because the instruments require high purity
reagents and chemicals. Graphite tube is required to change often, typically lasting for
only100-125 cycles.
These experiments will address the lack of experience using modern analytical
techniques among Food Science students; however, it might be too advanced for
some who didn’t have a background in analytical chemistry.
4 CHAPTER II
REVIEW OF LITERATURE
Food science and technology have had a great impact upon the food system. In the twentieth century, food science and technology were applied to transform raw ingredients into foods that were available year round (Ziegler et al., 2010). Then came advances in developing foods that were nutritious, of high quality and safe to eat. The production of such foods can account for the enormous interest in food quality among consumers and public institutions (Cifuentes, Dugo, & Fanali, 2013). In this regard, food manufacturers and distributors have to improve the quality and safety of their products.
There is, thus, a greater need for more sophisticated instruments and more methods that are able to offer more accurate quantitative and qualitative measurements while improving the precision and sensitivity of food analysis. It is important to incorporate analytical methods and instruments into the education and training of future food scientists. Effective training in the use of the current analytical instruments and methods would strengthen the quality of our food science educational program and properly develop the technical skills in the food industry workforce.
The Growth of Food Science in the Food Industry
Food science and technology have contributed to changes in the food industry throughout history. Two million years ago, our ancestors discovered cooking as a form of processing food. They learned to preserve, using salting, drying and fermenting and how to store food (Wrangham, 2009). These methods of food processing allowed humans to gather foods and survive. Many millennia later, humans learned to do more sophisticated methods of food processing. For example, in ancient Greece, bread, wine, and olive oil
5 were perishable and had an unpleasant flavor when spoiled; but these were transformed into safe, stable, and enjoyable foods (Floros, 2004). These simple processing methods led to advancements in food processing and the development of food science and technology.
In the early 1900s, the United States developed its own food system preservation techniques, which were used so food could be available year round (Arnold et al., 2000).
The United States also developed the agronomic system to help manage crop production
(Arnold et al., 2000). This allowed the U.S. to distribute food for Americans and other countries. Between 1945 and 1965, nutritional quality was an important consideration in food preservations; its importance was ultimately diminished (Arnold et al., 2000).
Instead, convenience took priority, particularly since, during this time, the role of women began to shift; more and more women joined the workforce (Arnold et al., 2000).
In the 1980s, food science and technology adapted to more social and economic changes. In 1981, the Food and Agriculture Organization (FAO) estimated that millions of people worldwide were malnourished or starving, and that the demand for food could double in the near future (Food and Agriculture Organization of the United Nations,
1981). To solve this problem, politicians, scientists, technologists, and planners considered the role of food science and technology (Food and Agriculture Organization of the United Nations, 1981). In 1985, the United Nations University published its Food and Nutrition Bulletin and Hans Meliczek, a Senior Officer in Agrarian and Rural
Development of FAO, again mentioned the crucial role of food science and technology in agricultural production (Meliczek, 1985); he noted that mass production could reduce food cost. Preservation, packaging, and storing were also considered.
6 Nutrient Enrichment and Fortification
The natural nutrient in most food is relatively low. Nutrient enrichment (replacing nutrient lost) and nutrient fortification (adding of nutrient) are often used to enhance levels of nutrients in certain foods (Fulgoni, Keast, Bailey & Dwyer, 2011). In the 20th century, food science and technology contributed to nutrient enrichment and fortification in food products (Schmidt, 2009). This was the result of research on micronutrients, such as iodine, iron, and calcium, in our diet during the early part of the 20th century, which showed the effects of deficiencies (Ziegler et al., 2010), This led to the development of the Recommended Dietary Allowances (RDA) for essential nutrients (i.e. nutrients that must come from the diet) (Ziegler et al., 2010). To achieve the RDAs for essential nutrients, food manufacturers enriched and/or fortified foods with micronutrients. The most commonly enriched and/or fortified foods include pasta, milk, salt, butter, and flour
(Ziegler et al., 2010).
Dietary Guidelines
The Dietary Guidelines provide information on the role of nutrition in health. The
Dietary Guidelines often identifies areas in addressing a public health need based on current scientific evidence (USDA, 2015). It focuses on foods and beverages that help maintain and achieve good health as well as preventing chronic disease (USDA, 2015).
The Dietary Guidelines is published jointly by the U.S. Department of Health and Human
Services (HHS) and the U.S. Department of Agriculture (USDA); it is revised every five years (USDA, 2015).
Modern Day Food Science and Technology Needs
In addition, dietary guidelines provide information on the role of nutrition in
7 health. Many food companies are committed to offering healthier products, containing whole grains, higher fiber content, and reduced trans-fat. At the same time, food science and technology often manufacture functional food products that are designed for people with specific health conditions. Some examples are sugar-free products for diabetics or low-sodium products for people with hypertension (Ziegler et al., 2010).
Today, food science and technology have a big part in the production-to- consumption food system. The food system is more complex now; it requires safe, tasty, low cost, and convenient but nutritious food products (Schmidt, 2009). Food manufacturers must apply modern food technologies to maintain or improve the quality of food and enhance the safety of mass-produced food. The growth of the food industry has increased the need for future food scientists with expertise in current trends and food technologies.
Application of Food Science in the Classroom
The national professional organization for food science and technology is the
Institute of Food Technologists (IFT). The IFT has always taken an active role in training future food scientists. Since 1939, the IFT has developed and revised curriculum standards for food science and technology education programs (Iwaoka, Britten & Dong,
1996). The purpose of revising the standard education curriculum was to support food science educators train future food scientists. By acquiring scientific knowledge, graduates of food science programs will be well prepared for a career.
In 1944, the IFT released the first educational standards for undergraduate food science programs. Basic courses in chemistry, biology, physics and math were strongly recommended, as was technical knowledge specific to the food science industry (Stewart,
8 1947). During those years, many IFT reports suggested that the program complement the core science curriculum to meet the needs of the food industry. In 1958, the first curriculum was issued for undergraduate food science education. Biochemistry and microbiology courses were added into the core sciences. The non-technical courses, such as English, speech, writing, and economics, were also part of the curriculum (Schaffner,
1958). In 1966, the minimum standards of food science education programs were established; it provided detailed guidelines for institutions. The minimum standard included program organization, budgets, faculty, and facilities (Anonymous, 1966). In
1977, the minimum standards were modified to make the programs less rigid; program organization and resources were minimized and the guidelines were more general
(Anonymous, 1977).
In 1990, the IFT Education Committee conducted a survey in both academia and industry. The results suggested that the minimum standards should include sensory evaluation, packaging, food law, ingredients technology, technical writing, experimental design, and marketing as required courses. Respondents believed that these courses would benefit students looking for specific industry experiences such as research and laboratory. In 1992, the minimum standards were again revised (Satterlee, 1992).
Besides improving standard education curricula, food science educators were concerned about the instructional pedagogy. In the past, most food scientists had been trained in an instructional paradigm of lectures, homework, and exams (Hartel, 1995).
Food scientists who had been educated under this paradigm found it difficult to find employment because of inadequate training (Iwaoka, Britten & Dong, 1996). At the 1993 and 1994 IFT annual meetings, participants suggested changes to food science education
9 (Hassel, 1993). A learning paradigm in which teachers design learning method and environment and students control learning and figure out how to apply it was proposed as a replacement for the instructional paradigm (Arnold, 1994). Many educators agreed that a learning paradigm should develop the talents and competencies that would make graduates of food science programs employable (Iwaoka, Britten & Dong, 1996). The
Curriculum Minimum Standards, which was revised in 1992, added critical thinking and problem solving to the list of professional skills.
Since 1992, the minimum standards have been used as guidelines for food science education programs; however, food science educators have complemented them in innovative ways. For example, faculty at the University of California, Davis use computer animation to teach the principles of enzymology and protein chemistry
(Martinez, Ramirez, Johnston, Smith, & Whitaker, 2005). A video technology, live demonstrations, and hands-on laboratory assignments have been found to be equally effective at Washington State University (Johnson, Trout & Luedecke, 2005). Several food science programs have reported that the new strategies have improved students’ learning.
Every five years, the IFT Educational Committee Task Force reviews and revises the Minimum Standards. In 2001, computer literacy and statistics were added to the standards along with coursework in quality control and sensory analysis (Hartel, 2006).
In 2006, the Higher Education Review Board (HERB), formed by the IFT Educational
Committee Task Force, learned that the 2001 IFT Standards were not being used to measure the educational standard in some of the approved programs. Those programs were then required to submit their annual reports. The annual reports were used to
10 conduct new guidelines for educational standards. These guidelines are able to incorporate the prevailing issues of food industry into Food Science Program.
Between 2005 and 2010, in the United States and internationally there have been declining enrollments in food science programs, especially at the graduate level. An estimated 2,700 positions are unfilled each year in food and agricultural science in the
United States (Weller, Robbins, Elmore & Wiedmann, 2015); and there is a shortage of more than half of the number of workers needed for the science and technology fields in the United Kingdom (Chikthimmah & Floros, 2007). Roberts, Robbins,
McLandsborough and Wiedmann (2010) discussed the factors that have led to the present shortage of qualified food scientists. According to researchers, high school students and guidance counselors have not been informed of the importance of the food science discipline, so students are not being encouraged to apply to programs in food science.
Since few students graduate from undergraduate food science programs, even fewer enter masters and doctoral programs. Further, researchers found that undergraduates with degrees in food science are satisfied with the jobs that were offered to them during their undergraduate internships; thus, many had no interest in further education (Weller,
Robbins, Elmore, & Wiedmann, 2015).
In an effort to help stimulate more student interest in food science careers, food science educators have been attempting to introduce high school students to careers in food science by having food science taught in home economics class (McEntire &
Rollins, 2007). Roberts, Robbins, McLandsborough, and Wiedmann (2010) also discussed the use of undergraduate and graduate scholarships in food science, as well as offering field trips to food companies and research laboratories to encourage students
11 interested in the profession. Researchers also discussed the advantages and opportunities that are available to students holding higher degrees in food science as a way to inspire students to pursue advanced educational programs.
Application of Analytical Chemistry in the Food Industry
Many students majoring in food science are not required to take courses in analytical chemistry. Employers have complained that students’ lack of preparation in analytical chemistry places them at a disadvantage (Weller, Robbins, Elmore &
Wiedmann, 2015). Therefore, it is important to have analytical chemistry experience for future food science students.
Chemistry is the study of a compound’s structure, chemical, and physical properties. There are five sub-disciplines of chemistry: biochemistry, organic chemistry, inorganic chemistry, physical chemistry, and analytical chemistry (Harvey, 2008).
Analytical chemistry, the qualitative and quantitative study of matter, is used in many fields such as medicine, clinical chemistry, toxicology, and environmental chemistry
(Hieftje, 2000). In the food industry, analytical chemistry is applied to study the components and properties of foods (McClements, 2003).
There are numerous reasons for analyzing food, including food safety, quality control, and product research and development (McClements, 2003). Government agencies have issued regulations and recommendations to maintain food quality, protect consumers’ rights and safety. The Food Inspection and Grading Service is the system that is used by the government to ensure that food products meet the regulations
(McClements, 2003). Food manufacturers and government agencies rely on analytical techniques to manage food safety and quality (McClements, 2013).
12 Food safety is one of the most important reasons for food analysis. Both food manufacturers and consumers have increased concerns about the safety of food and of the food supply. Foods that contain harmful microorganisms (Salmonella and Ecoli), toxic chemicals (pesticides and cleaning detergent), or immaterial matters (heavy metal and glass) are considered as unsafe (Pomeranz & Meloan, 2006). Food manufacturers do everything they can to eliminate harmful substances from foods in order to ensure that food is safe and wholesome. To achieve this, food manufacturers follow good manufacturing practices and extensively use analytical techniques to analyze food products routinely (McClements, 2003). The use of analytical techniques allows them to detect harmful substances at a very low levels. This ensures that food products are safe for the consumers (Pomeranz & Meloan, 2006).
The food industry is highly competitive, so food manufacturers want to ensure that their products are of high quality, nutritious, and inexpensive. The consistency of a final product is one of the most determinations of the food quality (Nielsen, 2010). The consistency of a food product requires the same overall properties, including texture, flavor, appearance, and shelf life (McClements, 2013). Food manufacturers understand that the raw materials and processing operations affect the properties of the final product
(Nielsen, 2010). To control these variations, food manufacturers analyze raw materials and monitor the properties of food during operation using analytical techniques
(Pomeranz & Meloan, 2006). By analyzing the incoming ingredients, this allows food manufacturers to predict the behaviors of the food products during and after processing in order to make necessary adjustments to produce desired products (Pomeranz & Meloan,
2006). During processing, food manufacturers use analytical techniques that can help to
13 detect problems quickly (Pomeranz & Meloan, 2006). This allows them to adjust the processing conditions and helps to retain product quality and reduce the amount of material and time wasted.
