Spectrophotometric Determination of Reducing and Non- Reducing Sugar in Commercial Sugar Samples

Spectrophotometric Determination of Reducing and Non- reducing Sugar in Commercial Sugar Samples

Mona Abdalgaum Alnour Ahmed

B.Sc. (Hons.) in Chemistry, University of Khartoum (2010)

A Dissertation

Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science

Chemistry Department of Applied Chemistry and Chemical Technology Faculty of Engineering and Technology

November, 2013

Spectrophotometric Determination of Reducing and Non- reducing Sugar in Commercial Sugar Samples

Mona Abdalgaum Alnour Ahmed

Supervision Committee :

Name Position Signature

Prof. Alnaeim Abdalla Ali Main Supervisor Dr. Mohammed Osamn Babiker Ahmed Co-supervisor

Date: November 2013

Spectrophotometric Determination of Reducing and Non- reducing Sugar in Commercial Sugar Samples

Mona Abdalgaum Alnour Ahmed

Examination Committee:

Name position Signature

Prof. Alnaeim ABdalla Mohamed Chair Person Dr. Shama Elamin Daw Elbeit External Examiner Dr. Mustafa Ohag Mohamed Internal Examiner

Date of Examination: 24 / 11 /2013

Dedication

To my Family

My father

Mother and sister

Brothers and all my friends

Acknowledgment

I send my heart full thanks to all those who contributed to the completion of this research especial thanks to my supervisor prof.

Alnaeim Abdalla for his great help in all steps of this research.

Also I want to thank the Biochemistry lab staff in Agricultural researcher corporation ,.

My thanks also extends to the faculty of Engineering &

Technology university of Gezira for offering me the chance to pursue post graduate studies. Eventually my thanks to my friend

Sara Mustafa for her help

Spectrophotometric Determination of Reducing and Non-Reducing Sugar in Commercial Sugar Samples Mona Abdalgaum Alnour Ahmed Master of Science in Chemistry 2014 Department of Applied Chemistry and Technology Faculty of Engineering and Technology University of Gezira Abstract Sucrose or table sugar is a non-reducing disaccharide containing the monosaccharides glucose and fructose linked by carbon atom 1 of glucose containing the aldhydic group and carbon atom 2 of fructose containing the ketonic group. The sucrose content of commercial sugar must not be less than 99.5% while the reducing sugar content must not be more than 0.1%. the aim of this work is the determination of the reducing sugars and non-reducing sugar in commercial sugar spectrophotometrically using the modified Nelson method in which the Cu+2 in alkaline solution was reduced quantitatively by glucose (reducing agent) to Cu+1 which reduced the Mo+6 in ammonium molybdate to Mo+5 to give a blue color. The spectrophotometer was used to determine the absorbance at 600 nm and from which the % of sugars was calculated. Four commercial sugar samples were collected from Wad Medani local market, two samples from Kenana and Gunied and the other two were imported from Brazil and India. The results indicated that the four samples contain 0.1 -0.23% reducing sugar and 99.27-99.9% sucrose in conformity with standard specifications of table sugar. The Gunied sample contains slightly higher percentage of reducing sugar compared to the other samples. The method is simple and easy to run and hence it is very suitable for determination of soluble sugars in commercial sugar samples and it is recommended that other sugar brands should be analyzed in the same way.

التحديد الطيفي للسكريات المختزلة وغير مختزلة في عينات السكر التجاري منى عبد القيوم النور أحمد ماجستير الكيمياء التطبيقية 4102م قسم الكيمياء التطبيقية وتكنولوجيا الكيمياء كلية الهندسة والتكنولوجيا جامعة الجزيرة ملخص الدراسة

List of Contents:

page Dedication III Acknowledgment IV English Abstract V Arabic Abstract VI List of contents VII List of tables IX List of figures X Chapter one 1-1 Carbohydrate compound 1 1-1-1 Carbohydrate chemical structure 2 1-1-2 Carbohydrate classification 2 1-2 Sugar 3 1-2-1 History of sugar 4 1-2-2 Chemistry of sugar 7 1-2-3 Natural polymer of sugar 7 1-2-4 Types of sugar 8 1-3 What is sucrose 11 1-3-1 Invert sugar 11 1-3-2 Hydrolysis of sucrose 12 1-3-3 Metabolism of sucrose 12 1-3-4 Physical and Chemical Properties of sucrose 13 1-3-5 Synthesis and biosynthesis of sucrose 14 1-4 Production of sugar 14 1-4-1 Sugar beet 14 1-4-2 Sugar cane 14 1-4-3 Sugar Processing 15 1-4-4 Refining between sugar beet & sugar cane 16 1-4-5 Forms and uses of sugar 16 1-5 Consumption of sugar 17 1-6 Health effect of sugar 18 1-7 Methods of analysis of carbohydrate 20 8

1-7-1 Chromatographic and Electrophoretic 20 methods 1-7-2 Chemical Methods 21 1-7-3 Physical Methods 24 The objective of the work 26 Chapter two Materials and methods 2-1 Sample collection and preparation 27 2-2 Apparatus 27 2-3 Chemicals 27 2-4 Preparation of solution 28 2-5 Procedure 30 Chapter three (Result and Discussion) Result of concentration of reducing sugar 33 Result of concentration of total soluble sugar 34 Soluble sugar in commercial sugars sample 34 Discussion 34 Conclusion 35 References 36

List of table

No Name of table Page 2-1 Types, colour, origin of sugar sample 27 2-2 Absorbance of the reference solution 31 3-1 Concentration of reducing sugars 33 3-2 Concentration of total soluble sugars 34 3-3 Soluble sugar in commercial samples 34

List of figures

No Name of figure Page 3-1 Calibration curve of standard solution of glucose 32

Chapter one Introduction 1-1 Carbohydrate compounds

Carbohydrates are the most abundant class of organic compounds found in living organisms. They originate as products of photosynthesis, an endothermic reductive condensation of carbon dioxide requiring light energy and the pigment chlorophyll.

n CO2 + n H2O + energy CnH2nOn + n O2

As noted here, the formulae of many carbohydrates can be written as carbon hydrates, Cn (H2 O)n hence their name. The carbohydrates are a major source of metabolic energy, both for plants and animals that depend on plants for food. Aside from the sugars and starches that meet this vital nutritional role, carbohydrates also serve as a structural material (cellulose), a component of the energy transport compound Adenosine Triphosphate(ATP), recognition sites on cell surfaces, and one of three essential components of Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). (Kovac and Saksena, 2008).

