Monday, January 23, 2012


  • Chemically extracted or plant-derived pesticides. These include the pyrethroids, rotenoids and nicotinoids.
  • Botanicals can be
    – A constructive chemical
    Present in plant and subsequently extracted from plant.
    – Inducible chemical
    Activated in plants as a response to insect activity
  • There are limited number of compounds commercially available. Actually there is a large number of compounds but due to many reasons such as stability, quantity, etc., only limited number is commercially available. Some are chemically modified after extraction to enhance insecticidal properties.


Alternatives for Pesticides

Alternatives to pesticides are available and include methods of cultivation, use of biological pest controls (such as pheromones and microbial pesticides), genetic engineering, and methods of interfering with insect breeding. Application of composted yard waste has also been used as a way of controlling pests. These methods are becoming increasingly popular and often are safer than traditional chemical pesticides. In addition, EPA is registering reduced-risk conventional pesticides in increasing numbers.

Cultivation practices include polyculture (growing multiple types of plants), crop rotation, planting crops in areas where the pests that damage them do not live, timing planting according to when pests will be least problematic, and use of trap crops that attract pests away from the real crop. In the U.S., farmers have had success controlling insects by spraying with hot water at a cost that is about the same as pesticide spraying.

Release of other organisms that fight the pest is another example of an alternative to pesticide use. These organisms can include natural predators or parasites of the pests. Biological pesticides based on entomopathogenic fungi, bacteria and viruses cause disease in the pest species can also be used.

Interfering with insects' reproduction can be accomplished by sterilizing males of the target species and releasing them, so that they mate with females but do not produce offspring. This technique was first used on the screwworm fly in 1958 and has since been used with the medfly, the tsetse fly, and the gypsy moth. However, this can be a costly, time consuming approach that only works on some types of insects.

Another alternative to pesticides is the thermal treatment of soil through steam. Soil steaming kills pest and increases soil health.

In India, traditional pest control methods include using Panchakavya, the "mixture of five products." The method has recently experienced a resurgence in popularity due in part to use by the organic farming community


Farmers and workers, Consumers, The public and pesticides

Farmers and workers

The World Health Organization and the UN Environment Programme estimate that each year, 3 million workers in agriculture in the developing world experience severe poisoning from pesticides, about 18,000 of whom die. According to one study, as many as 25 million workers in developing countries may suffer mild pesticide poisoning yearly. There have been many studies of farmers intended to determine health effects of occupational pesticide exposure. Associations between non-Hodgkin lymphoma, leukemia, prostate cancer, multiple myeloma, and soft tissues sarcoma have been reported in studies, with less associations found for other cancers.

Organophosphate pesticides have increased in use, because they are less damaging to the environment and they are less persistent than organochlorine pesticides. These are associated with acute health problems for workers that handle the chemicals, such as abdominal pain, dizziness, headaches, nausea, vomiting, as well as skin and eye problems. Additionally, many studies have indicated that pesticide exposure is associated with long-term health problems such as respiratory problems, memory disorders, dermatologic conditions, cancer, depression, neurological deficits, miscarriages, and birth defects. Summaries of peer-reviewed research have examined the link between pesticide exposure and neurologic outcomes and cancer, perhaps the two most significant things resulting in organophosphate-exposed workers.

According to researchers from the National Institutes of Health (NIH), licensed pesticide applicators who used chlorinated pesticides on more than 100 days in their lifetime were at greater risk of diabetes. One study found that associations between specific pesticides and incident diabetes ranged from a 20 percent to a 200 percent increase in risk. New cases of diabetes were reported by 3.4 percent of those in the lowest pesticide use category compared with 4.6 percent of those in the highest category. Risks were greater when users of specific pesticides were compared with applicators who never applied that chemical.


The examples and perspective in this article deal primarily with the United States and do not represent a worldwide view of the subject. Please improve this article and discuss the issue on the talk page.