Food analysis also has a significant role in product research and development. In recent years, food manufacturers have adapted to changes in consumers food preferences, including consumer desires to have foods that are more healthy, lower cost, and more interesting or exotic (Nielsen, 2010). To meet these preferences, many food scientists have carried out more new product development. In product development, food scientists improve the properties of existing products and methods of production to develop new products (Nielsen, 2010). Besides the information of the basic research, food scientists rely on food analysis to understand of the properties of foods such as flavor, texture, color, and shelf life (McClements, 2003). Scientists can then experiment with food composition and characteristics to improve a product and/ or design a new product.
The impact of the use of analytical techniques in food manufacturing is evident in safety, quality, and product developments. The demand for improved food safety, quality, and product development have increased the need for more sensitive and precise methods and instruments that can measure a food sample (Cifuentes, Dugo, & Fanali, 2013).
Cifuentes and Ibañez (2001) reviewed the use of analytical methods and instruments in food analysis. They stated that along with many biological and electrochemical techniques, spectroscopic and separation techniques have been used for more than 50 years.
Spectrophotometry
Spectroscopic techniques are used to examine the physical-chemical structure and
14 qualitative-quantitative of the compound based on the interaction between molecules/atoms and electromagnetic radiation, thus frequency or wavelength is detected through emission or absorbance. Spectroscopy is often used as a means of fast and direct measurement in food analysis. Spectroscopy can be used in process line because their solvents and reactants are not toxic. Among the sub-fields of spectrometry are atomic spectroscopy, infrared spectroscopy, nuclearmagnetic resonance, and mass spectrometry.
Atomic spectroscopy has been used for years to detect metals and non-metals.
Determining the concentration of individual elements such as calcium, sodium, and potassium, and trace elements such as iron, magnesium, copper and selenium, are important. Different kinds of atomic spectroscopy can be used to detect the elements, including flame atomic absorption spectrometry (F-AAS), graphite atomic absorption spectrometry (GF-AAS), and inductively coupled plasma atomic emission spectrophotometry (ICP-AES) (Cifuentes, Dugo & Fanali, 2011). GF-AAS is a classic form of atomic absorption spectrometry that can detect trace minerals as low as parts-per- million (ppm). GF-AAS is more sensitive and can detect as parts-per-billion (ppb); it is
100 times more sensitive than flame method. GF-AAS uses smaller samples than F-AAS, only 0.005 mL up to 0.1 mL for GF; the flame method requires about 5 mL (Price, 2008).
In addition, GF-AAS sample preparation and handling can be minimized than other methods. ICP-AES can give measurement to higher reproducibility and quantitative linear range; however, it is difficult to interpret and much more expensive than F-AAS and GF-AAS.
Separation Techniques
Separation technique contributes the most to food analysis. Separation techniques
15 are based on the partitioning of a sample between a moving (mobile) and a fixed
(stationary) phase (Neilsen, 2010). After 1970, many new separation techniques have appeared, based on classic chromatography; however, these are automated, faster, more sensitive, sensitivity, and have reduced sample matrix interferences (Cifuentes & Ibañez,
2001).
There are four kinds of separation: gas chromatography, liquid chromatography, supercritical fluid chromatography (SFC), and capillary electrophoresis. High- performance liquid chromatography (HPLC) is a specialized technique for liquid chromatography separation. HPLC is widely used to measure vitamins, amino acids, protein, carbohydrate, lipid, and additives (colorants, antioxidants, and preservatives)
(Harvey, 2008) in foods. HPLC has been developed for more specific methods of separating components containing biological activity (antibody/antigen, hormone, and toxin) (Cifuentes & Ibañez, 2001). HPLC has also been used to improve separation. For example, liquid chromatography-atomic absorption spectroscopy (LC-AAS) can operate for both separation of metallic species and to confirm their oxidation state altogether
(Cifuentes, Dugo & Fanali, 2013).
HPLC and AAS have been used worldwide for years (Cifuentes & Ibañez, 2001).
They offer an extended range of applications in many industries. The techniques are used in the food industry to ensure the quality of products, protect consumer health, and comply with safety regulations and standards.
Caffeine, Iron and Calcium in Food Products
Caffeine
Caffeine is a major alkaloid in more than 60 plants, including tea, cola nuts, and
16 coffee beans (Injac, Srdjenovic, Prijatelj, Boskovic, Karljikovic-Rajic, & Strukelj, 2008).
Cola nuts and cacao pods are often used to make flavors in soft drinks and chocolate products (Food and Drug Administration [FDA], 2007). More than 80% of the adult population worldwide consumes caffeine every day in one form or another (FDA, 2007).
Caffeine is often used as a central nervous system, cardiac, and respiratory stimulant. It is reported that there is little to no risk in consuming less than 300 mg of caffeine per day
(McCusker, Goldberger, & Cone, 2006). Research has been done to evaluate the effect and recommendations of caffeine limitations during pregnancy. Smith (2005) determined that less than 200 mg per day is recommended for pregnant women, or when someone feels anxiety or stress. However, McCusker, Goldberger, and Cone (2006) found that pregnant women whose caffeine intake was 71-140 mg/day had infants weighing 116 g less than did pregnant women who consumed less than 10 mg per day. Consuming larger amounts of caffeine (i.e. over 500 mg of caffeine per day) might cause a fast heart rate, anxiety, tremors, and diuresis (McCusker, Goldberger, & Cone, 2006).
Children are also vulnerable to excess caffeine intake (Temple, 2009). Dr.
Margaret Hamburg, the former Food and Drug Administration (FDA) commissioner, reported that children are at risk for possible health effects from ingestion of caffeine from what they eat and drink without even having any sense of the exposure (Hamburg,
2013). Temple (2009), found that 98% of children aged 5 to 18 years consumed caffeine on a weekly basis, and this was mostly derived from soft drinks. The study also found that many children consuming high caffeine concentrations experienced persistence of all headaches and interrupted sleep during the night.
According to the FDA regulatory requirements, caffeine must be listed among the
17 ingredients on the product label, though the amount of caffeine does not have to be disclosed (Injac, Srdjenovic, Prijatelj, Boskovic, Karljikovic-Rajic, & Strukelj, 2008). It has been reported that soft drinks can contain up to 105.7 mg/serving and energy drinks can contain up to 357 mg/serving (McCusker, Goldberger, & Cone, 2006). While there are no regulatory requirements to label the caffeine content of food, the caffeine concentration of commonly consumed beverages has been extensively been studied
(Injac, Srdjenovic, Prijatelj, Boskovic, Karljikovic-Rajic, & Strukelj, 2008; Temple,
2009; Wang, Helliwell, & You, 2000).
HPLC is of one the most popular techniques for measuring the amount of caffeine. It is used to analyze drug mixtures and other samples (Injac, Srdjenovic,
Prijatelj, Boskovic, Karljikovic-Rajic, & Strukelj, 2008). The separation of caffeine consists of a reverse-phase column C18 and an isocratic elution system of water/methanol/acid (Wang, Helliwell, & You, 2000). Injac and colleagues have proposed that HPLC is a quick and reproducible method for the routine simultaneous analysis of caffeine in foods and drinks.
Iron
Unlike caffeine, iron is a trace mineral that is essential for human health. In the body, iron is responsible for oxygen transport and storage, energy metabolism, antioxidant and pro-antioxidant, oxygen sensing, and DNA synthesis (Linus Pauling
Institute, 2015). As an essential trace mineral, iron must come from the diet; it can be obtained from vegetables, milk, fish, meat, and processed flours and cereals. Iron deficiency can result in anemia and can lead to inadequate oxygen in the bloodstream, impaired ability to do physical work in adults, and impaired intellectual development in
18 children (Durukan, Şahin, Şatıroğlu, & Bektaş, 2011). The recommended dietary allowance of iron is as follows: in children (ages 4-8) 10mg/day, (aged 9-14) 8mg/day; in adolescents 11mg/day for males and 15mg/d for females; in male adults 8mg/day and female adults 18mg/d. (Linus Pauling Institute, 2015). These recommendations prevent iron deficiency and maintain the iron storage.
Even though iron deficiency is the most common nutritional deficiency worldwide, the health effects of iron excess are now receiving increased attention.
Fortification and enrichment of foods with iron was originally implemented to reduce the rate of iron deficiency. Ninety percent of flour and bread sold in the United Sates is iron enriched and fortified (Swanson, 2003). The iron content is highest in cereals (43% and higher), meat, fish and poultry (22%), and other sources (9%) (Swanson, 2003). It is rare to have excess of iron from diets. However, 40% of adults in the U.S. use multivitamin and mineral supplements (Swanson, 2003). These people might have iron overload without realizing it.
Iron deficiency and iron overload have generated interest in the determination and monitoring of iron levels in the diet. AAS has been proposed as a way to detect minerals and trace minerals in food items in an instrumental analysis laboratory experiment.
Durukan, Şahin, Şatıroğlu, and Bektaş (2011) state that AAS is one of the simple, rapid and reliable methods to detect vitamin and mineral in food samples. This method of analysis is based on calculating the atom-absorbing energy, which is triggered by heat energy (Nielsen, 2010). The heat energy from the equipment processes molecules to atoms. By absorbing the energy, atoms are excited from the ground state (Nielsen, 2010).
The excited atoms emit energy of a specific wavelength as they drop from the excited
19 state to the ground state (Nielsen, 2010). The amount of energy emitting from the hallow cathode lamp then reaches the detector (Nielsen, 2010). Researchers determined iron concentration in vegetables and bread using AAS method (Durukan, Şahin, Şatıroğlu, &
Bektaş, 2011; Millikan, 2012). Their results exhibited accuracy, good precision and sensitivity of iron found in samples when compared to certified samples.
Calcium
Like iron, calcium is important for health. Calcium is a major constituent of bones and teeth; it is required for vascular contraction and vasodilation, nerve transmission, muscle function, as a second messenger of intracellular signaling, and hormonal secretion
(Linus Pauling Institute, 2015). Calcium deficiency may increase the risk of osteoporosis, cancer of the colon and rectum, elevated blood pressure, and cardiovascular diseases
(Linus Pauling Institute, 2015). The recommended dietary allowance for calcium is
1,000-1,200 mg/day for adults. This amount can be obtained from dairy products, green leafy vegetables, as well as fortified orange juice, soymilk, tofu, and cereal. According to the USDA Nutrient Data Base, one cup of cheese provides 771-890 mg of calcium.
Fortified products give 30-40% of the daily (DV) value per serving (equivalent to 300-
400 mg). Excess calcium intake from food alone is rare. However, excess intake may result from dietary intake combined with calcium supplementation, causing hypercalcemia. Hypercalcemia can lead to renal insufficiency, soft tissue calcification and kidney stones (Ross et al., 2011). Therefore, it is necessary to monitor calcium intake.
The human body needs adequate nutrition in order to help maintain health.
Inadequate intake, or malnourishment, can lead to a myriad of poor health consequences.
20 The Recommended Dietary Allowances (RDA) are established to set minimum and/ or maximum recommendations for maintaining health. The Federal Food and Drug
Administration (FDA) works standardize the fortification of essential nutrients as a means improving the nutritional quality of the food supply (Yamini, 2012). Fortification is also regarded as long-term approach to reducing the prevalence of certain nutrient deficiency and to meet a demonstrated public health need (Yamini, 2012). Since nutrient toxicity is very rare from consuming food alone, it is more likely associated with supplement, it is important for food manufacturers to monitor the added levels of nutrient fortification in food products.
Conclusion
Food analysis is a discipline that applies analytical procedures to identify and measure the properties and the constituents of foods. Foods are analyzed by ingredients suppliers, food manufacturers, government laboratories, and university research laboratories (Nielsen, 2010). In response to the demands of consumers, and to comply with national and international regulations, food analysts have used analytical instruments such as high performance liquid chromatography (HPLC), UV-Vis spectrophotometry, gas chromatography, fluorimetry, ion chromatography, gas chromatography mass spectrometry, and atomic absorption spectrophotometry (AAS) in order to preserve the quality and safety of the food supply. Food scientists need to possess expertise in food analysis techniques. Therefore, hands-on training in advanced analytical methods and instrumentation is necessary in order to increase the analytical skills and ability to perform and contribute important skills within our food industry.
Thus, it is necessary to provide up-to-date food analysis training for all who plan to be in
21 the food science fields.
22 CHAPTER III
METHODOLOGY
The purpose of this project was to design three experiments based on the analytical instruments HPLC and AAS.