Carbohydrates are one of the main types of nutrients. They are the most important source of energy for your body. Your digestive system changes carbohydrates into glucose (blood sugar). Your body uses this sugar for energy for your cells, tissues and organs. It stores any extra sugar in your liver and muscles for when it is needed.

Carbohydrates are called simple or complex, depending on their chemical structure. Simple carbohydrates include sugars found naturally in foods such as fruits, vegetables, milk, and milk products. They also include sugars added during food processing and refining. Complex carbohydrates include whole grain breads and cereals, starchy vegetables and legumes. Many of the complex carbohydrates are good sources of fiber. (Aldrich, et al. , 2008).

1-1-1 Carbohydrates –Chemical Structure:

Carbohydrates consist of the elements carbon (C), hydrogen (H) and oxygen (O) with a ratio of hydrogen twice that of carbon and oxygen.Carbohydrates include sugars, starches, cellulose and many other compounds found in living organisms. In their basic form, carbohydrates are simple sugars or monosaccharaides. These simple sugars can combine with each other to form more complex carbohydrates. The combination of two simple sugars is a disaccharide. Carbohydrates consisting of two to ten simple sugars are called oligosaccharides, and those with a larger number are called polysaccharides, (Meng, et al., 2008)

1-1-2 Carbohydrate Classification:

There are a variety of interrelated classification schemes. The most useful classification scheme divides the carbohydrates into groups according to the

- number of individual simple sugar units.

Monosaccharaides contain a single unit of sugar.

Disaccharides contain two sugar units

Polysaccharides contain many sugar units as in polymers (most contain glucose as the monosaccharide unit).

Carbohydrates Monosaccharide’s Disaccharides Polysaccharides Glucose Sucrose Starch Galactose Maltose Glycogen Fructose Lactose Cellulose Ribose Glyceraldehyde

Number of Carbons:

Monosaccharide’s can be further classified by the number of carbons present. Hexoses (6-carbones) are by far the most prevalent.

Number of Carbons Six = Hexose Five = Pentose Three = Triose Glucose Ribose Glyceradehyde Galactose Fructose

Functional Groups:

 Aldoses contain the aldehyde group – Monosaccharide’s in this group are glucose, galactose, ribose, and glyceraldehyde.  Ketoses contain the ketone group. The major sugar in this group is fructose.

 Reducing: Contain a hemiacetal or hemiketal group. Sugars include glucose, galactose, fructose, maltose, lactose.

 Non-reducing: contain no hemiacetal groups. Sucrose and all polysaccharides in this groups. (Pigman, et al., 1972)

1-2 Sugar:

Sugar is the generalized name for a class of chemically-related sweet-flavored substances, most of which are used as food. They are carbohydrates, composed of carbon, hydrogen and oxygen. There are various types of sugar derived from different sources. Simple sugars are called monosaccharaides and include glucose (also known as dextrose), fructose and galactose. The table or granulated sugar most customarily used as food is sucrose, a disaccharide (in the body, sucrose hydrolyses into fructose and glucose). Other disaccharides include maltose and lactose. Chemically-different substances may also have a sweet taste, but are not classified as sugars. Sugars are found in the tissues of most plants but are only present in sufficient concentrations for efficient extraction in sugarcane and sugar beet.( wikipedia.org/wiki/Sruga) 13

The average person consumes about 24 kilograms of sugar each year (33.1 kg in industrialized countries), equivalent to over 260 food calories per person, per day. Sugar provides energy but no nutrients. Since the latter part of the twentieth century, it has been questioned whether a diet high in sugars, especially refined sugars, is bad for health. Sugar has been linked to obesity and suspected of being implicated in diabetes, cardiovascular disease, dementia, macular degeneration and tooth decay. Numerous studies have been undertaken to try to clarify the position but the results remain largely unclear, mainly because of the difficulty of finding populations for use as controls that do not consume sugars. (Abbott, 2009).

1-2-1 History of Sugar:

Ancient times and middle Ages:

Sugar has been produced in the subcontinent since ancient times. It was not plentiful or cheap in early times and honey was more often used for sweetening in most parts of the world. Originally, people chewed raw sugarcane to extract its sweetness. Sugarcane was a native of tropical South Asia and Southeast Asia. Different species seem to have originated from different locations with Soccharum barberi originating in India and S. Edules and S. Officnarum coming from New Guinea.

One of the earliest historical references to sugarcane is in Chinese manuscripts dating back to 8th century BC which mention the fact that the use of sugarcane originated in India. It appears that in about 500 BC, residents of present-day India began making

sugar syrup and cooling it in large flat bowls to make crystals that were easier to store and transport than the cane itself.

Sugar remained relatively unimportant until the Indians discovered methods of turning sugarcane juice into granulated crystals that were easier to store and to transport. Crystallized sugar was discovered by the time of the Imperial Guptas, around 5th century AD. Indian sailors, who carried clarified butter and sugar as supplies, introduced knowledge of sugar on the various trade routes they travelled. China then established its first sugarcane plantations in the seventh century. Chinese documents confirm at least two missions to India, initiated in 647 AD, to obtain technology for sugar-refining. In south Asia, the Middle East and China, sugar became a staple of cooking and desserts. (George Rolph, 1873).

The triumphant progress of Alexander the Great was halted on the banks of the Indus River by the refusal of his troops to go further east. They saw people in the Indian subcontinent growing sugarcane and making granulated, slat-like sweet powder. On their return journey, the Macedonian solders carried the “honey bearing reeds” home with them Sugarcane remained a little known crop in Europe for over a millennium, sugar a rare commodity, and traders of sugar wealthy. Crusaders brought sugar home with them to Europe after their campaigns in the Holy Land, where they encountered caravans carrying “sweet slat”. Early in the 12th century, Venice acquired some villages near Tyre and set up estates to produce sugar for export to Europe, where it supplemented honey which had previously been the only available sweetener. Crusase chronicler William of Tyre, writing in the late 12th century, described sugar as “very necessary for the use and health of mankind”. In the 15th century, Venice was the chief sugar refining and distribution center in Europe. (Ahmed Y. Hassan, 2007).

