Analysis of vitamin C in orange juice

Vitamin C (or ascorbic acid) is found in many fruits and vegetables.

Vitamin C participates in reactions that are required for the formation of the protein collagen.

When collagen is produced a series of events occur inside and outside of the cell. Vitamin C is an active inside of the cell and hydroxylates to the amino acids proline and lysine.

These help form a precursor molecule called procollagen that is later modified into collagen outside of the cell.

Without vitamin C the formation of collagen is disrupted, causing a wide variety of problems throughout the body.

Collagen is found wherever tissues require strengthening, especially in those tissues with a protective, connective or structural function.

Collagen is critical to the maintenance of bone and blood vessels and is essential for the healing of wounds.

Vitamin C aids red blood cell formation helping preventing haemorrhaging and fighting bacterial infections. Vitamin C can act as an antioxidant by donating electrons and hydrogen ions and reacting with reactive oxygen species or free radicals.

Vitamin C is important for the absorption of iron and reduces ferric iron to its ferrous form and is beneficial in the treatment of those suffering from the iron deficiency anaemia. Vitamin C is vital for the function of the immune system especially for the function of lymphocytes.

Vitamin C can be prepared by synthesis from glucose or extracted from plant sources such as rose hips, blackcurrants or citrus fruits.

Humans are among the few mammals that can not synthesise their own vitamin C, and so must ingest it.

Many foods are known for their ability to provide a source of vitamin C, but a number of factors - such as processing method, storage conditions, and exposure to light and heat - determine how much is actually present by the time the product is consumed.

In addition to its nutritional benefits, vitamin C is used as a photographic developing agent in alkaline solutions and is used industrially as a reducing agent in metallurgy.

The history of vitamin C.

Vitamin C was first isolated in 1928 by the Hungarian biochemist Albert Szent-Gyorgyi.

In 1937, Szent-Gyorgyi received the Nobel Prize in physiology and medical science for discoveries in the area of biological combustion processes, particularly with regard to vitamin C and fumaric acid catalysis.

Linus Pauling was the first to realise the value of vitamin C towards the maintenance of a healthy immune system and, in 1970, proposed that a regular intake at far higher levels than the recommended daily allowance (RDA) could help prevent and shorten the duration of the common cold.

The medical community immediately voiced their strong opposition to this theory, but many ordinary people followed the advice of Pauling and noticed a great reduction in the frequency and severity of their colds.

Certain recent medical advice does seemingly confirm Pauling's original idea that vitamin C can markedly reduce the severity of a cold and help prevent secondary viral or bacterial complications.

Vitamin C intake.

The European Community RDA of vitamin C currently stands at 60 milligrams per day.

All of us require vitamin C, but some people need more than others - such as those that have low nutrient diets, who are at risk from vitamin C deficiency.

Many elderly men consume low levels of fruits and vegetables and are also at risk.

Studies conducted have shown that 20% of elderly men have low levels of vitamin C.

In addition, smokers and those individuals exposed to cigarette smoke generally have lower levels of vitamin C, and may need to consume roughly twice as much vitamin C as non-smokers. Good sources of vitamin C include broccoli, brussel sprouts, cauliflower, cabbage, green leafy vegetables, red peppers, parsley, blackcurrants, strawberries, kiwi fruit, guavas and citrus fruit.

The highest amounts of vitamin C are found in citrus fruits and green vegetables bu,t because vitamin C is quickly lost in cooking and processing, fresh fruits are often a better source of nutrients.

In food, vitamin C can be partially or completely destroyed by overcooking or long periods of storage, as it is sensitive to heat, light and oxygen.

Pauling recommended a vitamin C intake of 1000 milligrams per day or more as opposed to the current RDA.

One should realise that the RDA is not based on what is required for optimum health, but is the amount required to avoid the most obvious deficiency disease. Many eminent medical and scientific experts are of the opinion that the value is currently too low to provide optimum health and protection against disease, especially as evidence continues to emerge about the important health benefits at higher levels than those once considered adequate to prevent scurvy.

Vitamin C has been used as an alternative therapy for many years and numerous doctors do not hesitate to recommend doses of 1-5 grams per day for certain individuals.