This chapter presents the process of selecting, designing, and testing the three
Food Chemistry and Analysis experiments. Dr. Claudia Fajardo-Lira, the previous committee chair of this project and CSUN Food Science Coordinator, suggested that the three experiments should incorporate the use of two high precision analytical instruments
(the HPLC and AAS), and should reflect the food analysis methods taught in advanced
Food Chemistry courses. The HPLC and AAS instruments have been in the Food
Chemistry laboratory at California State University, Northridge (CSUN) for a long period of time, but have not been used because they were not functioning. After some troubleshooting, reinstallation of new software, and repairs, the instruments were restored to working condition. The instruments and the analytical methods were discussed with
Dr. Simon Garrett, an Analytical Chemistry instructor at CSUN. The three experiments, which were carefully reviewed and approved by committee members Dr. Claudia
Fajardo-Lira, Professor Julie Ellis, and Dr. Elizabeth Sussman, were chosen for inclusion in the FCS 401/501 laboratory manual. The following experiments were selected:
1. Determination of Caffeine in Energy Drinks, by HPLC
2. Iron Determination in Fresh Spinach and Breakfast Cereal using, GF-AAS
3. Detection of Calcium in Kale and Calcium Tablet, using GF-AAS
The goal of this project was to require food science students to perform more complex experiments in food analysis. Therefore, each experimental procedure included
23 detailed instructions on how to handle the chemicals, prepare reagents, and operate the instruments, as well as the background principle, and pre- and post- lab questions. Each section of the laboratory procedure was written to ensure that students understood what they were expected to learn from each experiment. Each experiment was designed including the following sections:
Objective: One sentence that states the purpose of the experiment.
Pre-Assignment: Students will be asked to answer two to three questions prior to the class. These questions are designed to introduce students to the instrument and method that they are going to learn in the experiment.
Background and Introduction: This section includes background theory and principle of the instrument and method used for food analysis. It also includes a brief description of the analyte which will be detected in the experiment.
Equipment and Materials: A detailed list of chemicals, reagents, ingredients, and accessories that are necessary for the experiment.
Procedure: A detailed instruction for sample preparation, standard solutions, equipment operation, sample analysis, and obtaining results.
Hazards: Instruction on how to handle and dispose of chemicals, and on precautions to take while working in the lab.
Calculations: Instruction on how to use obtained results to prepare the calibration curve and calculate the analyte in the sample.
Discussion and Questions: Students will be asked to discuss their obtained results and possible errors that might have led to inaccurate results. Several questions will require critical thinking and the ability to apply of food analysis to current issues
24 pertaining to the food industry and regulation. Discussion and questions will be part of each student’s laboratory report.
References: A list of sources that used to conduct the experiment. Students can consult the references when completing the discussion and questions section.
Each experiment was designed to reflect complex concepts of modern food analysis that would be encountered with the lecture of the food chemistry course.
Complex concepts were well researched and broken down in the experiment to give students a worthwhile educational experience in the lab.
Since most CSUN Food Science students who enrolled in the food chemistry course are not familiar with HPLC and AAS methods, each section of the experimental procedure was written concisely and comprehensively to provide students with clear directions on performing new methods, and to minimize hazardous incidents in the lab.
The HPLC experiment was tested twice and the results were consistence both times. Each AAS experiment was tested multiple times until desired and consistent results were achieved. The purpose of multiple testing not only was to get a desired result, but also provide smooth and clear laboratory instruction. In addition, the
Instruction on Running Experiments included in Appendix D were designed to help the instructor to be familiar with the methods and instruments. Due to time constraints, the three experiments were not able to be evaluated by students taking FCS 401 and FCS
501.
25 CHAPTER IV
RESULTS
The three experiments (Appendix A) were developed based on two current food analysis instrument techniques, one with High Performance Liquid Chromatography
(HPLC) and two with Atomic Absorption Spectrophotometry (AAS). The analytical instruments require the proper understanding of how to operate laboratory instruments and safely prepare chemicals and solutions. The three experiments were designed in consultation with analytical chemists. The HPLC experiment was tested twice and each
AAS experiment was tested many times. Appendix B contains the set of data and calculations for each experiment.
The instructor and students will be provided with the basic analytical formulas and conversions to create the standard solutions and calculate the concentration of the unknown (Appendix C). Appendix D contains supplemental materials on troubleshooting and on the use and maintenance of instruments. The instructor and students should be familiar with all the chemicals that they are going to work with; therefore, the material safety data sheets (MSDS) for each chemical is included in Appendix E. The results of the project are presented below.
The parameters of both instruments were first set as recommended in the manual and recommended by an analytical expert in order to obtain the best measurements and avoid errors. Table 1 shows the Graphite Furnace AAS (GF-AAS) instrumental setup parameters for Iron (Fe) and Calcium (Ca). Table 2 shows the HPLC instrumental setup parameters.
26 Table 1
GF-AAS Instrumental Setup Parameters Parameters Quantity Fe Ca
Argon flow rate 40psi Cooling water rate 1.5-2L/min Wavelength 248.3nm 0.2nm Band width 422.7nm 0.7nm Sheath gas 1.25 mL/min Internal flow 200 mL/min Mini flow 50 mL/min Ashing 1,250ºC 1,200ºC Atomization 2,300ºC 1,700ºC Drying step 250ºC 200ºC
Table 2
HPLC Instrumental Setup Parameters Parameters Quantity
Pump flow 1.00 ml/min Purge time 1.0 min Wavelength 278 nm Lamp D2 Sampling speed 15.0 µL/sec Run time 7 min
Each experiment was then performed. The results obtained from the three experiments are presented below.
Determination of Caffeine in Energy Drinks by HPLC
In HPLC, the limit of detection (LOD) is the minimum detectable amount of caffeine. The limit detection for HPLC is as low as parts-per-million (ppm). Table 3 shows the detection of standard solutions and standard calibration curve equation for caffeine analysis. The caffeine calibration curve, which was generated using the external standard curve method, was obtained by plotting the standards’ absorbance against
27 standards’ concentrations (Appendix B). The relationship between the absorbance and the caffeine concentration was expressed as a linear regression line (y=mx + b), where y is the absorbance measured by HPLC, x is the caffeine concentration (mg/ml), m is the slope, and b is the intercept. The correlation coefficient is r. The caffeine of the unknown
(Monster Energy) was calculated at 0.31899mg/ ml (318.99 ppm) and the caffeine listed on Monster Energy label is 0.29587 mg/ml (295.87 ppm).
The mobile phase was prepared at the ratio 65:35:1 (v/v/v), water (HPLC-grade): methanol (HPLC -grade): acetic acid. The caffeine peak appeared at 3.72 minutes using this ratio mobile phase.
Table 3
Caffeine Standard Solutions, Unknown and Calibration Curve Equation Samples Concentration (mg/ml) Area
Standard 1 0.05 8217 Standard 2 0.1 15478 Standard 3 0.15 23058 Standard 4 0.2 29217 Unknown (Monster Energy) 47288
Retention time: 3.72 mins
Caffeine Calibration curve equation: y= 146550x + 539 (r2=0.9978)
Iron Determination in Fresh Spinach and Breakfast Cereals Using GF-AAS
In the GFAAS method, the limit of detection (LOD) is the minimum detectable amount of iron (Fe). The limit detection for AAS is as low as parts-per-billion (ppb).
Table 4 shows the detection of Fe standard solutions and sample. The calibration curve was expressed as linear regression line (y=mx + b) as in the caffeine experiment. The amount of iron in each sample is shown in Table 5.
28 Table 4
Fe Standard Solutions, Unknown and Calibration Curve Equation Samples Concentration (ppb) Abs-sec
Standard 1(blank) 0 0.0334 Standard 2 10 0.0959 Standard 3 25 0.1795 Standard 4 50 0.3088 Standard 5 75 0.4011 Unknown (Cereal- diluted to 100ml then 1:10ml) 0.7414 Unknown (Spinach- diluted to 50ml then 1:25ml) 0.0749 Fe Calibration curve equation: y = 0.0049x + 0.0461 (r2=0.9918)
Table 5
Amount of Fe in Samples Cereal (Honey Kix) In 0.3447g Cereal Per Serving- 33g
Unk1 Sample: 141.897ppb Unk1 Sample: 13.58mg Label: 94ppb Label: 9mg USDA database: 12.61mg Spinach (Earthbound Farm) In 0.3927g Spinach Per Serving- 85g
Unk1 Sample: 5.877ppb Unk1 Sample: 1.59mg Label: 9.979ppb Label: 2.7mg USDA database: 2.3mg 1 Unknown
Detection of Calcium in Kale and Calcium Tablet
In the GFAAS method, the limit of detection (LOD) is the minimum detectable amount of Calcium (Ca). The limit detection for AAS is as low as parts-per-billion (ppb).
Table 6 shows the detection of Ca standard solutions and sample. The calibration curve was expressed as linear regression line (y=mx + b) as in the caffeine experiment. The amount of Ca in each sample is shown in Table 7.
Table 6
Ca Standard Solutions, Unknown and Calibration Curve Equation
29 Samples Concentration (ppb) Abs-sec
Standard 1(blank) 0 0.0719 Standard 2 5 0.1083 Standard 3 10 0.1609 Standard 4 25 0.2816 Standard 5 50 0.4364 Standard 6 75 0.6113 Unknown (Tablet- diluted to 100ml then 1:100-1:100-4:10ml) 0.6368 Unknown (Spinach- diluted to 50ml then 1:100-4:10ml) 1.3332 Fe Calibration curve equation: y = 0.0071x + 0.0824 (r2=0.9967)
Table 7
Amount of Ca in Samples Calcium Tablet 2 In 0.4276g Tablet Powder Per Tablet- 1.7477g
Unk1 Sample: 78.08ppb Unk1 Sample: 797.8mg Label: 58.72ppb Label: 600mg
Kale In 0.6466g Kale Per 100g
Unk1 Sample: 176.17ppb Unk1 Sample: 340.56mg USDA database: 150mg 1Unknown
2Target brand
30 CHAPTER V
DISCUSSION
The purpose of this project was to develop three experiments based on two methods of analysis methods: one using High Performance Liquid Chromatography
(HPLC) and two using Atomic Absorption Spectrophotometry (AAS). The food industry relies on these two analytical methods to detect components or trace elements in a food sample. The three experiments that were designed for this project were 1) the determination of caffeine concentration in energy drinks using HPLC; 2) the detection of iron in breakfast cereal and fresh spinach; and 3) the detection of calcium in calcium tablet and kale, using GFAAS. These experiments were designed to be taught in advanced food chemistry laboratory courses for both undergraduate (FCS 401) and graduate (FCS 501) students.
Even though the three experiments that were designed for this project were based on the literature reviews (Harris, 2007; McCusker, Goldberger, & Cone, 2006; Millikan,
2012; Nielsen, 2010; Injac, Srdjenovic, Prijatelj, Boskovic, Karljikovic-Rajic, & Strukelj,
2008), the methods were adjusted. The methods used for each experiment were adjusted in order to accommodate with the availability of the Food Chemistry lab at CSUN, including instrumentation and reagents. These adjustments significantly benefit the Food
Science Department.
Discussion of the Findings and Modifications
HPLC Experiment
The caffeine experiment, using HPLC, was the simplest experiment of the three. In this experiment, chromatography was used to separate caffeine, using the techniques described by Harris (2007). Compounds in a mixture are separated from each other based
31 on the chemical’s affinity for one of two different solvents in contact with each other. If a polar and non-polar solvent are brought into contact, polar molecules will prefer to be in the polar solvent and nonpolar molecules in the non-polar solvent (Nielsen, 2010).
The caffeine, which is non-polar, was separated while traveling through the column. The column is the stationary phase (non-polar), which adsorbs the caffeine on its surface and the isocratic mobile phase (polar, and a combined of water, methanol, and acetic acid at a ratio of 65:35:1), which pushes caffeine through the column. In the first trial, the caffeine was separated. The caffeine’s retention time, the interval between the instant of injection and the detection of caffeine, was approximately four minutes. The complete peak area of the caffeine showed during the retention time. The peak area and the retention time were compared to the literature review and they were very close to each other. In the second trial, the caffeine peak areas and the retention times were consistent.
Monster Energy was used as the unknown in the experiment and the result came out very close to the amount of caffeine listed on the label. The result from the experiment was
0.31899 mg/ml (318.99 ppm) and the caffeine listed on the Monster Energy was 0.29587 mg/ml (295.87 ppm). The difference from claim on the label is 23.12 ppm.
Even though the results obtained from the caffeine experiment were reliable and very close to the label, there were several limitations that can account for errors in the results.
The first limitation was that the HPLC experiment required HPLC graded water and reagents for the mobile phase. Methanol, which was used to make mobile phase, was not HPLC-grade and had been in stock since 2008.
The second limitation was that the mobile phase needed to be degased before being
32 run through the column because pressure might build up and disrupt the run. Therefore, the degased system should be calibrated for HPLC experiments.
AAS Experiments
Both iron and calcium detection experiments used the AAS method and the graphite furnace AAS (GF-AAS) instrument. In each experiment, the element was intentionally identified in the processed food and fresh produce (iron in cereal and fresh spinach and calcium in calcium tablet and fresh kale) in order to compare the differences in mineral content. In addition, the instructor compared the measurements with the label.