Modern History:

In August 1492, Christopher Columbus Stopped at La Gomera in the Canary Islands, for wine and water, intending to stay only four days. He became romantically involved with the Governor of the island, Breatriz d e Bobadilla y Ossorio, and stayed a

month. When he finally sailed she gave him cuttings of sugarcane, which became the first to reach the New World. Sugar was a luxury in Europe prior to the 18th century when it became more widely available. It then became popular and by the 19the century it was considered a necessity. This evolution of taste and demand for sugar as an essential food ingredient unleashed major economic and social changes. It drove, in part, colonization of tropical islands and nations where labor-intensive sugarcane plantations and sugar manufacturing could thrive. The demand for cheap and docile labor to perform the hard work involved in its cultivation and processing drove first, the slave trade from Africa ( in particular West Africa), followed by the indentured labor trade from south Asia (n particular India). Millions of slave and indentured laborers were brought into the Caribbean. Indian Ocean, Pacific Islands, East Africa, Natal, North and eastern parts of South America, and southeast Asia. The modern ethnic mix of many nations that have been settled in the last two centuries has been influenced by sugar. (Henry et al. , 2003). Sugar also led to some industrialization of former colonies. For example, Lieutenant J. Paterson, of the Bengal establishment, persuaded the British Government that sugar cane could be cultivated in British India with many advantages and at less expense than in the West Indies. As a result, a number of sugar factories were established in Bihar in eastern India (Moxham, and Roy., 2001). During the Napoleonic Wars, sugar beet production increased in continental Europe because of the difficulty of importing sugar at times in which shipping was subject to blockade. By 1880. Sugar beet was the main source of sugar in Europe though the United Kingdom continued to import the main part of its sugar from its colonies. Until the late nineteenth century, sugar was purchased in loaves, which had to be cut using implements called “nips” while in later years bags of sugar became more common. The first inventor of a process to make sugar in cube form was Moravian Jakub Krystof Rad, director of a sugar company in Dacice where sugar cube production began when he was granted a five-year patent for the invention on January 23, 1843. Henry Tate of Tate & Lyle was another early manufacturer of sugar cubes at his refineries in Liverpool and London. Tate prochased a patent for sugar cube manufacture from German 16

Eugen Langen, who had invented a different method of processing of sugar cubes in 1872. (Kenneth., 2012).

1-2-2 Chemistry of Sugar:

Scientifically, sugar loosely refers to a number of a carbohydrates, such as monosaccharide’s, disaccharides, or oligosaccharides. Monosaccharides are also called “simple sugars” the most important being glucose. Almost all sugars have the formula

CnH2nOn (n is between 3 and 7). Glucose has the molecular formula C6H12O6. The names of typical sugars end with “=ose.” As in “glucose”, “dextrose”, and “fructose”. Sometimes such words may also refer to any types of carbohydrates soluble in water. The acyclic mono-and disaccharides contain either aldehyde groups or ketone groups. These carbon-oxygen double bonds (C=O) are the reactive centers. All saccharides with more than one ring in their structure result from two or more monosaccharide’s joined by glycosidic bonds with the resultant loss of a molecule of water (H2O) per bond. Monosaccharide’s in a closed-chain can form glycosides bonds with other monosaccharide’s, creating disaccharides (such as sucrose) and polysaccharides (such as starch). Enzymes must hydrolyze or otherwise break these glycosidic bonds before such compounds become metabolized. After digestion and absorption the principal monosaccharide’s present in the blood and internal tissues include glucose, fructose, and glactose. Many pentose’s and hexoses can form ring structures. In these closed-chain forms, the aldehyde or ketone group remains non-free, so many of the reactions typical of these groups cannot occur. Glucose in solution exists mostly in the ring form at equilibrium, with less than 0.1% of the molecules in the open-chain form. (Mare Aronson and Marina Budhos, 2010).

1-2-3 Natural Polymers of Sugars:

Biopolymers of sugars are common in nature. Through photosynthesis plants produce glucose, which has the formula C6H12O6 and convert it for storage as an energy reserve in the form of other carbohydrates such as starch, or (as in cane and beet) as sucrose, , with the chemical formula C12 H22 O11. Starch, consisting of two different

polymers of glucose, is a readily degradable form of chemical energy stored by cells ,and can be converted to other types of energy . Another polymer of glucose is cellulose which is a liner chain composed of several hundred or thousand glucose units. It used by plant as structural component in their cell walls. Human can only digest cellulose to a very limited extent, though ruminants can do so with the help of symbiotic Bactria in their gut. DNA and RNA are built up of the monosaccharide deoxyribose and ribose respectively . deoxyribose has the formula (C5H10O4) and ribose the formula (C5H10O5). (Hynes, et al. 1991).

1-2-4 Types of Sugar:

Monosaccharides:

Glucose, fructose and glactose are all simple sugars, monosaccharaides, with the general formula C6 H12O6. They have five hydroxyl groups (-OH) and a carbonyl group (C=O) and are cyclic when dissolved in water. They each exist as several isomers with dextro- and laevo-rotatory forms which cause polarized light to diverge to the right or the left.

Glucose, dextrose or grape sugar occurs naturally in fruits and plant juices and is the primary product of photosynthesis. Most ingested carbohydrates are converted into glucose during digestion and it is the form of sugar that is transported round the bodies of animals in the bloodstream. It can be obtaind from starch by the addition of enzymes or in the presence of acids. Glucose syrup is a liquid form of glucose that is widely used in the manufacture of foodstuffs. It can be manufactured from starch by enzymatic hydrolysis.( Fred ,2006)

Fructose or fruit sugar occurs naturally in fruits, some root vegetables, cane sugar and honey and is the sweetest of the sugars. It is one of the components of sucrose or table sugar. It is used as high fructose syrup which is manufactured from hydrolyzed corn starch which has been processed to yield corn syrup, with enzymes then added to convert part of the glucose into fructose.