There are concerns that pesticides used to control pests on food crops are dangerous to people who consume those foods. These concerns are one reason for the organic food movement. Many food crops, including fruits and vegetables, contain pesticide residues after being washed or peeled. Chemicals that are no longer used but that are resistant to breakdown for long periods may remain in soil and water and thus in food.

The United Nations Codex Alimentarius Commission has recommended international standards for Maximum Residue Limits (MRLs), for individual pesticides in food.

In the EU, MRLs are set by DG-SANCO. In the United States, levels of residues that remain on foods are limited to tolerance levels that are established by the U.S. Environmental Protection Agency‎ and are considered safe. The EPA sets the tolerances based on the toxicity of the pesticide and its breakdown products, the amount and frequency of pesticide application, and how much of the pesticide (i.e., the residue) remains in or on food by the time it is marketed and prepared. Tolerance levels are obtained using scientific risk assessments that pesticide manufacturers are required to produce by conducting toxicological studies, exposure modeling and residue studies before a particular pesticide can be registered, however, the effects are tested for single pesticides, and there is little information on possible synergistic effects of exposure to multiple pesticide traces in the air, food and water.
A study published by the United States National Research Council in 1993 determined that for infants and children, the major source of exposure to pesticides is through diet. A study in 2006 measured the levels of organophosphorus pesticide exposure in 23 school children before and after replacing their diet with organic food (food grown without synthetic pesticides). In this study it was found that levels of organophosphorus pesticide exposure dropped dramatically and immediately when the children switched to an organic diet.

To reduce the amounts of pesticide residues in food, consumers can wash, peel, and cook their food; trim the fat from meat; and eat a variety of foods to avoid repeat exposure to a pesticide typically used on a given crop, however, many pesticides are systemic, which means they penetrate into the fruit and vegetable itself and cannot be washed off. Many pesticides are also by design created to be rain-proof.
Strawberries and tomatoes are the two crops with the most intensive use of soil fumigants. They are particularly vulnerable to several type of diseases, insects, mites, and parasitic worms. In 2003, in California alone, 3.7 million pounds (1,700 metric tons) of metam sodium were used on tomatoes. In recent years other farmers have demonstrated that it is possible to produce strawberries and tomatoes without the use of harmful chemicals and in a cost effective way.

The public

Exposure routes other than consuming food that contains residues, in particular pesticide drift, are potentially significant to the general public.

The Bhopal disaster occurred when a pesticide plant released 40 tons of methyl isocyanate (MIC) gas, a chemical intermediate in the synthesis of some carbamate pesticides. The disaster immediately killed nearly 3,000 people and ultimately caused at least 15,000 deaths.
In China, an estimated half million people are poisoned by pesticides each year, 500 of whom die.

Children have been found to be especially susceptible to the harmful effects of pesticides. A number of research studies have found higher instances of brain cancer, leukemia and birth defects in children with early exposure to pesticides, according to the Natural Resources Defense Council. Often used for ridding school buildings of rodents, insects, pests, etc., pesticides only work temporarily and must be re-applied. The poisons found in pesticides are not selectively harmful to just pests and in everyday school environments children (and faculty) are exposed to high levels of pesticides and cleaning materials. "No testing has ever been done specifically pertaining to threats among children"
Peer-reviewed studies now suggest neurotoxic effects on developing animals from organophosphate pesticides at legally tolerable levels, including fewer nerve cells, lower birth weights, and lower cognitive scores. The United States Environmental Protection Agency‎ finished a 10 year review of the organophosphate pesticides following the 1996 Food Quality Protection Act, but did little to account for developmental neurotoxic effects, drawing strong criticism from within the agency and from outside researchers.

Some scientists think that exposure to pesticides in the uterus may have negative effects on a fetus that may manifest as problems such as growth and behavioral disorders or reduced resistance to pesticide toxicity later in life.

A new study conducted by the Harvard School of Public Health in Boston, has discovered a 70% increase in the risk of developing Parkinson's disease for people exposed to even low levels of pesticides.