The GF-AAS was more difficult than HPLC. The GF-AAS is one of the most reliable techniques and instruments for mineral detection. The method is based on energy absorption and the absorption is triggered by heat (Nielsen, 2010). For absorption to happen, the machine has to perform a three-step process: drying, ashing, and atomization
(Harris, 2007). Many trials were performed to ensure the accuracy of the data for both experiments. The standard addition should be used to determine the concentration of the element using GF-AAS. However, the readings fluctuated and the results were inconsistent. The standard calibration curve, which expressed as a linear regression equation, showed a series of consistent results. The standard calibration curve method was used as the main method of obtaining the experimental results. The results were then compared to what was found in the review of literature (Millikan, 2012), the claim on the label, and to the USDA database for accuracy.
As shown in Table 5, the amount of iron (Fe) detected in 0.3447g of Honey Kix cereal was 141.897ppb, which was higher than the 94ppb stated on the label. As converted into per serving (33g), the Fe content in the sample was 13.58mg, which is close to the
33 USDA database of 12.61mg and the label claims of 9mg. In the same table, the amount of
Fe in 0.3927g of Earthbound Farm fresh spinach was 5.877ppb, less than the 9.979ppb stated on the label. In each serving size (85g), the sample tested was 1.59mg of Fe, but the label claimed 2.7mg, and the USDA database claim was 2.30mg. According to the literature review, the amount of Fe detected in cereal and fresh spinach ranged from
0.113mg/100g to 19.82mg/10g and from 0.88 to 2.64mg/100g of fresh spinach, respectively. In addition, the amounts of Fe detected in samples were similar to the amounts listed in the USDA database. The literature review and UDSA database confirm the accuracy of the Fe experiment.
Calcium commonly presents in different water and solution sources and this causes the contamination of the graphite. Lamp AAS should be used to detect Ca since Lamp AAS detects parts-per-million and the contamination can be prevented. Therefore, some modifications were made to the original setting of the GFAAS to run the calcium experiment. The calcium element produced many signals and was much more sensitive than iron. The atomization step was changed from 2400ºC to 1700ºC to reduce calcium’s signals and to increase the lifetime of the graphite tube. Table 7 shows the results of the calcium experiment. The amount of calcium in 0.4276g of calcium tablet sample was
78.08ppb which was higher than the label 58.72ppb. Each tablet (1.7477g) contains
797.8mg of calcium compared to 600mg on the label. There is no USDA or FDA database so the label is the only basis of comparison. In the same table (Table 7), the amount of calcium detected in 0.6466g of fresh kale was 176.17ppb. As converted into 100g,
340.56mg of calcium was detected in the sample; 150mg was reported in USDA database.
In the literature review, the mean concentration of calcium in leafy vegetables was
34 2580mg/kg (258mg/100g). The experimental results were higher than those found in the literature review (Millikan, 2012) and in the USDA database. Again, calcium is commonly contaminated in solutions. Nitric acid, which was used to digest the sample, was labeled containing calcium as ppm. The water used to dilute the solution showed high readings for calcium. These reasons might explain the higher amount of calcium in the detected samples.
GFAAS is useful in detecting trace elements at very low concentrations (parts-per- billion). However, the instrument is extremely sensitive and some factors can account for the errors in the experiments.
Both experiments required sample digestion. The digestion was done by a MARS microwave. It was convenient, requiring less time and labor. However, the sample should be used quickly after digestion because this method uses only HNO3. Without added HCl or H2SO4, the sample will not be stable for a long period of time. In addition, 0.1g of sample requires 7ml of HNO3 for digestion. It could cause over dilution for certain trace elements and result in low readings.
Another limitation is that since the instrument can detect elements as parts-per- billion, the graphite tube could become easily contaminated with calcium even in plain water. Once that happens, the tube requires heavy cleaning to remove residue. This will shorten the lifetime of the graphite tube.
AAS requires AAS-grade reagents and chemicals to run the experiments. The nitric acid, that was used to digest the samples, was old and labeled as containing detecting unspecified elements of up to parts-per-million.
The graphite tube lasts for only 100-125 cycles. The firing cycles must be
35 monitored and changed before reaching the maximum. Otherwise, this can cause fluctuating readings and may dirty the windows. Calcium is commonly present in water and in the standard solution. Therefore, calcium contamination is very common in graphite experiments. A separate graphite tube should be used for each element.
Discussion of the Expert Evaluation
The three experiments were developed based on the availability of the CSUN Food
Chemistry laboratory, equipment and reagents. There was not enough analytical glassware available in the laboratory and it could have led to erroneous results. The instruments had not been calibrated and maintained to assure their accuracy. Therefore, it is necessary to have these instruments serviced. Trained faculty should be able to perform regular calibration and maintenance.
Furthermore, experts should evaluate the quality of these experiments and, if necessary, modify them. The procedures and results of the experiments can be tested in an analytical laboratory.
Discussion of the Target Population Evaluation
Time constraints precluded evaluation of the three experiments by students taking
FCS 401 and FCS 501; neither of the classes was taught during the designing of the experiments created in this project. In the future, these three experiments should be pilot- tested in the FCS 401 and/or FCS 501 laboratory classes. For each experiment, students should complete an anonymous survey asking for their opinions and feedback for each section of the experiment. The feedback can be used to refine the experiments in order to improve the students’ ability to learn from them.
Implications
36 The three experiments were designed to introduce food science students to the most up-to-date methods and techniques in food chemistry and analysis. In becoming proficient with these types of analytical techniques, students would be able to learn to conduct the kind of experiments that they will need to use in the workplace. HPLC and
AAS have been used worldwide for years and have an extended range of applications in food industry (Cifuentes & Ibañez, 2001). Currently, students majoring in food science are not required to take courses in analytical chemistry. However, this is a skill that employers in the food science industry are demanding. Students’ lack of preparation in analytical chemistry places them at a disadvantage. Being familiar with these instruments would prepare them to enter the food science industry.
Conclusion
The purpose of this project was to conduct three experiments based on current food analysis methods. These experiments will be taught in Advanced Food Science laboratory courses FCS 401/ FCS 501. Two experiments that used AAS to detect iron and calcium in fresh produce (kale and spinach) and processed foods (supplement and breakfast cereal). The other experiment detected the caffeine content in an energy drink.
Analyses performed with HPLC and AAS were found to be accurate, precise and fast. These experiments will be able to give students practical experience in instrumental analysis and an understanding of current food regulations. In addition, the lab experiments in instrumental analysis might encourage their interest in research. The experiments would advance students’ knowledge and sustain them academically and professionally.
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43 APPENDIX A
Appendix A contains the three developed experiments, as follow:
1. Determination of Caffeine in Energy Drinks by HPLC
2. Iron Determination in Fresh Spinach and Breakfast Cereals using GF-AAS
3. Detection of Calcium in Kale and Calcium Tablet using GF-AAS
44 Determination of Caffeine in Energy Drinks by HPLC
I. Objective:
To determine the concentration of caffeine in various soft drinks utilizing high performance liquid chromatography (HPLC) method.
II. Pre-Assignment:
1. Briefly discuss three types of liquid chromatography: normal phase, reversed-
phase, and ion exchange.
2. Draw the structure of caffeine and explain if it is polar or non-polar.
III. Background & Introduction:
HPLC is the technique used to separate liquid mixtures in order to analyze the different components of the sample. It plays an important role in modern food analysis and can tolerate a wide range of sample concentrations. HPLC gives both qualitative and quantitative analysis with a high degree of precision and accuracy.
The method of liquid chromatography is simply based on the solubility of the compounds. Compounds in a mixture are separated from each other based on their preferences for one of two different solvents in contact with each other. If a polar and non-polar solvent are brought into contact, polar molecules will prefer to be in the polar solvent and nonpolar molecules in the non-polar solvent. In a HPLC experiment, one solvent is the stationary phase (non-polar) and other solvent is the mobile phase such as water, dilute buffer, methanol, or acetonitrile (polar).The column, which packed with small particles, is the stationary phase.
At a steady flow rate, a pump(s) deliver liquid mobile phase from the reservoir through
45 the system. Once the sample is injected, it flows with the mobile phase and reaches the column. In the column, the sample mixture is separated into bands. The bands then will be detected by detector. HPLC detector is UV absorption spectrum that measures the absorbance of the bands and generates peaks.
In this experiment, reversed-phase chromatography is used, meaning that the stationary phase is non polar. The caffeine will be separated using a non-polar C18 column and a water/methanol/acetic acid mobile phase remains constant “isocratic elution” during separation. The standards and sample of caffeine will be prepared and measured in order.
The standards’ peak areas and concentration are needed to generate a calibration curve.
The curve is used to determine the caffeine content in soft drink samples.
IV. Equipment and Materials:
A. Equipment
Analytical balance
HPLC Shimadzu Model
Reversed-phase cartridge column C18
B. Materials
Reagents:
o Mobile phase: Water(HPLC-grade): methanol (HPLC -grade): acetic acid,
65:35:1 (v/v/v)
o *Degas
o Filter if use DI water
46 Caffeine standard
Caffeinated soft-drink samples (degas and filter)
Sample vials for auto sampler
Disposable plastic syringe, 3-5ml (for filtering sample)
Disposable syringe filter assembly, 0.45 µm pore size
Volumetric pipettes
Volumetric flaks: 100ml and 10ml
V. Procedure:
A. Calibration Standards
1. Prepare a stock solution containing 20 mg caffeine/100 mL HPLC grade H2O (0.20 mg/mL). Weigh 20 mg caffeine in a 100 mL volumetric flask. Fill up to the line with
HPLC grade water. Transfer this solution to an Erlenmeyer flask and label it caffeine stock solution.
2. Degas the stock solution *
3. Standard solutions
Std. 1 (0.05 mg caffeine/mL): take 2.5 mL of stock solution and fill with HPLC
grade water to the mark of a 10ml volumetric flask
Std. 2 (0.10 mg caffeine/mL): take 5.0 mL of stock solution and fill with HPLC
grade water to the mark of a 10ml volumetric flask
Std. 3 (0.15 mg caffeine/mL): take 7.5 mL of stock solution and fill with HPLC
grade water to the mark of a 10ml volumetric flask
47 Std. 4 (0.20 mg caffeine/mL): use the stock solution
B. Preparation of Caffeinated Beverage
1. Obtain a sample for which you wish to measure the caffeine content. (Degas or simple open the drink sample the night before to remove the gas, but aware of dust)
2. Dilute the sample 1:100. Measure 0.5 mL sample beverage and transfer it in a 50 mL volumetric flask. Fill the volumetric flask up to the line with HPLC grade water.
3. Filter the sample using a disposable plastic syringe and a syringe filter (0.45 µm pore size).
a. Connect the syringe filter to the syringe barrel.
b. Remove the plunger and use a Pasteur pipette to transfer a portion of beverage
sample to the syringe barrel.
c. Insert and depress syringe plunger to force sample through the membrane filter
and into a clean test tube.
C. Analysis of Standards and Sample
1. Fill the HPLC vials with standard solutions and sample. Fill one extra vial with HPLC water as your blank. Make sure the samples reach the marked line on the vial and cap the vial with the lid provided.
2. Place vials in the auto-injection rack, starting from 0 for the blank and continuing with
48 1, 2, 3, and 4 for the standard solutions. Place the vial containing your sample in the 5 slot.
3. In the CLASS-VP 7.4 software, press single run button. Input your sample ID, MAKE
SURE YOU INCLUDE YOUR GROUP NAME (e.g. for the blank vial input blank_GROUP NAME, for standard 1 solution vial input std1_GROUP NAME, etc.).
4. Input the vial number to match with the number in the auto-sampler rack (e.g. put 0 for blank, 1 for standard 1 and so on).
5. Press start. The sample will run for 5-7 minutes.
6. Press Stop Run to stop the analysis.
7. To get the data, press Reports (on the left side of the screen). Click Area%. Record the data (peak area and retention time) in your lab notebook. Draw a baseline for the caffeine peak if appropriate.
8. Make two additional runs for the sample and record the data of each run.
VI. Hazards:
1. The standards and samples can be poured down the sink.
2. Discard the mobile phase waste in the jar labeled as “Caffeine experiment waste/
49 Methanol/Acetic acid”
V. Calculations:
A. Results
Report the standards and sample peak areas and retention time in the table below.
Sample Concentration (mg/ml) Retention Time (minute) Peak Area
0 0
Std.1 0.05
Std.2 0.1
Std.3 0.15
Std.4 0.2
Unknown
B. Calibration Curve
Prepare a plot of peak area versus caffeine concentration using the data obtained for your standard solutions and fit the data with a least-squares line (remember 0,0 is a data point).
C. Sample
From the equation for the least-square line and the peak are for your unknown, calculate the caffeine concentration (mg/ml).
From the label, calculate the caffeine concentration (mg/ml) in the can.