Calactose does not generally occur in the free State but is a constituent with glucose of the disaccharide lactose or milk sugar. It is less sweet than glucose. It is a component of the antigens found on the surface of red blood cells that determine blood groups. ( Kretchmer ,1991 )

Disaccharides:

Sucrose, maltose and lactose are all disaccharides, with the general formula

C12H22O11. They are formed by the combination of two monosaccharide molecules with the exclusion of a molecule of water.

Sucrose is found in the stems of sugar cane and roots of sugar beet. It also occurs naturally alongside fructose and glucose in other plants, particularly fruits and some roots such as carrots. The different proportion of sugars found in these foods determines the range of sweetness experienced when eating them. A molecule of sucrose is formed by the combination of a molecule of glucose with a molecule of fructose. After being eaten, 19

sucrose is split into its constituent parts during digestion by a number of enzymes known as sucrases.

Maltose is formed during the germination of certain grains, most notably barley which is converted into malt, the source of the sugar’s name. a molecule of maltose is formed by the combination of two molecules of glucose. It is less sweet than glucose, fructose or sucrose. It is formed in the body during the digestion of starch by the enzyme amylase and is itself broken down during digestion by the enzyme maltase.

Lactose is the naturally occurring sugar found in milk. A molecule of lactose is formed by the combination of a molecule of galactose with molecule of glucose. It is broken down when consumed into its constituent parts by the enzyme lactase during digestion. Children have this enzyme but some adults no longer form it and they are unable to digest lactose.(Garrett and Grisham.. 1999)

1-3 what is Sucrose?

Sucrose, the technical name for table sugar, cane sugar, or white sugar, is made of one glucose molecule and one fructose molecule bound together. It comes in powdered and granulated forms, the sugar is made from highly processed form of sugar beet or sugar cane plant extracts.

Table sugar is high in calories but has zero nutritional values, that is, it supplies energy, but virtually nothing else that builds or rebuilds the body. Excessive sugar and insulin can directly affect the strength of our immune and cardiovascular system, and lead to many health problems, including diabetes and heart disease. Sucrose, ordinary table sugar, is probably the single most abundant pure organic chemical in the world and the one most widely known to non-chemists. Whether from sugar cane (20% by weight) or sugar beets (15% by weight), and whether raw or refined, common sugar is still sucrose. (Peter.et al ,1995)

Unlike most other disaccharides, sucrose is not a reducing sugar and does not exhibit mutarotation. These facts imply that sucrose has no hemiacetal linkages and that glucose and fructose must both be glycosides. This can happen only if the two sugars are joined by a glycoside link between C1 of glucose and C2 of fructose. (Geneva, 2003).

1-3-1 Invert Sugar:

When sucrose is hydrolyzed it forms a 1:1 mixture of glucose and fructose. This mixture is the main ingredient in honey. It is called invert sugar because the angle of the

specific rotation of the plain polarized light changes from a positive to a negative value due to the presence of the optical isomers of the mixture of glucose and fructose sugars.

1-3-2 Hydrolysis of Sucrose:

In the hydrolysis of any di- or poly saccharide, a water molecule helps to break the acetal bond. The acetal bond is broken, the H from the water is added to the oxygen on the glucose.

1-3-3 Metabolism of sucrose:

In humans and other mammals sucrose is broken down into its constituent monosaccharide’s, glucose and fructose, by sucrase or isomaltase glycoside hydrolases, which are located in the membrane of the microvilli lining the duodenum. The resulting glucose and fructose molecules are then rapidly absorbed into the bloodstream. In bacteria and some animals, sucrose is digested by the enzyme invertase.

Sucrose is an easily assimilated macronutrient that provides a quick source of energy, provoking a rapid rise in blood glucose upon ingestion. Sucrose, as a pure carbohydrate, has an energy content of 3.94 kilocalories per gram (or 17 kilojoules per gram). Overconsumption of sucrose has been linked with adverse health effects. (Kaneko et at., 2008).

C12H22O11 +12 O2 →12 CO2 + 11H2O

1-3-4 Physical and chemical properties of sucrose:

Pure sucrose is most often prepared as a fine, odorless crystalline powder with a pleasing, sweet taste. Large crystals are sometimes precipitated from water solutions of sucrose onto a string (or other nucleation surface) to form rock candy, a confection.

Like other carbohydrates, sucrose has hydrogen to oxygen ratio of 2:1. It consists of two monosaccharaides, glucose and fructose, joined by a glycosidic bond between carbon atom 1 of the glucose unit and carbon atom 2 of the fructose unit. What is notable about sucrose is that unlike most polysaccharides, the glycosidic bond is formed between the reducing ends of both glucose and fructose, and not between the reducing end of one and the non-reducing end of the other. The effect of this inhibits further bonding to other saccharide units. Since it contains no free a numeric carbon atom, it is classified as a non- reducing sugar.

Sucrose melts and decomposes at 186 C to form caramel, and when combusted produces carbon, carbon dioxide, and water. Water breaks down sucrose by hydrolysis; however the process is so gradual that it could sit in solution for years with negligible change. If the enzyme sucrose is added however, the reaction will proceed rapidly.. For example, in the amateur rocket motor propellant called rocket candy it is the fuel together with the oxidizer potassium nitrate

. 48 KNO3 + 5 C12H22O11 → 24 K2CO3 + 24 N2 + 55 H2O + 36 CO2

Sucrose burns with chloric acid, formed by the reaction of hydrochloric acid and potassium chlorate:

8 HCIO2 + C12H22O11  11 H2O + 12CO2 + 8HCI

Sucrose can be dehydrated with sulfuric acid to form a black, carbon-rich solid, as indicated in the following idealized equation:

H2SO4(catalyst) + C12H22O11  12 C + 11 H2O + heat and H2O + SO3 as a result of heat. (Vasilarou and Georgiou, 2000)

1-3-5 Synthesis and biosynthesis of sucrose:

The biosynthesis of sucrose proceeds via the precursors Uriding diphosphate (UDP), glucose and fructose 6-phosphate, catalyzed by the enzyme sucrose -6- phosphate synthase. The energy for the reaction is gained by the cleavage of (UDP). Sucrose is formed by plants and cyanobacteria but not by other organisms. Sucrose is found naturally in many food plants along with the monosaccharide fructose. In many fruits, such as pineapple and apricot, sucrose is the main sugar. In other, such as grapes and pears, fructose is the main sugar. (Lemieux and Huber, 1953).