A 2008 study from Duke University found that the Parkinson's patients were 61 percent more likely to report direct pesticide application than were healthy relatives. Both insecticides and herbicides significantly increased the risk of Parkinson's disease.

One study found that use of pesticides may be behind the finding that the rate of birth defects such as missing or very small eyes is twice as high in rural areas as in urban areas. Another study found no connection between eye abnormalities and pesticides. In the United States, increase in birth defects is associated with conceiving in the same period of the year when agrichemicals are in elevated concentrations in surface water.

Pyrethrins, insecticides commonly used in common bug killers, can cause a potentially deadly condition if breathed in.


Health effects of Pesticides

Pesticides can be dangerous to consumers, workers and close bystanders during manufacture, transport, or during and after use.
The American Medical Association recommends limiting exposure to pesticides and using safer alternatives:
Particular uncertainty exists regarding the long-term effects of low-dose pesticide exposures. Current surveillance systems are inadequate to characterize potential exposure problems related either to pesticide usage or pesticide-related illnesses…Considering these data gaps, it is prudent…to limit pesticide exposures…and to use the least toxic chemical pesticide or non-chemical alternative.


Environmental effects of Pesticides (reduced nitrogen fixation)

Pesticide use raises a number of environmental concerns. Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including non-target species, air, water and soil. Pesticide drift occurs when pesticides suspended in the air as particles are carried by wind to other areas, potentially contaminating them. Pesticides are one of the causes of water pollution, and some pesticides are persistent organic pollutants and contribute to soil contamination.

In addition, pesticide use also reduces biodiversity and results in lower soil quality, reduced nitrogen fixation, contribute to pollinator decline, can reduce habitat, especially for birds, and can threaten endangered species.


Regulation of Pesticides ( bioaccumulative and toxic (PBT), persistent, very persistent and very bioaccumulative (vPvB) )

In most countries, pesticides must be approved for sale and use by a government agency. For example, in the United States, the Environmental Protection Agency (EPA) does so. Complex and costly studies must be conducted to indicate whether the material is safe to use and effective against the intended pest. During the registration process, a label is created. The label contains directions for proper use of the material. Based on acute toxicity, pesticides are assigned to a Toxicity Class.

Some pesticides are considered too hazardous for sale to the general public and are designated restricted use pesticides. Only certified applicators, who have passed an exam, may purchase or supervise the application of restricted use pesticides. Records of sales and use are required to be maintained and may be audited by government agencies charged with the enforcement of pesticide regulations.
In Europe, recent EU legislation has been approved banning the use of highly toxic pesticides including those that are carcinogenic, mutagenic or toxic to reproduction, those that are endocrine-disrupting, and those that are persistent, bioaccumulative and toxic (PBT) or very persistent and very bioaccumulative (vPvB). Measures were approved to improve the general safety of pesticides across all EU member states.

Though pesticide regulations differ from country to country, pesticides and products on which they were used are traded across international borders. To deal with inconsistencies in regulations among countries, delegates to a conference of the United Nations Food and Agriculture Organization adopted an International Code of Conduct on the Distribution and Use of Pesticides in 1985 to create voluntary standards of pesticide regulation for different countries. The Code was updated in 1998 and 2002. The FAO claims that the code has raised awareness about pesticide hazards and decreased the number of countries without restrictions on pesticide use.

Two other efforts to improve regulation of international pesticide trade are the United Nations London Guidelines for the Exchange of Information on Chemicals in International Trade and the United Nations Codex Alimentarius Commission. The former seeks to implement procedures for ensuring that prior informed consent exists between countries buying and selling pesticides, while the latter seeks to create uniform standards for maximum levels of pesticide residues among participating countries. Both initiatives operate on a voluntary basis.

Reading and following label directions is required by law in countries such as the United States and in limited parts of the rest of the world.

One study found pesticide self-poisoning the method of choice in one third of suicides worldwide, and recommended, among other things, more restrictions on the types of pesticides that are most harmful to humans.