50 VII. Discussion & Questions:
1. Discuss your obtained results and possible errors. Compare the caffeine concentration in the measured sample with the label.
2. Why was it important to filter and degas the mobile phase and the samples?
3. If the mobile phase composition was changed from 65:35:1(water:methanol:acetic acid) (v/v/v) to 75:25:1 and 65:35:1 to 55:45:1. How would that change the retention time and why?
4. What is the current caffeine regulation on labeling? (Hint: FDA website)
*Degas: Use vacuum filtering flask. Clean the nitrogen tube before degasing. Insert the nitrogen tube into the solution and observe for decreasing bubble (15minutes, longer if more solution). The Nitrogen tank is located next to the hood in the back left side of the room. This process needs to be done under the hood.
Sources Consulted:
Campbell-Platt, G. 2009. Food Science and Technology. 1st ed. John Wiley & Sons Ltd.
Harris, DC. 2007. Quantitative Chemical Analysis. 7th ed. New York: Freeman.
Nielsen, SS. 2010. Food Analysis. 4th ed. New York: Aspen Publishers.
51 Determination of Fe in Fresh Spinach and Breakfast Cereals using GF-AAS
I. Objective:
To determine the concentration of iron in breakfast cereal and fresh spinach using Atomic
Absorption Spectrophotometer (AAS) method.
II. Pre-Assignment:
1. Briefly discuss three types of atomic spectroscopy: flames, graphite furnace, and
inductively coupled plasma.
2. What is the basic wavelength and split required for parameter set up to detect
Iron?
3. How would you prepare the 50 ppb standard solution using 1000 ppm stock
solution? (Do not use volume less than 1ml)
III. Background & Introduction:
Atomic absorption spectroscopy is a technique that is used to detect a specific element in the sample at a very low concentration (from parts-per-million to parts-per-billion). The techniques has been used widely in food industry to detect the concentration of specific minerals/ trace elements in food samples.
Atomic absorption is a method based on atom absorbing energy, energy absorption is triggered by heat energy. The heat energy from the equipment has processed molecules to atoms. By absorbing the energy, atoms are excited from the ground state to the excited state. The atoms emit energy of a specific wavelength as they drop from the excited state to the ground state. The amount of energy emitting from the hallow cathode lamp then
52 reaches the detector.
In graphite furnace, the atomization process is as follow steps: (1) Drying step is around
100-150ºC, this step is to remove solvent by evaporation. (2) Ashing (pyrolysis or charring) step is round 500-1300ºC, this sept oxidizes organic matter and matrix. (3)
Atomization step is around 1800-2400ºC, this step vaporize and atomize sample.
Iron is a trace mineral and the body doesn’t require much to function. However, iron deficiency is the most common nutritional deficiency worldwide and in the United States, the health effects of iron excess have recently received increased attention. Fortification and enrichment of foods with iron were undertaken as an intervention to reduce the prevalence of iron deficiency.
In this experiment, graphite furnace is used to detect iron in breakfast cereal and fresh spinach. The standard solutions and unknown samples will be prepared and measured in ascending order. The absorbance of standards and concentrations are needed to generate a calibration curve. The curve is used to determine the iron content in cereal and fresh spinach.
IV. Equipment and Materials:
A. Equipment
Analytical balance
Graphite Furnace Atomic Absorption Spectroscopy- Buck Scientific
53 Microwave digestion MARS
B. Materials
Chemical
o Nitric acid (HNO3) (Trace Analysis Graded). Handle with care; use
glove, eyes protection, work under fume hood, and avoid breathing in.
Nitric acid is concentrated and corrosive.
o Fe standard solution.
0.5% HNO3
Millipore Deionized (DI) Water
Breakfast cereal
Fresh spinach
Sample vials for auto sampler
Volumetric flasks and volumetric pipettes to prepare standard solutions and
sample.
Weight boats/ paper
Ceramic mortar and pestle
V. Procedure:
A. Sample Digestion and Preparation
Each digestion tube is strictly at 0.1g of sample in 7.0ml of nitric acid (HNO3) for a complete sample digestion. We can digest more than one vessel to obtain desired sample amount.
54 1. Grind up the cereal and spinach in separate ceramic mortar and pestle.
2. Weight out 0.1g of cereal and place in digestion tube (repeat and make a total of three digestion tubes). Make sure to record exact weight for each sample (three decimal) if you can’t get exactly to 0.1g.
3. Repeat step 2 for spinach.
4. CAREFULLY add 7.0ml of nitric acid (HNO3) into the tube. Work under the fume hood and protective gear is a must.
5. Place the plastic stopper on top of the tube, then use the cap to seal the tube tightly.
6. Place the tubes evenly in the digestion vessel. Cereal and spinach can be digested at the same time. Make sure to record the slot numbers of your samples.
7. The instructor will show how to operate the microwave digestion.
8. After digestion, wait for it to cool. Then take these tubes out.
B. Preparation of Samples and Standard Solutions
Samples
1. Cereal (diluted to 100ml then 1:25ml): carefully transfer your digested cereal samples
55 into a 100ml volumetric flask and fill up to the line with Millipore DI water. Mix well, take 1.0 ml of this solution then dilute into 25ml volumetric flask with Millipore DI water.
2. Spinach (diluted to 50ml then 1:10ml): carefully transfer your digested spinach samples into a 50ml volumetric flask and fill up the line with Millipore DI water. Mix well, take 1.0 ml of this solution then dilute into 10ml volumetric flask with Millipore DI water.
Standard Solutions
1. Carefully prepare a 100ppb from 100ppm stock solution provided by laboratory instructor (Use 0.5% HNO3).
2. Then carefully prepare a set of standard solutions 10, 25, 50, and 75ppb from a prepared 100ppb (Use 0.5% HNO3).
*Note: Do not use volume less than 1.0ml aliquot for these dilutions. Use the same volume of the volumetric flasks and volumetric pipets to prepare standard solutions. For example, use all 25ml volumetric flasks to make the standard solutions. Do not use 10ml volumetric flask to make 10ppb and 25ml volumetric flask to make 25ppb. Your instructor must approve your method of making standard solutions.
C. Equipment Check
1. Turn on the Argon gas. The pressure should be set at ~40psi.
56
2. Turn on the water (for cooling purpose) and it should be set at 1.5-2L per minute.
These should be turned on before operating Graphite Furnace AAS. Make sure the pressure is consistent and there is no bubble on the water pipe, especially at the graphite chamber. If there is bubble that means the cooling process is interrupted and it might cause problems such as the graphite tube will burn, or the heat sink will explode.
3. Turn on all the equipment including graphite furnace, detector, auto sampler, and printer. The lamp and graphite should be on at least 5-10 minutes before running the sample. Check for the rinsing water on the right of the furnace, it should be filled with the same water that used to dilute the standard (Millipore DI water in this case).
4. Check if the right lamp (Fe in this case) is in the position. You can rotate the turret to get the right lamp.
5. If the element of interest doesn’t appear on the detector screen, go to library and select the element.
6. On the right side of the detector, adjust the wavelength and slit to maximize energy sample and energy background.
D. Graphite Furnace AAS Operation and Sample Analysis
1. Go to [Sample] to adjust the injection amount to 20µL (modifier 1 and 2 should be set
57 at 0).
2. Pour the samples prepared on step V.B above into vials (shake the sample well before pour into the vial). Then place the vial in the autosampler tray beginning at position 1.
Start with blank (0.5% HNO3), then in ascending order 10ppb, 25ppb, 50ppb, 75ppb, spinach (last diluted solution 1:25), and cereal (last diluted solution 1:10).
3. Press [Start] to run the sample. Check for the printer during run, it might disrupt the run.
4. The screen will show the sample cup position, you need to change to the appropriate position. Press [Del] and enter the number matching the cup position. *Do not press
[Esc], it will abort the run. Run each sample twice. If the readings are fluctuated, notify your instructor immediately.
5. Record the Abs-Sec readings, the readings for blank should be in the range of 0-0.06.
6. When data acquisition is completed, allow the instrument to turn off. Remove your samples, and turn off the argon gas tank and cooling water.
VI. Hazards:
Discard samples and standard solution into the bin labeled “Acid Nitric/HNO3”
VII. Calculations:
A. Results
58 Report the standards and samples Abs-sec in the table below.
Sample Concentration (ppb) Abs-sec
Std.1 (Blank-0.5% HNO3) 0
Std.2 10
Std.3 25
Std.4 50
Std.5 75
Unknown (Cereal)
Unknown (Spinach)
B. Addition Calibration Curve
1. Prepare a standard calibration curve by plotting the Abs-sec versus the concentration using the standard obtained for your standard solutions.
2. Fit these data points with a linear least squares line.
C. Sample
1. Calculate the concentration of Fe in the dilute solution from the equation for the standard calibration curve and the dilution factor associated with the graphite furnace analysis.
2. Calculate the concentration (in mg) of Fe in solid cereal and fresh spinach samples.
Calculate the concentration of Fe per serving.
59 VIII. Discussion & Questions:
1. Discuss your results and compare to the Fe on the label.
2. What is the likely source of error in this experiment?
3. What would be the advantages of having an atomic absorption unit that had a graphite furnace (vs. a flame)?
4. Compare the Iron values for breakfast cereal and fresh spinach to those reported in the
U.S. Department of Agriculture Nutrient Database for Standard Reference. What does it tell you about the results obtained?
Sources Consulted:
Harris, DC. 2007. Quantitative Chemical Analysis. 7th ed. New York: Freeman.
Nielsen, SS. 2010. Food Analysis. 4th ed. New York: Aspen Publishers.
60 Determination of Ca in Kale and Calcium Tablet using GF-AAS
I. Objective:
To determine the concentration of calcium in vitamin tablet and fresh kale using Atomic
Absorption Spectrophotometer (AAS) method.
II. Pre-Assignment:
1. Briefly discuss the advantages and disadvantages between Flame and Graphite
Furnace AAS.
2. What is the basic wavelength and split required for parameter set up to detect Ca?
3. There are two types of interferences that are encountered in AAS: spectral
interference and nonspectral interference. Briefly discuss these interference.
4. How would you prepare the 50 ppb standard solution using 1000 ppm stock
solution? (Do not use volume less than 1ml)
III. Background & Introduction:
Atomic absorption spectroscopy is a technique that is used to detect a specific element in the sample at a very low concentration (from parts-per-million to parts-per-billion). The techniques has been used widely in food industry to detect the concentration of specific minerals/ trace elements in food samples.
Atomic absorption is a method based on atom absorbing energy, energy absorption is triggered by heat energy. The heat energy from the equipment has processed molecules to atoms. By absorbing the energy, atoms are excited from the ground state to the excited state. The atoms emit energy of a specific wavelength as they drop from the excited state
61 to the ground state. The amount of energy emitting from the hallow cathode lamp then reaches the detector.
In graphite furnace, the atomization process is as follow steps: (1) Drying step is around
100-150ºC, this step is to remove solvent by evaporation. (2) Ashing (pyrolysis or charring) step is round 500-1300ºC, this sept oxidizes organic matter and matrix. (3)
Atomization step is around 1800-2400ºC, this step vaporize and atomize sample.
Calcium is a major constituent of bones and teeth, it is required for vascular contraction and vasodilation, nerve transmission, muscle function, second messenger of intracellular signaling, and hormonal secretion. Calcium deficiency can cause osteoporosis, cancer of the colon and rectum, elevated blood pressure, and cardiovascular diseases.
The recommended dietary allowance for calcium is 1,000-1,200 mg/day for adults. This amount can be obtained from dairy products (especially cheese and milk), green leafy vegetables, and fortified orange juice, soymilk, tofu and cereal. According to USDA
Nutrient Data Base, one cup of cheese provides 771-890 mg of calcium. Fortified products give 30-40% daily value per serving (equivalent to 300-400 mg). People who are taking dietary supplements are at risk of calcium. Excessive calcium intake can cause renal insufficiency, soft tissue calcification and kidney stones. Therefore, it is necessary to monitor calcium intake.
In this experiment, graphite furnace is used to detect calcium in vitamin tablet and fresh
62 kale. The standard solutions and unknown samples will be prepared and measured in ascending order. The absorbance of standards and concentrations are needed to generate a calibration curve. The curve is used to determine the calcium content in vitamin tablet and fresh kale.
IV. Equipment and Materials:
A. Equipment
Analytical balance
Graphite Furnace Atomic Absorption Spectroscopy- Buck Scientific
Microwave digestion MARS
B. Materials
Chemical
o Nitric acid (HNO3) (Trace Analysis Graded). Handle with care; use glove,
eye protection, work under the fume hood, and avoid breathing in. Nitric
acid is concentrated and corrosive.
o Ca standard solution.
0.5% HNO3
Millipore Deionized (DI) Water
Calcium supplement
Fresh kale
Sample vials for auto sampler
63 Volumetric flasks and volume metric pipettes to prepare standard solutions and
sample.
