Description
Abstract
A UV method for the analysis of ascorbic acid with methanol as solvent to prepare a sample has been developed and applied. The effect of copper(II) concentrations on the oxidation of ascorbic acid in aqueous solution has been studied in detail, and the regularities of ascorbic acid oxidation in methanol, USP phosphate buffer (pH 2.50) and de-ionized water have been found. Upon experiments ascorbic acid has been found to dissolve in methanol, and its solubility in it has been measured to be 81.0mg/ml at room temperature (22 degrees C). The ascorbic acid bulk material from a manufacturer has been assayed to be 89.34% with this method, in good agreement with the assay value (89.58%) from the titration method. The ascorbic acid granule and tablet content uniformity also has been tested using this method. This method is simple, rapid, accurate and reliable, and can be adopted for the routine determination of ascorbic acid in its granule and tablet formulations.
CHAPTER ONE
- INTRODUCTION
- BACKGROUND OF THE STUDY
- OBJECTIVE OF THE STUDY
- PURPOSE OF THE STUDY
- PURPOSE OF THE STUDY
- IMPORTANCE OF ASCORBIC ACID
CHAPTER TWO
2.1 LITERATURE REVIEW
2.2 REVIEW OF ULTRAVIOLET–VISIBLE SPECTROSCOPY
2.3 METHODOLOGY
2.4 MATERIALS AND METHODS
2.5 STANDARD PREPARATION
2.6 SAMPLE PREPARATION
2.7 HPLC-UV METHOD
2.8 CALIBRATION PLOTS
2.9 METHOD VALIDATION
CHAPTER THREE
3.1 RESULTS AND DISCUSSION
3.2 CONCENTRATION OF ASCORBIC ACID IN BH EXTRACTED WITH DIFFERENT SOLVENTS
3.3 VALIDATION OF THE METHOD
CHAPTER FOUR
4.1 CONCLUSION
4.2 SUMMARY
4.3 REFERENCES
CHAPTER ONE
2.0 INTRODUCTION
2.1 BACKGROUND OF THE STUDY
Ascorbic acid or vitamin C is mostly found in natural products such as fruits and vegetables. High performance liquid chromatography (HPLC) method has been developed and validated to compare the ascorbic acid content in Benincasa hispida (Bh) fruit extract with three different extraction solvents; i) 3% metaphosphoric acid, ii) 3% citric acid and iii) distilled water. The compound has been detected and quantified by the use of HPLC coupled with UV-Vis detector. The amount of ascorbic acid detected in Bh fruit extract prepared with different extraction solvents; 3% metaphosphoric acid, 3% citric acid and distilled water were 13.18, 7.91 and 9.42 mg/100g respectively. Total run time was 6 min and the retention time was 2.60 min. Calibration curve was linear with the concentration range 1.00 – 16.00 μg/ml. Limits of detection was 0.24 μg/ml, limit of quantification was 0.81 μg/ml and recovery was 93.52%. The result showed ascorbic acid content is higher in Bh fruit extract with 3% metaphosphoric acid, followed by extract with distilled water and 3% citric acid. Thus, Bh is another novel fruit/ vegetable potentially used as food ingredient as it contains a good source of ascorbic acid that can be good for one’s health.
Benincasa hispida (Bh) commonly known as ash gourd or winter melon is from the Cucurbitaceae family. It has been valued as a healthful vegetable as it is good source of natural sugars, amino acids, organic acids, mineral elements and vitamins (Zaini et al., 2010). Nutritionally, Bh fruit contains major contents of water with minor amounts of protein, fat and ash. Edible portion of Bh flesh recorded the highest vitamin C and riboflavin content. Previous study showed the presence of triterpenes, catechins, carotenes, tannins, uronic acids and polyphenols in Bh extracts (Gill et al., 2010)
Fruits have large amounts of potentially interfering compounds. Analysis of ascorbic acid (Figure 1) from specific plant tissue types need great caution in the use of methods that have been developed (Davey et al., 2000). Ascorbic acid will be oxidized under alkaline conditions. The use of a high ionic strength, acidic extraction solvent is required to block metabolic activity upon disruption of the cell and to precipitate proteins.