1-4 Production of Sugars:

1-4-1 Sugar beet: Sugar beet (Beta vulgaris) is an annual plant in the family Amaranthaceae, the tuberous root of which contains a high proportion of sucrose. It is cultivated in temperate regions with adequate rainfall and requires a fertile soil. The crop is harvested mechanically in the autumn and the crown of leaves and excess soil removed. The roots do not deteriorate rapidly and may be left in a clamp in the field for some weeks before being transported to the processing plant. Here the crop is washed and sliced and the sugar extracted by diffusion. Milk of lime is added to the raw juice and carbonated in a number of stages in order to purify it. Water is evaporated by boiling the syrup under a vacuum. The syrup is then cooled and seeded with sugar crystals. The white sugar which crystallized out can be separated in a centrifuge and dried. It requires no further refining (SKIL. Reterieved, 2012).

1-4-2 Sugar Cane:

Sugarcane ( Soccharum spp.) is a perennial grass in the family poaceae. It is cultivated in tropical and sub=tropical regions for the sucrose that is found in its stems. It requires a frost-free climate with sufficient rainfall during the growing season to make

full use of the plant’s great growth potential. The crop is harvested mechanically or by hand, chopped into lengths and conveyed rapidly to the processing plant.

Here it is either milled and the juice extracted with water or the sugar is extracted by diffusion. The juice is then clarified with lime and heated to kill enzymes. The resulting thin syrup is concentrated in a series of evaporators after which further water is removed by evaporation in vacuum containers. The resulting supersaturated solution is seeded with sugar crystals and the sugar crystallizes out, is separated from the fluid and dried. Molasses is a byproduct of the process and the fiber from the stems, known as bagasse, is burned to provide energy for the sugar extraction process. The crystals of raw sugar have sticky brown coating and can either be used as they are or can be bleached by suphur dioxide or treated in a carbonatation process to produce a whiter product. (USAID, 2006)

1-4-3 Sugar Processing:

Sugar or more specifically sucrose is a carbohydrate that occurs naturally in every fruit and vegetable. It is the major product of photosynthesis, the process by which plant transom the sun’s energy into food. Sugar occurs in greatest quantities in sugar cane and sugar beets from which it is separated for commercial use.

In the first stage of processing the natural sugar stored in the cane stalk or beet root is separated from the rest of the plant material by physical methods. For sugar cane, this is accomplished by:

a) Pressing the cane to extract the juice containing the sugar. b) Boiling the juice until it begins to thicken and sugar begins to crystallize. c) Spinning the sugar crystals in a centrifuge to remove the syrup, producing raw sugar; the raw sugar still contains many impurities. d) Shipping the raw sugar to a refinery where it is washed and filtered to remove remaining non=sugar ingredients and color. e) Crystallizing, drying and packaging the refined sugar.

Beet sugar processing is similar, but it is done in one continuous process without the raw sugar stage. The sugar beets are washed, sliced and soaked in hot water to separate the sugar-containing juice from the beet fiver. The sugar-laden juice is purified, filtered, concentrated and dried in a series of steps similar to cane sugar processing (Clarke, 1988).

1-4-4 Refining:

Cane sugar requires further processing in provide the free flowing white table sugar required by the consumer. The sugar may be transported in bulk to the country where it will be used and the refining process often takes place there. The first stage is known as affixation and involves immersing the sugar crystals in concentrated syrup which softens and removes the sticky brown coating without dissolving them. The crystals are then separated from the liquor and dissolved in water. The resulting syrup is either treated by a carbonization or a phosphatation process. Both involve the precipitation of a fine solid in the syrup and when this is filtered out, a lot of the impurities are removed at the same time. Removal of color is achieved by either using a granular activated carbon or an ion-exchange resin. The sugar syrup is concentrated by boiling and then cooled and seeded with sugar crystals causing the sugar to crystallize out. The liquor is spun in a centrifuge and the white crystals are dried in hot air, ready to be packaged or used. The surplus liquor is made into refiners’ molasses. The international commission for Uniform Methods of Sugar Analysis sets standards for the measurement of the purity of refined sugar, known as ICUMSA numbers; lower numbers indicate a higher level of purity in the refined sugar. (SKIL. Retrieved, 2012).

1-4-5 Forms and Uses of Sugar:

Granulated sugars are used at the table to sprinkle on foods and to sweeten hot drinks and in home baking to add sweetness and texture to cooked products. They are also used as a preservative to prevent micro-organisms growing and perishable food from spoiling as in jams, marmalades and candied fruits.

Milled sugars are ground to a fine powder. They are used as icing sugar, for dusting foods and in baking and confectionery.

Screened Sugars are crystalline products separated according to the size or the grains. They are used for decorative table sugars, for blending in dry mixes and in baking and confectionery.

Brown sugars are granulated sugars with the grains coated in molasses to produce a light, dark of demerates sugar. They are used in baked goods, confectionery and toffees.

Sugar cubes are white or brown granulated sugars pressed together in block shape. They are used to sweeten drinks.

Liquid sugars are strong syrups consisting of 67% granulated sugar dissolved in water. They are used in the food processing of a wide range of products including beverages, ice cream and jams.

Invert sugars and syrups are blended to manufacturer’s specifications and are used in breads, cakes and beverages for adjusting sweetness, aiding moisture retention and avoiding crystallization of sugars.

Syrup and treacle’s are dissolved invert sugars heated to develop the characteristic flavors. Treacle have added molasses. They are used in a range of baked goods and confectionery including toffees and licorice.

Low calorie sugars and sweeteners are often made of maltodextrin with added sweeteners. Maltodextrin is an easily digestible synthetic polysaccharide consisting of short chains of glucose molecules and is made by the partial hydrolysis of starch. The added sweeteners are often aspartame, saccharin, stevia or sucralose.

1-5 Consumption of Sugar:

In most parts of the world, sugar is an important part of the human diet, making food more palatable and providing food energy. After cereals and vegetable oils, sugar

derived from sugar cane and beet provided more kilocalories per capita per day on average than other food group. According to the Food and Agriculture Organization of the United Nations (FAO), an average of 24 kilograms (53 Ib) of sugar, equivalents to over 260 food calories per day, was consumed annually per person of all ages in the world in 1999. Even with rising human populations, sugar consumption was expected to increase to 25.1 kilograms (55 Ib) per person per year by 2015. (Taubes Gary, 2011).