Weight boats/ paper
Ceramic mortar and pestle
V. Procedure:
A. Sample Digestion and Preparation
Each digestion tube is strictly at 0.1g of sample in 7.0ml of nitric acid (HNO3) for a complete sample digestion. We can digest more than one vessel to obtain desired sample amount.
1. Grind up the tablet and kale in separate ceramic mortar and pestle.
2. Weight out 0.1g of cereal and place in digestion tube (repeat and make a total of four digestion tubes). Make sure to record exact weight for each sample (three decimal) if you can’t get exactly to 0.1g.
3. Repeat step 2 for kale, you want to make a total of 6 tubes instead of four (fresh kale might not contain much Ca compared to vitamin tablet).
4. CAREFULLY add 7.0ml of nitric acid (HNO3) into the tube. Work under the fume hood and protective gear is a must.
5. Place the plastic stopper on top of the tube, then use the cap to seal the tube tightly.
64
6. Place the tubes on the digestion vessel in balance and even. Cereal and spinach can be digested at the same time. *Make sure to record the slot numbers for your samples.
7. The instructor will show how to operate the microwave digestion.
8. After digestion, wait for it to cool. Then take these tubes out.
B. Preparation of Samples and Standard Solutions
Samples
1. Calcium Tablet (diluted to 100ml then 1:100ml, 1:100ml, and 4:10ml): carefully transfer your digested tablet samples into a 100ml volumetric flask and fill up the line with Millipore DI water (1). Mix well, take 1.0 ml of this solution 1 then dilute into another 100ml volumetric flask with Millipore DI water (2). Mix well, take 1.0 ml of solution 2 then dilute into another 100ml volumetric flask with Millipore DI water (3).
Mix well, take 4.0 ml of solution 3 then dilute into 10ml volumetric flask with Millipore
DI water (4). Total of four dilutions.
2. Kale (diluted to 50ml then 1:100ml, and 1:10ml): carefully transfer your digested kale samples into a 50ml volumetric flask and fill up the line with Millipore DI water (1). Mix well then take 1.0 ml of solution 1 then dilute into 100ml volumetric flask with Millipore
65 DI water (2). Mix well, take 1.0 ml of solution 2 then dilute into 10ml volumetric flask with Millipore DI water (3). Total of three dilutions.
Standard Solutions
1. Carefully prepare a 100ppb from 100ppm stock solution provided by laboratory instructor (Use 0.5% HNO3).
2. Then carefully prepare a set of standard solutions 5, 10, 25, 50, and 75ppb from a prepared 100ppb (Use 0.5% HNO3).
*Note: Do not use volume less than 1.0ml aliquot for these dilutions. Use the same volume of the volume metric flasks and volume metric pipets to prepare standard solutions. For example, use all 25ml volumetric flasks to make the standard solutions. Do not use 10ml volumetric flask to make 10ppb and 25ml volumetric flask to make 25ppb.
Your instructor must approve your method of making standard solutions.
C. Equipment Check
1. Turn on the Argon gas. The pressure should be set at ~40psi.
2. Turn on the water (for cooling purpose) and it should be set at 1.5-2L per minute.
These should be turned on before operating Graphite Furnace AAS. Make sure the pressure is consistent and there is no bubble on the water pipe, especially at the graphite
66 chamber. If there is bubble that means the cooling process is interrupted and it might cause problems such as the graphite tube will burn, or the heat sink will explode.
3. Turn on all the equipment including graphite furnace, detector, auto sampler, and printer. The lamp and graphite should be on at least 5-10 minutes before run the sample.
Check for the rinsing water on the right of the furnace, it should be filled with the same water that used to dilute the standard.
4. Check if the right lamp (Ca in this case) is in the position. You can rotate the turret to get the right lamp.
5. If the element of interest doesn’t appear on the screen detector, go to library and select the element.
6. On the right side of the detector, adjust the wavelength and slit to maximize energy sample and energy background.
D. Graphite Furnace AAS Operation and Sample Analysis
1. Go to [Sample] to adjust the injection amount to 20µL (modifier 1 and 2 should be set at 0)
2. Pour the samples prepared on step V.B above into vials (shake the sample well before
67 pour into the vial). Then place the vial in the autosampler tray beginning at position 1.
Start with blank (0.5% HNO3), then in ascending order 5ppb, 10ppb, 25ppb, 50ppb,
75ppb, tablet (last diluted solution 4:10ml), and kale (last diluted solution 4:10ml.
3. Press [Start] to run the sample. Check for the printer during run, it might disrupt the run.
4. The screen will show the sample cup position, you need to change to the appropriate position. Press [Del] and type in the number matching the cup position. *Do not press
[Esc], it will abort the run. Run each sample twice. If the readings are fluctuated, notify your instructor right immediately.
5. Record the Abs-Sec readings, the readings for blank should be in the range of 0-0.07.
6. When data acquisition is completed, allow the instrument to turn off. Remove your samples and turn off the argon gas tank and cooling water.
VI. Hazards:
Discard samples and standard solution into the bin labeled “Acid Nitric/HNO3”
V. Calculations:
A. Results
Report the standards and samples Abs-sec in the table below.
68 Sample Concentration (ppb) Abs-sec
Std.1 (Blank-0.5% HNO3) 0
Std.2 5
Std.3 10
Std.4 25
Std.5 50
Std.6 75
Unknown (Tablet)
Unknown (Kale)
B. Addition Calibration Curve
1. Prepare a standard calibration curve by plotting the Abs-sec versus the concentration using the standard obtained for your standard solutions.
2. Fit these data points with a linear least squares line. Since there are six data points, one or two outliner can be eliminated.
C. Sample
1. Calculate the concentration of Ca in the dilute solution from the equation for the standard calibration curve and the dilution factor associated with the graphite furnace analysis.
2. Calculate the concentration (in mg) of Ca in solid tablet and fresh kale samples.
69 Calculate the concentration of Ca per serving.
VII. Discussion & Questions:
1. Discuss your results and compare to the Ca that claims on the label. What is the likely source of error in this experiment?
2. What is the major concern in sample preparation for specific mineral analysis? How can this concern be addressed?
3. Explain the significance of energy transitions in atoms and of atomization for the techniques of atomic absorbance spectrophotometer (AAS) and atomic emission spectrophotometer (AES). And describe the similarities and differences between AAS and AES for mineral analysis
4. What is the current regulation on vitamin supplement?
5. Compare the Ca value for fresh kale to reported in the U.S. Department of Agriculture
Nutrient Database for Standard Reference. What does it tell you about the results obtained?
Sources Consulted:
Harris, DC. 2007. Quantitative Chemical Analysis. 7th ed. New York: Freeman.
Nielsen, SS. 2010. Food Analysis. 4th ed. New York: Aspen Publishers.
70 APPENDIX B
Appendix B includes data analysis and calculation for each experiments.
1. Caffeine in Energy Drink
2. Iron in Fresh Spinach and Breakfast Cereal
3. Calcium in Kale and Calcium Tablet
71 Determination of Caffeine in Energy Drinks by HPLC
Sample Concentration (mg/ml) Peak Area
0 0
Std.1 0.05 8217
Std.2 0.1 15478
Std.3 0.15 23058
Std.4 0.2 29217
Unknown (Monster Energy) undil. 47288
Caffeine Standard Curve 35000
30000 y = 146550x + 539 R² = 0.9978 25000
20000
15000
10000
Concentration(mg/ml) 5000
0 0 0.05 0.1 0.15 0.2 0.25 Peak Area
*r square of 0.98 and up is indicate a good calibration curve, 0.95 and down is
poor y=14655x + 539
47288=146550x + 539
=>x= (47288 - 539)/ 146550= 0.31899 mg/ml
72 0.31899 mg/ml x 1000 ml/L =318.99 mg/L =318.99 ppm
The sample wasn’t diluted.
*It is recommend in the lab manual that the sample should be diluted to prevent/ minimize column contamination. If your sample was diluted, the concentration that is calculated from the calibration curve will be multiplied for the dilution volume.
Monster Energy label: 70mg per 8Fl oz. = 236.588ml
Convert into ppm: 70mg /236.588ml x 1000ml/L= 295.8729 ppm
73 Iron Determination in Fresh Spinach and Breakfast Cereals using GF-AAS
Sample Concentration (ppb) Abs-sec
Std.1 (blank) 0 0.0334
Std.2 10 0.0959
Std.3 25 0.1795
Std.4 50 0.3088
Std.5 75 0.4011
Unknown (Honey Kix Cereal) 0.7414
Unknown (Earthbound Farm Spinach) 0.0749
Iron Calibration Curve
0.5
0.4 y = 0.0049x + 0.0461 R² = 0.9918 0.3
0.2
0.1 Concentration(ppb) 0 0 10 20 30 40 50 60 70 80 Abs-Sec
*r square of 0.98 and up is indicate a good calibration curve, 0.95 and down is poor y = 0.0049x + 0.0461
Fe in cereal calculation
Fe in 0.3447g cereal
74 0.3447g of Honey Kix cereal was digested, then diluted to 100ml, then diluted 1:10ml y= 0.7414
0.7414= 0.0049x + 0.0461
=> x= (0.7414 – 0.0461)/ 0.0049 = 141.89ppb
Convert amount of Fe from ppb into mg:
141.89ppb /1000= 0.14189ppm= 0.14189mg/L or µg/ml
0.14189µg/ml x (10x100)ml (dilution factor)= 141.89 µg/1000=0.14189mg of Fe in 0.3447g cereal
Fe in per serving (33g)
0.14189mg of Fe in 0.3447g cereal
Fe in per serving= (33g cereal x 0.14189mg Fe)/ 0.3447g cereal= 13.58mg of Fe
Amount of Fe in label
The label claims 50% per serving, 50% of Fe per serving equal 9mg, 50%= 9mg of Fe
33g -> 50%=9mg
Fe in 0.3447g cereal = (0.3447g x 9mg of Fe) / 33g of cereal= 0.094mg of Fe
Fe in 0.3447g cereal as claimed in the label = 0.094mg of Fe / (100ml x 10ml)
= 9.4x10^-5mg/ml x 1000ml/L
= 0.094 ppm= 94ppb
USDA database: 33g of General Mills, Honey Kix contains 12,62mg of Fe
Fe in spinach calculation
Fe in 0.3927g spinach
0.3927g of Earthbound Farm fresh spinach was digested, then diluted to 50ml, then diluted 1:25ml y= 0.0749
0.0749= 0.0049x + 0.0461
75 => x= (0.0749 – 0.0461)/ 0.0049 = 5.877ppb
Convert amount of Fe from ppb into mg:
5.877ppb /1000= 5.877x10^-3ppm= 5.877x10^-3mg/L or µg/ml
5.877x10^-3µg/ml x (25x50)ml (dilution factor)= 7.346 µg/1000=0.007346mg of Fe in 0.3927g spinach
Fe in per serving (85g)
0.007346mg of Fe in 0.3927g cereal
Fe in per serving= (85g cereal x 0.007346mg Fe)/ 0.3927g cereal= 1.59mg of Fe
Amount of Fe in label
The label claims 15% per serving, 15% of Fe per serving equal 2.7mg, 15%= 2.7mg of Fe
85g -> 51%=2.7mg
Fe in 0.3927g cereal = (0.3927g x 2.7mg of Fe) / 85g of cereal= 0.01247mg of Fe
Fe in 0.3927g cereal as claimed in the label = 0.01247mg of Fe / (50ml x 25ml)
= 9.979x10^-6mg/ml x 1000ml/L
= 9.9792x10^-3 ppm= 9.979ppb
USDA database: 85g of raw spinach contains 2.30mg of Fe
76 Detection of Calcium in Kale and Calcium Tablet using GF-AAS
Sample Concentration (ppb) Abs-sec
Std.1 (blank) 0 0.0719
Std.2 5 0.1083
Std.3 10 0.1609
Std.4 25 0.2816
Std.5 50 0.4364
Std.6 75 0.6113
Unknown (Tablet Target Brand) 0.6368
Unknown (Fresh Kale) 1.3332
Calcium Calibration Curve 0.7 0.6 y = 0.0071x + 0.0824 0.5 R² = 0.9967 0.4 0.3 0.2
Concentration(ppb) 0.1 0 0 10 20 30 40 50 60 70 80 Abs-Sec
*r square of 0.98 and up is indicate a good calibration curve, 0.95 and down is poor y = 0.0071x + 0.0824
Ca in tablet calculation
77 Ca in 0.4276g tablet powder
0.4276g of tablet powder was digested, then diluted to 100ml, then diluted 1:100ml, 1:100ml, and final di dilution 4:10ml y= 0.6368
0.6368= 0.0071x + 0.0824
=> x= (0.6368 – 0.0824)/ 0.0071 = 78.08ppb
Convert amount of Ca from ppb into mg:
78.08ppb /1000= 0.07808ppm= 0.07808mg/L or µg/ml
0.07808µg/ml x ((10/4)x100x100x100)ml (dilution factor)= 195,211.2676 µg/1000=195.211mg of Ca in
0.4276g tablet powder
Ca in per tablet (1.7477g)
195.211mg of Ca in 0.4276g tablet
Ca in per tablet= (1.7477g tablet x 195.211mg Ca)/ 0.4276g cereal= 797.87mg of Fe
Amount of Ca in label
The label claims 600mg of Ca per tablet (1.7477g)
Ca in 0.4276g tablet = (0.4276g x 600mg of Ca) / 1.7477g of cereal= 146.79mg of Ca
Fe in 0.4276g tablet as claimed in the label = 146.79mg of Ca / (100ml x 100mlx 100ml x (10/4)ml)
= 5.872x10^-5mg/ml x 1000ml/L
= 0.05872 ppm= 58.72ppb
Ca in fresh kale calculation
Fe in 0.6466g kale
0.6466g of fresh kale was digested, then diluted to 50ml, then diluted 1:100ml, and final dilution
4:10ml
78 y= 1.3332
1.3332= 0.0071x + 0.0824
=> x= (1.3332 – 0.0824)/ 0.0071 = 176.169ppb
Convert amount of Ca from ppb into mg:
176.169ppb /1000= 0.176169ppm= 0.176169mg/L or µg/ml
0.176169µg/ml x ((10/4)x100x50)ml (dilution factor)= 2,201.11 µg/1000=2.202mg of Ca in 0.6466g of fresh kale
Ca in per serving (100g)
2.202mg of Ca in 0.6466g kale
Ca in per serving= (100g kale x 2.202mg Ca)/ 0.6466g kale= 340.55mg of Ca
USDA database: 100g of raw kale contains 150mg of Fe
79 APPENDIX C
Appendix C includes analytical math support (parts-per-million and parts-per-billion and serial dilutions)
80 ANALYTICAL MATH SUPPORT
Parts-Per-Million and Parts-Per-Billion
The definitions of parts-per-million (ppm) and parts-per-billion (ppb):
1 ppm= 1g /1x106 g 1 ppb= 1g /1x109 g
Derivation of other expression:
1 ppm= 1g /1x106 g = 1g /1x106 mL = 1g /1x103 L = 1mg /1L
1 ppb= 1g /1x109 g = 1g/1x109 mL = 1g /1x106 L = 1µg /1L
Example 1: How many g of analyte are there in 12.00 mL of a 314 ppm solution?