Uses of Pesticides (bioaccumulation, Pest Management Regulatory Agency (PMRA))

Pesticides are used to control organisms considered harmful. For example, they are used to kill mosquitoes that can transmit potentially deadly diseases like west nile virus, yellow fever, and malaria. They can also kill bees, wasps or ants that can cause allergic reactions. Insecticides can protect animals from illnesses that can be caused by parasites such as fleas. Pesticides can prevent sickness in humans that could be caused by mouldy food or diseased produce. Herbicides can be used to clear roadside weeds, trees and brush. They can also kill invasive weeds that may cause environmental damage. Herbicides are commonly applied in ponds and lakes to control algae and plants such as water grasses that can interfere with activities like swimming and fishing and cause the water to look or smell unpleasant. Uncontrolled pests such as termites and mould can damage structures such as houses. Pesticides are used in grocery stores and food storage facilities to manage rodents and insects that infest food such as grain. Each use of a pesticide carries some associated risk. Proper pesticide use decreases these associated risks to a level deemed acceptable by pesticide regulatory agencies such as the United States Environmental Protection Agency (EPA) and the Pest Management Regulatory Agency (PMRA) of Canada.

Pesticides can save farmers' money by preventing crop losses to insects and other pests; in the U.S., farmers get an estimated fourfold return on money they spend on pesticides. One study found that not using pesticides reduced crop yields by about 10%. Another study,conducted in 1999, found that a ban on pesticides in the United States may result in a rise of food prices, loss of jobs, and an increase in world hunger.

DDT, sprayed on the walls of houses, is an organochloride that has been used to fight malaria since the 1950s. Recent policy statements by the World Health Organization have given stronger support to this approach. Dr. Arata Kochi, WHO's malaria chief, said, "One of the best tools we have against malaria is indoor residual house spraying. Of the dozen insecticides WHO has approved as safe for house spraying, the most effective is DDT." However, since then, an October 2007 study has linked breast cancer from exposure to DDT prior to puberty. Poisoning may also occur due to use of DDT and other chlorinated hydrocarbons by entering the human food chain when animal tissues are affected. Symptoms include nervous excitement, tremors, convulsions or death. Scientists estimate that DDT and other chemicals in the organophosphate class of pesticides have saved 7 million human lives since 1945 by preventing the transmission of diseases such as malaria, bubonic plague, sleeping sickness, and typhus. However, DDT use is not always effective, as resistance to DDT was identified in Africa as early as 1955, and by 1972 nineteen species of mosquito worldwide were resistant to DDT. A study for the World Health Organization in 2000 from Vietnam established that non-DDT malaria controls were significantly more effective than DDT use. The ecological effect of DDT on organisms is an example of bioaccumulation.


Analysis of an antacid tablet

Most people at one or another have suffered from indigestion or heartburn. In many people, these symptoms arise when the pH of the stomach falls below 3.0. The observation suggests that one way to avoid or overcome these distressing symptoms is to neutralize any excess acid with a non toxic chemical reagent that maintains the contents of the stomach at pH above 3.0.

A number of readily available commercial preparations have been designed to fill this need. One of the most familiar is milk of magnesia. Your objective of this part of the experiment is to determine the magnesium hydroxide content in these tablets. You, like the manufacturer, assume that stomach acid is approximately 0.10M HCl acid. In the stomach, some of the hydrogen ions combine with other species and keep the pH above 3.0. The procedure involves dissolving a weighed milk of magnesia tablet in an excess of simulated stomach acid whose volume has been carefully measured. The antacid tablet neutralizes a specific amount of the acid. You then determine the volume of unneutralised acid by titrating the resulting solution with standard NaOH solution.


For magnesium hydroxide

pKb1 = pKb2

When an excess standard solution of HCl is added, to a sample of milk of magnesia,


Weigh the antacid tablet. Grind the tablet and put all the powdered material into a beaker. Dissolve this in approximately 200mL of 0.1M HCl acid. The solution may contain a small amount of undissolved, unreactive residue but this does not interfere with the analysis. Filter the solution in to a 250 mL volumetric flask and dilute up to the mark with 0.1M HCl. Pipette 25.00 mL into a titration flask, add few drops of a suitable indicator and titrate with 0.1M NaOH solution.