1-6 Health effects of Sugar:

Some studies involving the health impact of sugars are effectively inconclusive. The World Health Organization (WHO) and FAO meta studies have shown directly contrasting impacts of sugar in refined and unrefined forms and since most studies do not use a population who are not consuming any “free sugars’ at all, the baseline is effectively flowed. Hence there are articles such as Consumer Reports on Health that said in 2008, ‘ Some of the supposed dietary dangers of sugar have been overblown. Many studies have debunked the idea that it causes hyperactivity, for example. Despite this, the article continues to discuss other health impacts of sugar. Other articles and studies refer to the increasing evidence supporting the links between refined sugar and hyperactivity. The WHO and FAO meta- study suggests that such inconclusive results are to be expected when some studies do not effectively segregate or control for free sugars as opposed to sugars still in their natural form (entirely unrefined) while others do.

Blood glucose levels:

It used to be believed that sugar raised blood glucose levels more quickly than did starch because of its simpler chemical structure. This has been disproved and there is no longer a reason to segregate these two substances when controlling blood glucose levels in diabetics. This has bed to carbohydrate counting, a method used by diabetics for planning their meals. However, studies have shown that the consumption of sugar and starch have different impacts on oral health with the ingestion of starch have different impacts on oral health with the ingestion of starchy foods and fruit causing low levels of dental caries.

Obesity and diabetes:

Studies on the link between sugars and diabetes are inconclusive, with some suggesting that eating excessive amounts of sugar does not increase the risk of diabetes, although the extra calories form consuming large amounts of sugar can lead to obesity, which may itself increase the risk of developing this metabolic disease. Other studies show correlation between refined sugar (free sugar) consumption and the onset of diabetes, and negative correlation with the consumption of fiber. These included a 2010 met-analysis of eleven studies involving 310.819 participants and 15.043 cases of type 2 diabetes. This found that “SSBs (sugar-sweetened beverages) may increase the risk of metabolic syndrome and type 2 diabetes not only through obesity but also by increasing dietary glycemic load, leading to insulin resistance, B-cell dysfunction, and inflammation”. (Malik, et al,. 2010).

Cardiovascular disease:

A number of studies in animals have suggested that chronic consumption of refined sugars can contribute to metabolic and cardiovascular dysfunction. Some experts have suggested that refined fructose is more damaging than refined glucose in terms of cardiovascular risk. Cardiac performance has been shown to be impaired by switching from a carbohydrate diet including fiber to a high-carbohydrate diet.

Switching from saturated fatty acids to carbohydrates with high glycemic index values shows a statistically-significant increase in the risk of developing coronary heart disease is decreased by adopting a diet high in polyunsaturated fatty acids but low in sugar whereas as low fat, high carbohydrate diet brings no reduction. This suggests that consuming a diet with a high glycemic load typical of the “junk food” diet, is strongly associated with an increased risk of developing coronary heart disease. (Siri Tarion, et al, 2010).

Alzheimer’s disease:

It is suggested that Alzheimer’s disease is linked with the western diet, which is characterized by high intakes of red meat, sugary foods, high fat foods and refined grains. It has been hypothesized that dementia could be prevented by taking mono-supplements of specific vitamins or drugs, but studies have shown that this approach does not show appreciable results.

One group of experimenters compared a normal rodent diet (19% protein, 5% fat and 60% complex carbohydrate) with free access to water against the same diet but with free access to 10% sucrose solution. The pathogenesis of Alzheimer disease and suggest that controlling the consumption of sugar-sweetened beverages may be an effective way to curtail the risk of developing the disease (Berrino, 2002).

Tooth decay:

In regard to contributions to tooth decay, the role of free sugars is also recommended to be below an absolute maximum of zero. There is “ convincing evidence from human intervention studies, epidemiological studies, animal studies and experimental studies, for an association between the amount and frequency of free sugars intake and dental caries” while other sugars (complex carbohydrate) consumption is normally associated with a lower rate of dental caries. Lower rates of tooth decay have been seen in individuals with hereditary fructose. (Mohnihan and Peterson, 2004)

1-7 Methods of analysis of carbohydrates:

A large number of analytical techniques have been developed to measure the total concentration and type of carbohydrates present in food.

1-7-1 Chromatographic and Electrophoretic Methods:

Chromatographic methods are the most powerful analytical techniques for the analysis of the type and concentration of monosaccharide’s and oligosaccharides in food. Thin layer chromatography (TLC), Gas chromatography (GC) and High Performance

Liquid chromatography (HPLC) are commonly used to separate and identify carbohydrates. Carbohydrates are separated on the basis of their differential absorption characteristics by passing the solution to be analyzed through a column. Carbohydrates can be separated on the basis of their partition coefficients, polarities or sizes, depending on the type of column used. HPLC is currently the most important chromatographic method for analyzing carbohydrates because it is capable of rapid, specific, sensitive and precise measurements, In addition GC requires that the samples be volatile , whereas in HPLC samples can often be analyzed directly. HPLC and GC are commonly used in conjunction with Nuclear magnetic resonance spectroscopy (NMR) or mass spectrometry so that the chemical structure of the molecules that make up the peaks can also be identified.

Carbohydrates can also be separated by electrophoresis after they have been derivitized to make them electrically charged, e.g, by reaction with borates. A solution of the derivitized carbohydrate is applied to a gel and then a voltage is applied across it the carbohydrates are then separated on the basis of their size; the smaller the size of a carbohydrate molecule, the faster it moves in an electrical field.

1-7-2 Chemical methods:

A number of chemical methods used to determine monosaccharaides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components to yield precipitates or colored complexes which can be quantified. The concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by titration. Non-reducing carbohydrates can be determined using the same methods if they are first hydrolyzed to make them reducing. It is possible to determine the concentration of both non-reducing and reducing sugars by carrying out an analysis for reducing sugars before and after hydrolyzation. Many different chemical methods are available for quantifying carbohydrates. Most of these can be divided into three categories; titration, gravimetric and colorimetric. An example of each of these different types is given below.