Ascorbic acid, also known as vitamin C (C6H8O) is widely distributed in nature, typically rich in fresh fruits and leafy vegetables such as guava, mango, papaya, cabbage, mustard leaves and spinach (Tee et al., 1997). Ascorbic acid is an antioxidant but it is the least stable of all vitamins and is easily oxidized during processing and storage. Juices are good foods to be fortified with ascorbic acid because their acidity reduces ascorbic acid destruction. Exposure to oxygen, prolonged heating in the presence of oxygen, and the presence of oxidizing enzyme, contact with minerals (iron and copper) and exposure to light are destructive to the ascorbic acid content of foods. Dehydroascorbic acid is an oxidized form of ascorbic acid. In this study, only ascorbic acid was measured since at harvest, dehydroascorbic acid represents less than 2% of total vitamin C (Wills et al., 1984).
Figure 1. Chemical structure of ascorbic acid
Normally, the method used to determine ascorbic acid is based on the reduction of the blue dye 2,6 dichlorophenolindophenol by ascorbic acid (AOAC, 1999). The endpoint of the titration is indicated by the appearance of the pink acid form of the dye. This is a simple and fast method (De Assis et al., 2001). Alternatively, analysis of ascorbic acid by HPLC allows the determination of ascorbic acid in an easy, fast, and precise method. HPLC is considered a sensitive and selective method and therefore suitable for active substance determination; it is also suitable for the evaluation of stability in formulations in the pharmaceutical and cosmetic industries (Marshall et al., 1995). In addition, some studies reported liquid chromatography can avoid the problem of non-specific interference and ion-pair (Ke et al., 1994) NH2 bonded-phase (Silva, 2005) and reverse phase (Franke et al., 2004) techniques.
OBJECTIVE OF THE STUDY
Ascorbic acid, also known as vitamin C (C6H8O) is widely distributed in nature, typically rich in fresh fruits and leafy vegetables such as guava, mango, papaya, cabbage, mustard leaves and spinach. The objective of this work is to determined ascorbic acid in some fruits such as guava, mango, papaya, cabbage, mustard leaves and spinach
PURPOSE OF THE STUDY
The purpose of this study was to determine, develop and validate a uv method for the quantitation of ascorbic acid in Bh extract in different extraction solvents.
APPLICATIONS OF THE STUDY
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.
- Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the colour and changes the wavelength of maximum absorption (λmax).
- Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
- While charge transfer complexes also give rise to colours, the colours are often too intense to be used for quantitative measurement.
The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.
A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response assumed to be proportional to the concentration. For accurate results, the instrument’s response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the response factor.
The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule. The Woodward-Fieser rules, for instance, are a set of empirical observations used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones. The spectrum alone is not, however, a specific test for any given sample. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectrum. Experimental variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the spectrum. To apply UV/Vis spectroscopy to analysis, these variables must be controlled or accounted for in order to identify the substances present.
UV-Vis spectroscopy is also used in the semiconductor industry to measure the thickness and optical properties of thin films on a wafer. UV-Vis spectrometers are used to measure the reflectance of light, and can be analyzed via the Forouhi-Bloomer dispersion equations to determine the Index of Refraction (n) and the Extinction Coefficient (k) of a given film across the measured spectral range.
IMPORTANCE OF ASCORBIC ACID
Ascorbic acid, also known as vitamin C, is a nutrient that plays a crucial role in maintaining optimal health. Vitamin C is often present in multivitamin supplements and abundant in many fruits and vegetables such as strawberries, oranges and orange juice, grapefruit and grapefruit juice, kiwi fruit, cantaloupe, red and green peppers, broccoli, cabbage, Brussels sprouts and tomato juice. Getting plenty of vitamin C daily is a must for your body to function properly. Vitamin C plays a role in protecting against the following:
Heart Disease
Results of scientific studies on whether vitamin C is helpful for preventing heart attack or stroke are mixed. Vitamin C doesn’t lower cholesterol levels or reduce the overall risk of heart attack, but evidence suggests that it may help protect arteries against damage.
Some studies — though not all — suggest that vitamin C, acting as an antioxidant, can slow down the progression of atherosclerosis (hardening of the arteries). It helps prevent damage to LDL (“bad”) cholesterol, which then builds up as plaque in the arteries and can cause heart attack or stroke. Other studies suggest that vitamin C may help keep arteries flexible.
In addition, people who have low levels of vitamin C may be more likely to have a heart attack, stroke, or peripheral artery disease, all potential results of having atherosclerosis. Peripheral artery disease is the term used to describe atherosclerosis of the blood vessels to the legs. This can lead to pain when walking, known as intermittent claudication.