I. Calculate the percentage of magnesium hydroxide in the tablet
II. Why magnesium hydroxide cannot be titrated directly with HCl? (Hint: think of solubility, rate of the reaction and indicators)


Back titrations key words: activation energy, kinetics, antacid

When the reaction involves in a titration does not satisfies the conditions for a direct titration to be performed, it is possible to carryout a back titration.

If the reaction A & B’s slow (poor kinetics due to high activation energy), it cannot be analyzed using a direct titration. In such a case back titration is utilized by adding a known amount of the standard solution of A in excess to B and the excess of A is subsequently determined by using another titrant E, Where the reaction between A and E satisfies all conditions for a direct titration.

This process is called a back titration. Back titrations are very useful when the indicators recommended in the procedure are not available.


The general shape of the titration curves obtained by titrating 10.00 mL of a 0.01M solution of a metal ion M with 0.01M EDTA solution & relevent F A Q s

The apparent stability constant of various metal-EDTA complexes are indicated at the extreme right of the curves. It is evident that the greater the stability constant, the sharper is the end point provided the pH is maintained constant (the apparent stability constant is dependent on the pH)

In EDTA titrations, the end point is generally detected by a metal ion sensitive indicator (metalochromic indicator). Such indicators (which contain type of chelated groupings and generally posses resonance systems typical; of dyestuff) form complexes with specific metal ions, which differ in color from the free indicator and produce a sudden color change at the equivalence point.

Constant ph in EDTA titration is also important to maintain the proper function of many metalochromoc indicators because the color exhibited by them is dependant on pH.
eg: Eriochrome Black T indicator (EBT). This is represented as H3In

This reaction will proceed if the metal indicator complex [M-In] is less stable than the [metal-EDTA]. [M-In] dissociate during the titration and the free metal ions are progressively complexed by the EDTA until ultimately the metal is displaced from the [M-In] complex to leave the free indicator In.

a)Determination of Calcium and Magnesium in a mixture


Pipette 25.00mL of the mixture into a titration flask, dilute with 25 mL of distilled water, add 5 mL pH =10 buffer solution and mix by swirling. Add 6 drops of EBT indicator. Titrate with the 0.05M EDTA solution to a pure blue end point.

Color change at the end point : red to blue

b)Determination of the Calcium


Pipette two 25.00mL portions of the mixture into two separate titration flasks and dilute each with about 25 mL of distilled water. To the first flask add 4mL of 8 M KOH solution (a precipitate of magnesium hydroxide may be noted here), and allow to stand for 3-5 minutes with occasional swirling. Add about 50 mg of the Patton and Reeders indicator mixture and titrate with 0.05M EDTA until the color changes from red to blue. Run in to second flask from a burette a volume of EDTA solution equal to that required to reach the end point less 1mL. now add 4 mL of the KOH solution, mix well and complete the titration as with the first sample; record the exact volume of EDTA solution used.

I. Determine the concentration of magnesium and calcium ions in solution separately.

II. In experiment b, why can’t we use EBT instead of Patton and Reeder’s indicator?

All magnesium ions are precipitated as magnesium hydroxide by addition of 8M KOH. Only calcium ions remain in the solution. Calcium ions form a relatively stable complex with EDTA. With calcium ions alone no sharp end point can be obtained with EBT and the transition color from red to pure blue is not observed.

III. In experiment b, we should add KOH solution first and then the indicator. Why is this?

To determine calcium ion concentration, magnesium ions must be precipitated and removes first. Then add Patton and Reeder’s indicator. It works above pH 12

IV. Explain temporary hardness and permanent hardness. Give a method to determine total hardness and calcium hardness.

Temporary hardness is due to calcium and magnesium ions which are dissolved as bicarbonates.
Permanent hardness is due to magnesium, calcium, ferric, chloride and sulphate ions.