Titration Methods:

The Lane-Eynon (1972) method is an example of a titration method of determining the concentration of reducing sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to a flask containing a known amount of boiling copper sulfate solution and a methylene blue indicator. The reducing sugars in the carbohydrate solution react with the copper sulfate present in the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution required to reach the end point is recorded. The reaction is not stoichiometric, which means that it is necessary to prepare a calibration curve by carrying out the experiment with a series of standard solutions of known carbohydrate concentration.

The disadvantages of this method are (i) the results depend on the precise reaction times, temperatures and reagent concentrations used and so these parameters must be carefully controlled; (ii) it cannot distinguish between different types of reducing sugar, and (iii) it cannot directly determine the concentration of non-reducing sugars, (iv) it is susceptible to interference from other types of molecules that act as reducing agents

Gravimetric methods:

The Munson and Walker(1906) method is an example of a gravimetric method of determining the concentration of reducing sugars in a sample. Carbohydrates are oxidized in the presence of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate.

2+ Reducing sugar + Cu + base  oxidized sugar + CuO2 (precipitate)

The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial sample. The amount of precipitate present can be determined gravimetrically (by filtration, drying and weighing), or titrimetrically (by redissolving the precipitate and titrating with a suitable indicator). This method suffers

from the same Disadvantages as the Lane-Eynon method; nevertheless, it is more reproducible and accurate.

Colorimetric Methods:

The Anthrone method is an example of a colorimetric method of determining the concentration of the total sugars in a sample. Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at 620 nm. There is a linear relationship between the absorbance and the amount of sugar that was present in the original sample. This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is non-stoichiometric and therefore it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

Another method of a colorimetric method of determining the concentration of the total sugars in a sample, based on determination of it is capacity to reduce Cu (ii) in alkaline solution is the nelsons test(1944). In this test, the amount of curpous oxide formed is determined calorimetrically by addition of arsenomolybdic acid, which is quantitatively reduced by the Cu (i) to arsenomolybdous acid, the intense blue color of which is then measured calorimetrically.

The phenol-sulfuric acid method is an example of a colorimetric method that is widely used to determine the total concentration of carbohydrate the sample is placed in a test-tube, and then phenol and sulfuric acid are added. The solution develops a yellow- orange color as a result of the interaction between the carbohydrates and the phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present. This method is non- stoichiometric and so it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

1.7.3 Physical Methods:

Many different physical methods have been used to determine the carbohydrate concentration in foods. These methods rely on there being a change in some physicochemical characteristics of a food as its carbohydrate concentration varies. Commonly used methods include polarimetry, refractive index RI,infrared IR, and density.

Polarimetry:

Molecules that contain an asymmetric carbon atom have the ability to rotate plane polarized light. A polarimeter is a device that measures the angle that plane polarized light is rotated on passing through a solution. A polarimeter consists of a source of monochromatic light, a polarizer, a sample cell of known length, and an analyzer to measure the angle or rotation. The overall angle of rotation depends on the temperature and wavelength of light used and so these parameters are usually standardized to 20 c and 589.3 nm. A calibration curve of angle of rotation versus concentration is prepared using a series of solution with known concentration, or the value of is taken from the literature if the type of carbohydrates present is known. The concentration of carbohydrate in an unknown sample is then determined by measuring its angle of rotation and comparing it with the calibration curve.

Refractive Index:

The refractive index (n) of a material is the velocity of light in a vacuum divided by the velocity of light in the material (n=c/cm). The refractive index of a material can be determined by measuring the angle of refraction (r) and angle of incidence (i) at a boundary between it and another material of know refractive index (Snells Law, sin (i)/sin ( r) = n2/n1). In practice, the refractive index of carbohydrate solutions is usually measured at a boundary with quartz. The refractive index of a carbohydrate solution increases with increasing concentration and so can be used to measure the amount of

carbohydrate present. The R1 is also temperature and wavelength dependent and so measurement is usually made at a specific temperature (20 c) and wavelength (589.3 nm). This method is quick and simple to carry out and can be performed with simple hand- held instruments. It is used routinely in industry to determine sugar concentration of syrup, honey, molasses, tomato products and jams.

Density:

The density of material is its mass divided by the volume. The density of aqueous solutions increases as the carbohydrate concentration increases. Thus the carbohydrate concentration can be determined by measuring density, e.g., using density bottles or hydrometers. This technique is routinely used in industry for determination of carbohydrate concentration of juices and beverage.

Infrared:

A material absorbs infrared radiation due to vibration or rotation of molecular groups. Carbohydrate contain molecular groups that absorb infrared radiation at wavelengths where none of the other major food constituents absorb, consequently their concentration can be determined by measuring the infrared absorbance at these wavelengths. By carrying out measurement s at a number of different specific wavelengths it is possible to simultaneously determine the concentration of carbohydrate, proteins, moisture and lipids. Measurement are normally carried out by measuring the intensity of an infrared wave reflected from the surface of a sample the greater the absorbance, the lower the reflectance. Analytical instruments based on infrared absorbance are non-destructive and capable of rapid measurements and are therefore particularly suitable for on-line analysis or for use in a quality control laboratory where many samples are analyzed routinely.

More sophisticated instrumental method is capable of providing information about the molecular structure of carbohydrates as well as their concentration, e.g., NMR or mass spectrometry. (Browne and Zerban, 1991)

The Objective of the work:

1. To test validity of the method in determination of sugar, in commercial samples. 2. To compare the amounts of reducing sugars and non-reducing sugars (sucrose) in different commercial samples.

Chapter Two Materials and Methods 1-1 Sample collection and preparation:

Four samples of commercial sugar were collected from local markets (Wad Medani City). Two of the samples which were Sudanese product and the other two samples were one from India and the other from Brazil.

Table 2-1 shows the types color, origin of sugars samples collected for analysis.