314 mg/1.00 L = 314 mg/1000 mL = 314 µg/1 mL
Mass in 12.00 mL= (314 µg/1 mL) x 12mL= 0.00377 g
Example 2: What is the concentration (in ppb) of a solution that contains 0.00233 g of analyte in 980 mL of solution of density 0.920 g/mL?
Mass = Volume x (Mass /Volume) = 980 mL x (0.920 g/1 mL) = 901.6 g
Concentration = (0.00233 g/901.6 g) x (1ppb/1x10-9 g) = 2,580 ppb
Making Serial Dilutions
During the experiments, there will be preparing of a series of known concentration
(standards) from on provided solution of known concentration (stock solution). There are several ways to prepare these solution but the most efficient way is to perform a serial dilution (this means using one solution to prepare the next, more dilute solution) using this formula: M1 x V1 = M2 x V2
For example, you are asked to prepare at least 10 mL of three solutions between 100 and 1 ppm from a stock solution of 400 ppm.
81 Step1: To make our first (most concentrated) solution we’ll take 5 mL of the stock solution to make 25-mL of the first solution.
M1 = 400ppm, M2 =?ppm, V1 =5mL,V2 = 25mL
M2= (M1 x V1)/ V2 = (400 ppm x 5 mL)/ 25 mL = 80 ppm
Step 2: To make a second (less concentrated) solution, we’ll take 10 mL of the first solution and make 25-mL of new solution.
M1 =80ppm, M2 =?ppm, V1 =10mL,V2 = 25mL
M2= (M1 x V1)/ V2 = (80 ppm x 10 mL)/ 25mL = 32 ppm
Step 3: To make the final (least concentrated) solution, we’ll take 1 mL of the second solution and make 10-mL
M1 =32ppm,M2 =?ppm,V1 =1mL,V2 =10mL
M2= (M1 x V1)/ V2 = (32 ppm x 1 mL)/ 10mL = 3.2 ppm
82
APPENDIX D
Appendix D includes:
1. Instruction on running experiments
2. Supplemental material for Graphite Furnace
83
Instructions on Prepare and Running Experiments
Determination of Caffeine in Energy Drinks by HPLC
Reagents preparation:
-Mobile phase: Water (HPLC-grade): methanol (HPLC -grade): acetic acid, 65:35:1
(v/v/v) *HPLC graded water and solutions should be used to make mobile phase. The impurity of regular water and solution can damage the column.
-Degas mobile phase with either N2, Ar, or He for at least15 minutes in vacuum filtering flask. Cover the solution carefully and aware of dust particles can get in.
-The reservoirs of both pumps should be filled with mobile phase at all time (fill the bottle to at least half).
-The rinsing pipe from auto injector should be filled with the same solution as mobile phase.
-Lab assistant should prepare the caffeine stock solution (0.20 mg/ml as instructed in the lab manual) prior to class to prevent contamination and waste.
-Sample (unknown preparation): open the drink (overnight) to remove the gas.
-Everything goes through the column should be filtered (samples and standard solutions)
Before running HPLC:
-Turn on the instruments, including system controller, liquid chromatograph (pump/s), detector, auto injector, and the remote software.
- Purge the system for 10-15 minutes to clean out the residues. Press [Purge] on the liquid chromatograph. If experience high pressure, turn the knob on the right to release the pressure. Remember to close the knob when running sample.
84 Running the sample:
-The instructor can create a new caffeine method or use the existing one.
When turn on the CLASS-VP 7.4 software. It will ask if you want to create a new method.
New method or existing method should have the below set up for caffeine
Pumps
Mode: Isocratic
Constant: Flow
Pump Flow rate: 1.000 ml/minute
Autosampler
Model: SIL-10ADvp
Sample Rack: 2 (or just simply press on Detect Rack)
Rinsing Volume: 200 µL/sec (can adjust)
Needle Stroke: 41 nm (as recommended)
Rinsing Speed: 35 µL/sec
Sample Speed: 15.0 µL/sec
Purge Time: 1.0 (can adjust)
SPD-10Avp
Model: SPD-10Avp
Click on Channel 1
Wavelength Ch 1: 278 nm
Lamp D2
Polarity: Positive
85 Response: 0.5 sec (as recommended)
Sampling: Frequency 2 Hz
Run Time: 5-7 minutes (for Caffeine)
- Press “Single run button” then input sample ID and group name.
- Input the vial number to match with the number in the auto-sampler rack (e.g. put 0 for blank, 1 for standard 1 and so on).
- Press “Start”. The sample will run for 5-7 minutes.
- Press “Stop Run” to stop the analysis.
- To get the data, press “Reports” (on the left side of the screen). Click “Area%”. Record the data in your lab notebook. Draw a baseline for the caffeine peak if appropriate.
- Make two additional runs for the sample and record the data for each run.
-Sample calculation is included in Appendix B
Cleaning up HPLC:
After each lab, lab instructor should rinse the column and the needle of the autosampler
- Column: press “Pump” on LC-10ATVP, the solution will run through the column. Run for 15 minutes.
- Autosampler’s needle: mobile phase solution can be used as rinsing solution. Turn off all the devices but leave the autosampler on, then press “Rinse” and rinse for 10 minutes.
86 Detection of Iron in Cereal and Spinach using GF-AAS Materials & Reagents preparation:
- When purchasing cereal and fresh spinach make sure the package has the nutritional label.
- Concentrated Nitric acid (HNO3)-Trace analysis graded is needed for digestion in order to minimize the contamination.
- Make 0.5% HNO3 for dilution and blank because we used HNO3 to digest samples, therefore, 0.5% HNO3 will make the solutions consistent with the unknown.
- 0.5% HNO3 =5ml of concentrated HNO3 (the same HNO3 used to digest the samples) in
1000ml volumetric flask and fill with Millipore DI water.
- Lab instructor should prepare 100ppm from the stock solution to avoid contamination.
Fe stock solution is 1000µg/ml=1000ppm. To make 100ppm from stock solution: 10ml of stock solution (1000ppm) into 100ml volumetric flask and fill with 0.5% HNO3.
- Student is required to make 100ppb from 100ppm provided solution. There are many different ways to make this solution. Make sure the student makes enough 100ppb solution in order to make their standard solutions.
-100ppb from 100ppm: use M1V1=M2V2
100ppm=100,000ppb
1ml of 100,000ppb into 100ml volumetric flask and fill with 0.5% HNO3 to make 100ml of 1,000ppb (100,000ppbx1ml=100mlx1,000ppb)
87 Then 10ml of 1,000ppb into 100ml volumetric flask and fill with 0.5% HNO3 to make
100ml of 100ppb (100ppbx10ml=100mlx100ppb)
- Student is also required to make a set of standard solutions: 10, 25, 50, and 75ppb.
100ppb, which was prepared above, is used to make standard solutions. *It is extremely important that student needs to use the same volume of volumetric flasks for these solutions. For example; all the solutions can be made in the set of 25ml volumetric flasks or the set of 50ml volumetric flasks but not 10ppb in 25ml volumetric flask and others in
50ml volumetric flasks. The consistent usage of volumetric pipettes is also important to avoid error (these can cause fluctuate readings).
- Standard solutions prepared in 25ml volumetric flasks
10ppb: 2.5ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 10ppb (100ppbx2.5ml=25mlx10ppb)
25ppb: 6.25ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 25ppb (100ppbx6.25ml=25mlx25ppb)
50ppb: 12.5ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 50ppb (100ppbx12.5ml=25mlx50ppb)
75ppb: 18.75ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 75ppb (100ppbx18.75ml=25mlx75ppb)
Sample Preparation (Suggestion)
88 Lab instructor should have one group of students work on digestion and do the first dilution of the sample after digestion. The diluted sample solution should be distributed to the other groups so they can dilute further.
Equipment Check
- Turn on the Argon gas. The pressure should be set at ~40psi.
-Turn on the water (for cooling purpose) and it should be set at 1.5-2L per minute.
These should be turned on before operating Graphite Furnace AAS. Make sure the pressure is consistent and there is no bubble on the water pipe, especially at the graphite chamber. If there is bubble that means the cooling process is interrupted and it might cause problems such as the graphite tube will be burn down, or the heat sink will explode.
- Turn on all the equipment including graphite furnace, detector, auto sampler, and printer. The lamp and graphite should be on at least 5-10 minutes before run the sample.
Check for the rinsing water on the right of the furnace, it should be filled with the same water that used to dilute the standard (Millipore DI water in this case).
- Check if the right lamp (Fe in this case) is in the position. You can rotate the turret to get the right lamp.
- If the element of interest doesn’t appear on the screen detector, go to library and select the element.
- On the right side of the detector, adjust the wavelength and slit to maximize energy sample and energy background. Get the bar go to the right as much as you can. Then press Auto zero [A/Z].
Operation and Analysis
- Keep the original set up for this element.
89 - Main screen “Active Analysis”
Lamp 2
Name: CS-Fe-248.3-lib3
Lamp: Fe Buck Sci
Method: Graphite Furn
Slit: 0.2nm
Wvl (wavelength): 248.3nm
- Make sure “Peak Timing” is at “Fixed”
Press [Cntls] -> “Measurement Controls” ->”Furnace/Vapor Controls”-> “Fixed” (using the up and down arrow).
- Go to [Sample] to adjust the injection amount to 20µL (modifier 1 and 2 should be set at 0)
- Pour the samples prepared into vials (shake the sample before pour into the vial). Then place the vial in the autosampler tray beginning at position 1. Start with blank (0.5%
HNO3), then in order 10ppb, 25ppb, 50ppb, 75ppb, spinach, and cereal.
- Press [Start] to run the sample. Check for the printer during run, it might disrupt the run.
Keep feeding the paper to the printer. Make sure the “Absorbance” on the screen back to zero or close to zero before the next sample analysis.
- The screen will show the sample cup position, change to the appropriate position. Press
[Del] and type in the number matching the cup position. *Do not press [Esc], it will abort the run. Run each sample twice.
- Record the Abs-Sec readings, the readings for blank should be in the range of 0-0.06.
- If wish to look at the graph, press on [Cntls]-> “Furnace/Vapor Controls”->”Replot Last
90 Peak”
- Graphite tube (coated graphite tube for Fe): Keep track of the frying cycle because each graphite last up to 100 cycles or less depend on the temperature of element. The graphite needs to be changed before reaching its maximum 100 cycles, confirm with manufacturer. Otherwise, the graphite tube will burn and cause fluctuated readings while also dirtying the windows.