V. What would be the color change that you would observe if the method (a) is altered as follows
To a 25.00 mL of the test sample added 50.00mL of EDTA, 5mL of pH=10 buffer, 6 drops of EBT indicator and titrated with 0.05 M standard magnesium solution.

Purple to blue. (Red color is seen through blue)

VI. With calcium alone, no sharp end point can be obtained with EBT and a common procedure is to add a small amount of magnesium chloride to the EDTA solution before it is titrated. Explain why is that?

Calcium forms a relatively stable complex with EDTA but magnesium forms a less stable complex with EDTA.

VII. What is meant by a back titration? Explain why we determine Ni 2+ generally by a back titration

Some metal ions precipitate in the given pH or form inert complexes or suitable indicators are not available, so have to go for back titrations.
Nickel complexation reaction is very slow so it need to be titrated via a back titration.


Complexometric titrations (key words: EDTA) & Stability with respect to pH of some metal-EDTA complexes

This particular experiment is concerned with the way in which complexation reactions can be employed in titrimetry, especially for determining the proportions of individual cations in mixtures.


Ethylenediaminetetraacetic acid (EDTA) is the most widely used complexing agent in Complexometric titrations.

The pKa values for EDTA are pKa1 = 2.0, pKa2 = 2.7, pKa3 = 6.2 and pKa4 = 10.3 at 20 degrees Celsius.
These values suggest that it behaves as a dicarboxylic acid with two strongly acidic hydrogens. Na2H2Y.2H2O is most commonly used in the preparation of EDTA solution because of its high solubility.

The molecule has six potential sites for co-ordination and forms 1:1 complex with most metal ions.

water and posses almost the same colors as aqueous metal ion.

The metal complexes are soluble in It is obvious from this equation that the stability of the complex is dependent upon the pH of the medium etc. therefore EDTA titrations are generally carried out in buffered media.

Stability with respect to pH of some metal-EDTA complexes


F A Q s of Determination of iodate and iodide in a mixture

I. How could you determine the concentration of potassium dichromate in a solution quantitatively by an iodometric titration?

 II. Why cannot you titrate the oxidizing agent directly with the thiosulphate solution?

The oxidizing agent reduces thiosulphate to sulphur.

III. Explain why you do not add starch at the beginning of the titration.

In the beginning iodine concentration is too high and starch form a stable complex with iodine and blue color do not fades wawy even at the end point.
IV. Why is it necessary to do iodometric titrations in acidic medium as quickly as possible?
Acidic medium: Many weak oxidizing anions are completely reduced by iodide if their reduction potentials are raised considerably by the presence of large amount of acid in the medium.


How could you determine the concentration of potassium dichromate in a solution quantitatively by an iodometric titration?


Determination of iodate and iodide in a mixture


(i) Pipette 10.00 mL of the solution into a 100mL titration flask which contains 50 mL of water and add 5mL of 2M sulfuric acid. Titrate liberated iodine with 0.1M sodium thiosulphate solution using starch the indicator.

(ii) Pipette 25.00mL of solution in to a separating funnel, add 15mL of methelene chloride and 5mL of 1M sulfuric acid into it. Shake well to dissolve liberated iodine in organic layer. When the two layers separate remove organic layer into the stoppered bottle which contains 50 mL of water. Reextract liberated iodine usind two portions of 15mL of methelene chloride and transfer both organic layers into same bottle. Titrate extracted iodine with 0.1M sodium thiosulphate. Divide the aqueous layer in to two portions. Add a little KI solution into one and a little potassium iodate solution into the other and record your observations. Explain the observations. Determine the concentration of potassium iodate and KI in the mixture.