No. of samples Type of sugar Color of sugar Made

1 White sugar White Brazil

2 White sugar White Sudan (Kenana)

3 White sugar White India

4 Raw sugar Whiteish Sudan (Gunied)

2-2 Apparatus:

- glass ware - water bath - refluxing device - spectrophotometer (model 21 D mitonroy)

2-3 Chemicals:

- D-glucose

Molar Mass – 180.16 g/mol

- Copper Sulphate (CuSo4. 5H2O) Molar mass 249.68 g/mol Assay 98.5 -101 %

- Sodium Potassium tartrate (KNaC4H4O4.H2O) Minimum assay 99%

Sodium Carbonate (Na2CO3) Molar mass 105.99 g/mol Minimum assay 99.5%

- Sodium bicarbonate (NaHCO3 ) Molar mass 84.01 g/mol Minimum assay 99.5% - Potassium oxalate

- Analar Ammonium Molybdate (NH4)6Mo7 O24. H2O Molar mass 1235.9 g/mol Minimum assay 99%

- Sulfuric acid (H2SO4) Molar mass 60.05 g/mol Minimum assay 99.71% Density 1.049 g/cm3

- Sodium Arsenate (Na2HAsO4 7H2O) Molar Mass 105.99 g/mol Minimum assay 99.5%

- Oxalic acid (COOH)2H2O) Molar mass 126.07 g/mol Minimum assay 99.8%

2-4 Preparation of Solutions:

Glucose Standard solution:

1.0gm. glucose was dissolved in distilled water and diluted to 1000 ml to get 1000 ug/ml glucose solution. 1.00 ml of the standard solution was diluted to 10 ml to get 100

ug/ml, from it 0.00, 0.20, 0.40, 0.60, 0.80 and 1.00 ml were taken and diluted to 1.0 ml to get 0.00, 20.00, 40.00, 60.00, 80.00 and 100ug glucose for constructing the standard curve.

Copper Reagent:

1.5 gm of CuSO4. 5H2O was dissolved in 250 distilled water.

Alkaline tarirate reagent:

2gm of sodium potassium tartrate, 5 gm Na2CO3, 6.25gm NaHCO3 and 4.5 gm potassium oxalate were dissolved in 250 ml distilled water.

Nelson Reagent:

12.5 gm of analar ammonium molybdate was dissolved in 225 ml distilled water and then 10.5 ml of concentrated H2SO4 were added slowly. In separate beaker 1.5 gm of hydrated sodium arsenate was dissolved in 12.5 ml distilled water and then added to the first solution. Finally the mixture was allowed to stand at 37c for two days in brown bottle.

Oxalic acid (2.0%):

2 gm. of oxalic acid were dissolved in 100ml distilled water.

2-5 Procedure:

A. Preparation of sample solution:

1.0 gm. of sugar from the different samples was dissolved in 100 ml distilled water. From this solution an aliquot was taken for determination of reducing sugars.

B. hydrolysis of sugar samples:

From the solution in (A) 20 ml taken and then 20 ml of oxalic acid were added and hydrolyzed by refluxing for 2 hours. And then the volume made to 100 ml with distilled water, and aliquot was taken for the determination of total soluble sugars (reducing + non reducing sugars).

C. Determination of reducing sugars:

Reducing sugars were determined spectrophotomerically by a modification of Asatoor and kings (1954) adaptation of the cupurimetric method of Nelson (1944).

1 ml of either sample under test, glucose standard and distilled water for blank was taken in attest tubes, to it 1.0 ml of copper reagent was added followed by 1.0 ml alkaline tartrate reagent mixed well and heated in boiling water bath for 10 minutes. After cooling 1.0ml of Nelson reagent was add then mixed well to dissolve deposited cuprous oxide and then the volume was made to 10.0 ml with distilled water and allowed to stand for 10 minutes for color development. Finally the absorbance was read in spectrophotometer using 1 cm glass cell at wave length 600 nm.

Glucose + Cu+2 Cu+1 + mixture of sugar acids

Cu+1 + Mo+6  Cu+2 + Mo+5

The standard curve was constructed to obtain values of sugars in samples under test.

Table (2.2) absorbance of the reference solution by use spectrophotometer.

Concentration references solution (mg/ml) Absorbance

0 0

20 0.2

40 0.4

60 0.6

80 0.8

100 1.0

Chapter Three

Results and Discussion In this chapter the obtained amount of reducing sugar (glucose / fructose) and non-reducing sugar (sucrose) in commercial sugar using Nelson’s reagent and standard spectrophotometric techniques will be shown.

Table 3-1 : show the result of concentration of reducing sugars in the samples:

No. of sample Absorbance Concentration (mg/ml) Percentage 1 0.1 10 0.1%

2 0.11 10 0.1%

3 0.1 10 0.1%

4 0.232 23 0.23%

% = * * =

X = concentration of reducing sugar (gm/ml)

Table 3-2 : show the result of concentration total soluble sugars in the samples:

No. of sample Absorbance Concentration (mg/ml) Percentage 1 1.00 100 100%

2 0.998 99.8 99.8%

3 0.997 99.7 99.7%

4 0.995 99.5 99.5%

% = * * * =

X = concentration of total soluble sugar (gm/ml)

Table 3-3 : show the amount of soluble sugar in commercial sugar samples:

No. of sample Percentage of reducing Percentage of Percentage of sugar total soluble sucrose sugar

1 0.1 100 99.9%

2 0.1 99.8 99.7%

3 0.1 99.7 99.6%

4 0.23 99.5 99.27%

Percentage of sucrose = percentage of total soluble sugar- percentage of reducing sugar.

The standard specification of sugar states that the percentage of sucrose must be equal or more than (99.5%) and the percentage of reducing sugar are equal or less than (0.1%). 43

The results obtained in table (3-1) an (3-3) show that sample 1.2 and 3 reducing sugar and non-reducing sugar content is 0.1 and more than 99.5% respectively so is agree with the standard specification sugar but sample four found that the percentage of reducing sugar is more than 0.1% (0.23%) and the percentage of sucrose is less than 99.5% (99.27%) that because samples four consider as raw sugar with high purity which have layer of molasses coated the crystals of sugar, this layer make a sugar brown color and more sweetness.

In table 3-3 the result show that sample 1 have full percent of soluble sugar but sample 2,3,4 have 0.2 , 0.3 , 0.5 , % respectively other thing may be ache or other mineral.

Conclusion :

The nelson’s methods can be used to calculate the a mount of reducing sugar and non-reducing in all commercial – table sugar samples

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