- When data acquisition is completed, allow the instrument to turn off. Remove your samples, and turn off the argon gas tank and cooling water.
91 Detection of Calcium in Kale and Calcium Tablet using GF-AAS Materials & Reagents preparation:
- When purchasing fresh kale make sure the package has the nutritional label.
- Concentrated Nitric acid (HNO3)-Trace analysis graded is needed for digestion in order to minimize the contamination.
-*Ca contamination is common. Before making solution, we should check which water contains less Ca. We can check distilled water and Millipore DI water. Check the Ca content of the water by run the water with GFAAS, which one give lower Abs-sec reading should be used to make all the solution used for the entire experiment.
- Make 0.5% HNO3 for dilution and blank because we used HNO3 to digest samples, therefore, 0.5% HNO3 will make the solutions consistent with the unknown.
- 0.5% HNO3 =5ml of concentrated HNO3 (the same HNO3 that used to digest the samples) in 1000ml volumetric flask and fill with Millipore DI water.
- Lab instructor should prepare 100ppm from the stock solution to avoid contamination.
Fe stock solution is 1000µg/ml=1000ppm. To make 100ppm from stock solution: 10ml of stock solution (1000ppm) into 100ml volumetric flask and fill with 0.5% HNO3.
- Student is required to make 100ppb from 100ppm provided solution. There are many different ways to make this solution. Make sure the student makes enough 100ppb solution in order to make their standard solutions.
-100ppb from 100ppm: use M1V1=M2V2
100ppm=100,000ppb
92 1ml of 100,000ppb into 100ml volumetric flask and fill with 0.5% HNO3 to make 100ml of 1,000ppb (100,000ppbx1ml=100mlx1,000ppb)
Then 10ml of 1,000ppb into 100ml volumetric flask and fill with 0.5% HNO3 to make
100ml of 100ppb (100ppbx10ml=100mlx100ppb)
- Student is also required to make a serial of standard solutions: 5, 10, 25, 50, and 75ppb.
100ppb, which was prepared above, is used to make standard solutions. *It is extremely important that student needs to use the same volume of volumetric flasks for these solutions. For example; all the solutions can be made in the set of 25ml volumetric flasks or the set of 50ml volumetric flasks but not 10ppb in 25ml volumetric flask and others in
50ml volumetric flasks. The consistence usage of volumetric pipettes is also important to avoid error (these can cause fluctuate readings).
5ppb: 1.25ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 5ppb (100ppbx1.25ml=25mlx5ppb)
10ppb: 2.5ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 10ppb (100ppbx2.5ml=25mlx10ppb)
25ppb: 6.25ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 25ppb (100ppbx6.25ml=25mlx25ppb)
50ppb: 12.5ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 50ppb (100ppbx12.5ml=25mlx50ppb)
75ppb: 18.75ml of 100ppb into 25ml volumetric flask and fill with 0.5% HNO3 to make
25ml of 75ppb (100ppbx18.75ml=25mlx75ppb)
93 Sample Preparation (Suggestion)
Lab instructor should have one group of students work on digestion and do the first dilution of the sample after digestion. The diluted sample solution should be distributed to the other groups so they can dilute further.
Equipment Check
- Turn on the Argon gas. The pressure should be set at ~40psi.
-Turn on the water (for cooling purpose) and it should be set at 1.5-2L per minute.
These should be turned on before operating Graphite Furnace AAS. Make sure the pressure is consistent and there is no bubble on the water pipe, especially at the graphite chamber. If there is bubble that means the cooling process is interrupted and it might cause problems such as the graphite tube will be burn down, or the heat sink will explode.
- Turn on all the equipment including graphite furnace, detector, auto sampler, and printer. The lamp and graphite should be on at least 5-10 minutes before run the sample.
Check for the rinsing water on the right of the furnace, it should be filled with the same water that used to dilute the standard.
- Check if the right lamp (Ca in this case) is in the position. You can rotate the turret to get the right lamp.
- If the element of interest doesn’t appear on the detector screen, go to library and select the element.
- On the right side of the detector, adjust the wavelength and slit to maximize energy sample and energy background. Get the bar go to the right as much as you can. Then press Auto zero [A/Z].
Operation and Analysis
94 - Graphite tube (coated graphite tube for Ca, use separate graphite tube for Ca): Keep track of the frying cycle because each graphite last up to 100 cycles or less depend on the temperature of element. The graphite needs to be changed before reaching its maximum
100 cycles, confirm with the manufacturer. Otherwise, the graphite will burn and cause fluctuated readings while also dirtying the windows.
- Ca naturally presents in different water or reagent sources, therefore, Ca contamination is common. GFAAS is detect at parts-per-billion, the detector would receive a lot of Ca signals and give high readings (Abs-sec reading can be 2.0-6.0 for blank). We need to adjust the program to get the Abs-sec for blank around 0.0-0.6. The following steps should be done
1. Adjust “Furnace Program”
Press [Cntls]->”Furnace /Vapor Controls”->Furnace Program
Then adjust the temperature of step 8 “Atomize-Ramp”, 9 “Atomization”, 10 “Int-Flow-
On”, and 11 “Burn” from 2400 down to 1800.
2. Adjust Peak Delay and Peak Duration
Press [Cntls]-> “Furnace /Vapor Controls”-> “Peak Timing: Fixed”->Peak Delay: 0.50S and Peak Duration: 2.50S (Peak Delay and Peak Duration can be changed to get best readings).
Run the water or blank after these two steps above has done. If the number still high, we can run the furnace without sample (run dry) couple of times, and or run “Furnace Clean- up Cycle” under “Furnace /Vapor Controls”. These can be done to clean the graphite tube if suspect contamination.
- Main screen “Active Analysis”
95 Lamp 1
Name: Ca-Furn3-422.7
Lamp: Ca Buck Sci
Method: Graphite Furn
Slit: 0.7nm
Wvl (wavelength): 422.7nm
- Make sure “Peak Timing” is at “Fixed”
Press [Cntls] -> “Measurement Controls” ->”Furnace/Vapor Controls”-> “Fixed” (using the up and down arrow).
- Go to [Sample] to adjust the injection amount to 20µL (modifier 1 and 2 should be set at 0)
- Pour the samples prepared into vials (shake the sample before pour into the vial). Then place the vial in the autosampler tray beginning at position 1. Start with blank (0.5%
HNO3), then in order 5ppb, 10ppb, 25ppb, 50ppb, 75ppb, tablet, and kale.
- Press [Start] to run the sample. Check for the printer during run, it might disrupt the run.
Keep feeding the paper to the printer. Make sure the “Absorbance” on the screen back to zero or close to zero before the next sample analysis.
- The screen will show the sample cup position, change it the appropriate position. Press
[Del] and type in the number matching the cup position. *Do not press [Esc], it will abort the run. Run each sample two to three times.
- Record the Abs-Sec readings, the readings for blank should be in the range of 0-0.06.
- If wish to look at the graph, press on [Cntls]-> “Furnace/Vapor Controls”->”Replot Last
Peak”
96 - When data acquisition is completed, allow the instrument to turn off. Remove your samples and turn off the argon gas tank and cooling water.
97 Supplemental Material for Graphite Furnace
More detail is provided in the 210-211-Users-Manual GFAAS
Selecting the lamp from library and align the wavelength
1) Install desired lamp in the upper-most position of the lamp turret, this is the operating position. Plug the lamp connector for this lamp position (as designated by the number on the cap) onto the lamp.
2) Press the [lib] button to enter the library. Press [sel] button until the lamp number (top of screen) matches the turret position you are using. Press the [up/down] arrows until the desired metal and method are shown in the library window. Make sure you have selected an absorbance/flame file (flame files are designated as Xx-D2-wavelength-lib3). Press
[2] to load the method then [enter] then [esc]. The file you selected will now appear in the top active analysis window. Alternatively you can press [1] for enter a library name then enter the first letter or two of the atomic symbol of the element you wish to run (you can access the letters on the keyboard by pressing and holding either the [upper case] or
[lower case] button. Press [enter] then either the [up/down] arrow to select the exact file you wish to run then press [esc]. Turn the slit selector knob to the position specified by the library. Turn wavelength knob to the correct wavelength. Press [align] to display the bargraphs.
3) The bargraph indicates the amount of energy from the lamp reaching the detector. “0” on the scale represents the amount of energy you had after pressing align. Very slowly turn the wavelength knob in any direction. If the bar graph goes off screen to the right press [align] to center it. If the bar graph goes off scale to the left turn the wavelength knob in the other direction. Repeat this procedure until you get the HIGHEST reading
(bar graph farthest to the right) you can. Pay attention to the numeric value for energy.
98 This runs on a scale from 0 to 6 and you want to get it as high as possible. The bargraph will follow the energy reading (ie: as the bargraph moves to the right the energy value will increase. Most lamps will give an energy between 2.5 and 4.5. If you have trouble aligning the lamp, press [Bkgnd] to turn the background lamp off and try again. Press
[Align] when finished. The wavelength is now set.
4) On the top left of the instrument are 2 knobs for lamp alignment (front knob for horizontal, rear knob for vertical). Rotate either of the two knobs in any direction watching the sample bargraph, again trying to get the most energy you can in a manner similar to the wavelength alignment.
5) When the energy has been maximized, move to the other knob and repeat the procedure. Go back and forth between the two knobs until you can’t get any more energy. (NOTE: There will not be nearly the change in energy as there was with the wavelength). The lamp is set when you can’t get any more energy. Press [A/Z] to zero and exit to the analysis screen.
Align the burner (vertical)
vertical adjustment —> right knob (burner moves up & down)
vertical adjustment —> left knob (burner moves front to back)
6) With the flame and gasses off place a business card or similar surface on top of the burner so that you can see the lamp image on the card. Rotate the vertical adjust knob so that the bottom edge of the light at the focal point is approximately 4mm from the top of the burner (best position for most analysis). Adjust the horizontal if necessary to get the image over the burner head slot, this is only a rough adjustment for the horizontal.
(NOTE: Some elements may require different height settings especially when using
99 nitrous oxide, consult the standard conditions section for these instances). Another way to set the vertical position is to lower the burner head until it is clearly not blocking the beam. Perform an autozero by pressing the [a/z] key. Slowly raise the burner head while watching the absorbance display. When the burner head intersects the beam the number will suddenly go positive. As soon as the reaction is noticed, stop and lower the burner about 2 turns of the dial.
Gas Flow Setting
Note: The furnace must be activated using the Read or Start key before flows may be adjusted.
The Buck 220GF Power Unit has 4 flow meters on the front:
1) Sheath Gas is Argon flow outside the tube. It’s purpose is to prevent the Oxygen in the atmosphere from causing the rapid combustion of the Graphite Tube. The Sheath Gas should be set to 1.25 mL/min, or about half way up the scale.
2) Internal Flow is Argon flow inside the tube. The purpose is to remove unwanted compounds from the furnace tube as they are vaporized or ashed. The Internal Flow should be set to 200 uL/min. The internal flow is turned off during Atomize to allow data collection.
3) Mini-Flow is also Argon gas flow in the tube, but the flow remains ON during atomize. Mini-Flow is used to reduce the sensitivity of the Analyte and/or peak duration.
The flow is normally set to 50 uL/min to start, then increased to obtain the desired peak.
4) Alternate Internal Flow is the optional flow of another gas (possibly Oxygen or Air) used during the analysis of special types of samples.
100
Peak Area (Abs-sec) does not appear and replot last peak
You might want to change peak time from auto to fixed or vice versa, also set peak delay and set peak duration if experience no Abs-sec show on the screen.
In the [cntls] page the item furnace/vapor controls has been added as described below:
Peak Timing: The options are auto, armed and fixed.
Auto: The software will automatically determine the integration window for the peak during calibration. Armed: The software will set the window for the next reading. For best results, this setting should never be used for calibration. Fixed: The operator sets the peak delay and duration manually by observing the largest peak. Fixed settings are preferred for calibration when maximum sensitivity is needed.
Set Peak Delay (in seconds): The time from the Y-axis at which the integration begins.
Set peak Duration (in seconds): The length of time the integration will last.
Note: If you want to set your own values for delay and duration peak timing must be set to fixed
If you are setting the peak timing manually using the fixed timing mode, make sure that
Peak Timing is set to Fixed in the Furnace/Vapor Controls menu. Read your high standard using the [read] button. After the furnace program ends, Press [esc], then [cntls],
then highlight Furnace/Vapor Controls and press [enter]. Use the Replot Last Peak
function to examine the peak and select the proper peak delay and duration so that the
entire peak is integrated including the tail. Press [esc] then enter the values in for peak
delay and duration. You may return to replot last peak to check to see if the white bar,
which denotes the area of the curve which will be integrated, covers the entire peak.
101 APPENDIX E
This Appendix includes the MSDS sheets for all the chemical and reagents used in the experiments.
102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
125 126 127 128 129 130 131 132
133