Determination of Iodide


Pipette 25.00mL of iodide solution into stoppered bottle. Add 25 mL of water, 60 mL of conc. HCl and 5mL of methelene chloride into it. Allow the solution to aquire room temperature. Add a portion of 0.025M potassium iodate with a burette and shake well (organic layer becomes purple due to iodine). Add small portions of potassium iodate occasionally, after each addition shake well. When organic layer changes purple to light color, add potassium iodate drop by drop and shake well. At the end point purple color disappears completely.


IODOMETRY Key words: Iodimetry, Andrew's condition

Iodometry refers to the titration of iodine liberated in chemical reaction (eq 1& 2). Titration with a standard solution of iodine is called an iodimetric titration (eq 3)

In this experiment you will be using KIO3. It is a very strong oxidizing agent and its oxidizing power depends on the conditions under which it is used.
In moderately acidic solution (0.1 -2.0M HCl) with reducing agents such as iodide ions reaction stops when the iodate is reduced to iodine (eq 4). With more powerful reductants like Ti(III) the iodate is reduced to iodide. (eq 5)

In most strongly acidic medium (3-6M HCl) reduction occurs to iodine monochloride. This is the most widely used condition and is known as Andrew’s conditions.

In the experiments given below you will be using strong HCl acid medium and the reaction proceeds through several steps.


Alternatives for Pesticides

Alternatives to pesticides are available and include methods of cultivation, use of biological pest controls (such as pheromones and microbial pesticides), genetic engineering, and methods of interfering with insect breeding. Application of composted yard waste has also been used as a way of controlling pests. These methods are becoming increasingly popular and often are safer than traditional chemical pesticides. In addition, EPA is registering reduced-risk conventional pesticides in increasing numbers.

Cultivation practices include polyculture (growing multiple types of plants), crop rotation, planting crops in areas where the pests that damage them do not live, timing planting according to when pests will be least problematic, and use of trap crops that attract pests away from the real crop. In the U.S., farmers have had success controlling insects by spraying with hot water at a cost that is about the same as pesticide spraying.

Release of other organisms that fight the pest is another example of an alternative to pesticide use. These organisms can include natural predators or parasites of the pests. Biological pesticides based on entomopathogenic fungi, bacteria and viruses cause disease in the pest species can also be used.

Interfering with insects' reproduction can be accomplished by sterilizing males of the target species and releasing them, so that they mate with females but do not produce offspring. This technique was first used on the screwworm fly in 1958 and has since been used with the medfly, the tsetse fly, and the gypsy moth. However, this can be a costly, time consuming approach that only works on some types of insects.

Another alternative to pesticides is the thermal treatment of soil through steam. Soil steaming kills pest and increases soil health.

In India, traditional pest control methods include using Panchakavya, the "mixture of five products." The method has recently experienced a resurgence in popularity due in part to use by the organic farming community


Wednesday, January 11, 2012

Ninhydrin Test Key words Ruhemann's purple ,proline

Ninhydrin Test

Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is evolved. Ninhydrin is most commonly used to detect fingerprints, as amines left over from peptides and proteins (terminal amines or lysine residues) sloughed off in fingerprints react with ninhydrin.