The best thing to do is get enough vitamin C through your diet. That way, you also get the benefit of other antioxidants and nutrients contained in food. If you have low levels of vitamin C and have trouble getting enough through the foods you eat, ask your doctor about taking a supplement.
High Blood Pressure
Population based studies (which involve observing large groups of people over time) suggest that people who eat foods rich in antioxidants, including vitamin C, have a lower risk of high blood pressure than people who have poorer diets. Eating foods rich in vitamin C is important for your overall health, especially if you are at risk for high blood pressure. The diet physicians most frequently recommend for treatment and prevention of high blood pressure, known as the DASH (Dietary Approaches to Stop Hypertension) diet, includes lots of fruits and vegetables, which are loaded with antioxidants.
Common Cold
Despite the popular belief that vitamin C can cure the common cold, the scientific evidence doesn’t support the notion. Taking vitamin C supplements regularly (not just at the beginning of a cold) produces only a small reduction in the duration of a cold (about 1 day). The only other piece of evidence supporting vitamin C for preventing colds comes from studies examining people exercising in extreme environments (athletes such as skiers and marathon runners, and soldiers in the Arctic). In these studies, vitamin C did seem to reduce the risk of getting a cold.
Cancer
Results of many population based studies (evaluating groups of people over time) suggest that eating foods rich in vitamin C may be associated with lower rates of cancer, including skin cancer, cervical dysplasia (changes to the cervix which may be cancerous or precancerous, picked up by pap smear), and, possibly, breast cancer. But these foods also contain many beneficial nutrients and antioxidants, not only vitamin C, so it’s impossible to say for certain that vitamin C is protecting against cancer. Taking vitamin C supplements, on the other hand, has not been shown to have any helpful effect.
In addition, there is no evidence that taking large doses of vitamin C once diagnosed with cancer will help your treatment. Moreover, some doctors are concerned that large doses of antioxidants from supplements could interfere with chemotherapy medications. More research is needed. If you are undergoing chemotherapy, talk to your doctor before taking vitamin C or any supplement.
Osteoarthritis
Vitamin C is essential for the body to make collagen, which is a part of normal cartilage. Cartilage is destroyed in osteoarthritis (OA), putting pressure on bones and joints. In addition, some researchers think free radicals — molecules produced by the body that can damage cells and DNA — may also be involved in the destruction of cartilage. Antioxidants such as vitamin C appear to limit the damage caused by free radicals. However, that said, no evidence suggests that taking vitamin C supplements will help treat or prevent OA. What the evidence does show is that people who eat diets rich in vitamin C are less likely to be diagnosed with arthritis.
Taking nonsteroidal anti-inflammatory drugs can lower your levels of vitamin C. If you take these drugs regularly for OA, you might want to take a vitamin C supplement.
Age-related Macular Degeneration
Vitamin C (500 mg) appears to work with other antioxidants, including zinc (80 mg), beta-carotene (15 mg), and vitamin E (400 IU) to protect the eyes against developing macular degeneration (AMD), the leading cause of legal blindness in people over 55 in the United States. The people who seem to benefit are those with advanced AMD. It isn’t known whether this combination of nutrients helps prevent AMD or is beneficial for people with less advanced AMD. This combination includes a high dose of zinc, which should be taken only under a doctor’s supervision.
Pre-eclampsia
Some studies suggest that taking vitamin C along with vitamin E may help prevent pre-eclampsia in women who are at high risk. Pre-eclampsia, characterized by high blood pressure and too much protein in the urine, is a common cause of premature births. Not all studies agree, however.
Asthma
Studies are mixed when it comes to the effect of vitamin C on asthma. Some show that low levels of vitamin C are more common in people with asthma, leading some researchers to think that low levels of vitamin C might increase the risk for this condition. Other studies seem to show that vitamin C may help reduce symptoms of exercise-induced asthma.
Other
Although the information is limited, studies suggest that vitamin C may also be helpful for:
- Boosting immune system function
- Maintaining healthy gums
- Improving vision for those with uveitis (an inflammation of the middle part of the eye)
- Treating allergy-related conditions, such as asthma, eczema, and hay fever (called allergic rhinitis)
- Reducing effects of sun exposure, such as sunburn or redness (called erythema)
- Alleviating dry mouth, particularly from antidepressant medications (a common side effect from these drugs)
- Healing burns and wounds
- Decreasing blood sugar in people with diabetes
- Some viral conditions, including mononucleosis — Although scientific evidence is lacking, some doctors may suggest high-dose vitamin C to treat some viruses
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