A team of researchers at the National Institute of Health in the United States recently completed a study to determine the vitamin C requirement of healthy, young adults.

It was found that a minimum intake of 1000 milligrams per day was required to completely saturate the blood plasma with vitamin C.

The studies recommended that the vitamin C should be taken in several doses throughout the day, as urinary excretion increased rapidly for doses above 500 milligrams.

The benefits of vitamin C.

The Scottish physician James Lind first advocated the use of fresh vegetables and ripe fruits back in 1753 to prevent scurvy, with the British Navy adopting his advice some 40 years later.

The navy men were later nicknamed Limeys as they took lime juice on long sea voyages to ward off scurvy.

A severe deficiency of vitamin C can lead to the onset of scurvy, a disease that causes swollen gums, loose teeth, a tendency of wounds to not heal and excessive bleeding as well as bone malformations in infants.

Fortunately, scurvy is rare nowadays in the UK due to the wide availability of foods that are rich sources of vitamin C.

Research conducted by the National Institute on Ageing reported that elderly people who took vitamin C and E supplements had a 50% lower risk of dying prematurely from disease than those individuals that did not use such supplements.

Numerous studies have shown that an adequate intake of vitamin C is effective in lowering the risk of developing cancers of the breast, cervix, colon, rectum, lung, mouth, prostate, and stomach.

Pauling and his colleagues at the Vale of Leven Hospital in Scotland carried out clinical studies on cancer patients subjecting them to large daily doses of vitamin C in addition to their regular treatment. They concluded that cancer patients who received doses of greater than 10 grams per day lived longer, suffered less pain and had in general, a better quality of life than similar patients who did not receive the vitamin C.

Pauling believed that vitamin C combats cancer by promoting collagen synthesis and thus preventing growing tumours from invading adjacent tissue.

Many researchers now believe that vitamin C prevents cancer by deactivating free radicals before they can damage DNA and initiate tumour growth, while others think that vitamin C acts as a pre-oxidant helping the bodies own free radical defence mechanism destroy tumours in their early stages.

Whatever the mechanism, it seems apparent that vitamin C may be effective in preventing cancer, alleviating its symptoms and in certain cases halting its progress for some individuals.

The work of Pauling and his peers concerning the benefits of vitamin C has not been without controversy, and for many years has divided medical opinion and it is not unreasonable to suggest that the debate will continue for many more years to come.

Research published in the medical journal Lancet found that a 500 milligram daily supplement of vitamin C could significantly reduce high blood pressure in hypertensive patients.

High blood pressure is a serious health problem in much of the world and a key risk factor in heart disease and strokes.

The studies, performed by Boston University School of Medicine and Linus Pauling Institute at Oregon University, found that vitamin C supplements reduced blood pressure without side effects, were inexpensive, and could yield reductions comparable to certain prescription drugs used to treat hypertension.

Patients were given 500 milligrams of vitamin C daily and after a month the systolic, diastolic and mean blood pressures had decreased by about 9%, enough to reduce a moderately high blood pressure back to a normal level. Voltammetry - a brief overview.

Jaroslav Heyrovsky first introduced the concept of polarography circa 1922.

The term voltammetry is applied to designate the current-voltage measurement obtained at a given electrode.

Polarography is a special case of voltammetry referring to the current-voltage measurement acquired using a dropping mercury electrode with a constant flow of mercury drops.

With the Metrohm Multi Mode Electrode (MME), the mercury is hermetically sealed in the reservoir and suffices for around 200,000 drops - ensuring low laboratory running costs.

The Dropping Mercury Electrode (DME) is an electrode mode of the MME.

It is the classical mercury electrode where the mercury flows freely from the glass capillary until the mercury drop is knocked off by a tapping mechanism after each voltage step time set in the measurement mode.

The polarographic determination of vitamin C.

Polarography is suitable not only for the quantitative determination of organic substances but also for a large number of organic compounds.

The determining factors as to whether organic substances can be determined in an aqueous medium depend primarily on the functional groups that characterise the redox properties.

These ascertain whether a redox reaction is possible at a given potential window of the mercury drop and whether the determination is based on the reduction or oxidation of the compound.

The determination is based on the oxidation of ascorbic acid to dehydroascorbic acid:.

C6H8O6 - C6H6O6 + 2e- + 2H+.

The redox potential depends on the pH and without adequate buffering the pH at the electrode surface can be displaced by the oxidation reaction of the ascorbic acid leading to peak broadening. Degradation of vitamin C in orange juice.

A report conducted by the Journal of the American Dietetic Association recommended that orange juice should be consumed as soon as possible after purchase or making a can from frozen concentrate, as the vitamin C content decreases the closer one gets to the expiry date.

The study was performed by Arizona State University on different brands of orange juice in different types of containers: with screw top caps, those which open and close like traditional milk cartons, and the frozen kind that are reconstituted with water. The analysis found that generally frozen reconstituted orange juice contained more vitamin C than ready to drink type juice, and screw top containers contained more vitamin C at opening than that found in milk-like containers.

The level of nutrient from frozen concentrate fell from 65 milligrams per serving to 45 milligrams two weeks later, and after four weeks to about 36 milligrams - a fraction of the figure stated on the label. Fortunately orange juice has plenty of other nutrients that do not degrade so quickly, including folic acid, potassium, vitamin A, and trace minerals, so plenty of other nutritional value is being added to the human diet as orange juice is consumed by a large majority of the western world on a regular basis.

The study concluded that the vitamin C content of orange juice was variable and dependant on the variety and maturity of the oranges, fresh fruit handling, processing factors and packaging.

Pasteurised, ready to serve orange juices were found to contain typically 25% less vitamin C per serving than frozen concentrates, caused in part by heat destroying the vitamin C.

It is a fact the majority of food manufacturers add considerably more vitamin C than the labelled value to compensate for the losses that occur due to degradation.

Method for analysis of vitamin C in orange juice. 10ml of deionised water, 1ml of acetate buffer (pH 4.64) and 0.5ml of orange juice sample were added to the reaction vessel in the Metrohm 757 VA Computrace.

The role of the electrolyte and additional solutions in voltammetry is crucial as many determinations are pH dependent and the electrolyte can increase the conductivity and selectivity of the solution.

The solution was then degassed with nitrogen for a period of five minutes to remove the electrochemically active oxygen, before the vitamin C content was determined with two standard additions using the DME. Automation with voltammetry.

For a large numbers of samples it is possible to automate the process of voltammetry through the use of an autosampler, pump units and liquid dosing units known as dosimats.

The Metrohm 813 compact autosampler allows fully automatic precise, reproducible analysis of multiple samples of a similar nature and can accommodate a maximum of 18 samples that are transferred using a peristaltic pump to the measuring vessel of the Computrace.

The analyte concentration is determined by means of automatic standard additions carried out by Metrohm 765 Dosimats.

After each sample the measurement vessel is emptied and rinsed by two Metrohm 772 pump units.

The sample data is entered into a sample queue that is automatically processed by the 757 software.

Conclusion of analysis of vitamin C in orange juice. The determination of vitamin C in a variety of food related matrices has been well documented for the likes of fruit juices, fresh produce and canned products.

The polarographic method is straightforward, requiring no sample extraction or handling unless the sample is in a solid form, saving the user time as the usual procedure is simply to dilute the sample and then run the voltammogram.

Often these types of food products will contain coloured material or dyes that generally do not interfere with voltammetric analyses.

Over recent years, voltammetry has undergone a tremendous surge in popularity and today represents a refined, clean simple technique that offers outstanding limits of detection and is now the fastest growing analytical technique for trace analysis and is eminently suited to the vitamin C analysis of orange juice.

Voltammetry is an increasingly popular technique that in many instances offers unrivalled detection limits even when compared to vastly more expensive analytical techniques.

For samples of orange juice, then, voltammetry often requires little or no sample preparation and the result determined by standard addition obtained in less than ten minutes.

The advantage of using standard addition as a means of calibration and quantification is that matrix effects present in the sample are taken into account.

Voltammetry requires no specialist laboratory infrastructure like expensive fume extraction; all that is required is a sturdy bench top on which to mount the instrument and a regulated flow of an inert gas.

The running and maintenance costs of voltammetry are minimal, ensuring a cost effective analytical solution to surpass the demands required by those organisations interested in quantifying the levels of vitamin C present in orange juice.