Ninhydrin can also be used to monitor deprotection in solid phase peptide synthesis (Kaiser Test). When the growing peptide chain is deprotected, a ninhydrin test yields blue. If the next peptide residue is coupled then the test is colorless or yellow.
Ninhydrin is also used in amino acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with ninhydrin except proline; Also, certain amino acid chains are degraded. Therefore, separate analysis is required for identifying such amino acids that either react differently or don't react at all with ninhydrin. The rest of the amino acids are then quantified colorimetrically after separation by chromatography.
A solution suspected of containing the ammonium ion can be tested by ninhydrin by dotting it onto a solid support (such as silica gel); treatment with ninhydrin should result in a dramatic purple color if the solution contains this species. In the analysis of a chemical reaction by thin layer chromatography (TLC), the reagent can also be used. It will detect, on the TLC plate, virtually all amines, carbamates and also, after vigorous heating, amides.
When ninhydrin reacts with amino acids, the reaction also releases CO2. The carbon in this CO2 originates from the carboxyl carbon of the amino acid. This reaction has been used to release the carboxyl carbons of bone collagen from ancient bones[3] for stable isotope analysis in order to help reconstruct the palaeodiet of cave bears.[4]
A ninhydrin solution is commonly used by forensic investigators in the analysis of latent fingerprints on porous surfaces such as paper. Amino acid containing fingermarks, formed by minute sweat secretions which gather on the finger's unique ridges, are treated with the ninhydrin solution which turns the amino acid finger ridge patterns purple and therefore visible. [
The carbon atom of a carbonyl bears a partial positive charge, so the central carbon of a 1,2,3-tricarbonyl is less stable and more electrophilic than a simple ketone. In most compounds, a carbonyl is more stable than the dihydroxy (hydrate) form. However, ninhydrin is a stable hydrate of the central carbon because this form does not have the destabilizing effect of adjacent carbonyl partial-positive centers. Indane-1,2,3-trione reacts readily with nucleophiles.
Note that in order to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yellow-orange in color.

Cilck on the image if you cant see it clearly


Add about 2 mg of the sample to 1 mL of a solution of 0.2 g of ninhydrin (1,2,3indanetrione monohydrate) in 50 mL of water. The test mixture is heated to boiling for 15-20 sec; This reaction is important not only because it is a qualitative test, but also because it is the source of the absorbing material that can be measured quantitatively by an automatic amino acid analyzer. This color reaction is also used to detect the presence and position of amino acids after paper chromatographic separation.


Xanthoproteic Test key words tyrosine, tryptophan

Xanthoproteic Test

This test will determine if residues of tyrosine or tryptophan are present. The solution to be tested is treated with concentrated nitric acid, which will nitrate the benzene riugs of those residues. The nitrated aromatic ringR are yellow in color and are (:alled Xanthoproteic acids (Xantho, = yellow, Greek). If base is added. the color becomes more intense and the color will shift more to orange.


Carefully add 1 ml of concentrated nitric acid to 2 ml of amino acid solution in a test tube. Stir well with a glass stirring rod and heat.


Tuesday, January 10, 2012


Add 2 ml of Hopkins-Cole Reagent to 1 ml of amino acid solution and mix.
VERY SLOWLY AND CAREFULLY pour this down the side of an inclined test tube containing 2 ml of concentrated sulfuric acid, so that it forms a layer on top of the acid. Allow the test tube to stand without mixing.


Isolation of Casein and Lactose from Milk- Proteins and Carbohydrates. Isolation of Casein and Lactose from Milk

Proteins and Carbohydrates. Isolation of Casein and Lactose from Milk


Milk is the most nutritionally complete food found in nature. All kinds of milk, human or animal, contain vitamins (principally thiamine, riboflavin, pantothenic acid, and vitamins A, B12, and D), minerals (calcium, potassium, sodium, phosphorus, and trace metals), proteins (mostly casein), carbohydrates (principally lactose), and lipids (fats). The amounts of these nutrients present in different types of milk differ greatly, however. Cows' milk and goats' milk are almost identical in every respect. Human milk contains less than half of the proteins and minerals of cows' or goats' milk, but almost 1.5 times as much sugar. Horses' milk is quite low in proteins and fats compared with the others, whereas reindeer milk is very high in proteins, fats, and minerals, but quite low in carbohydrates. The average composition of whole cows' milk is 87.1% water, 3.4% protein, 3.9% fats, 4.9% carbohydrates, and 0.7% minerals. The only important nutrients lacking in milk are iron and vitamin C.

Whole milk is an oil-in-water emulsion, containing its 3.9% fat dispersed as micron-sized globules. The fat emulsion is stabilized by complex phospholipids and proteins that are adsorbed on the surfaces of the globules. Because the fat in milk is so finely dispersed, it is digested more easily than fat from any other source. The globules are lighter than water, and thus coalesce on standing and eventually rise to the surface of the milk as cream. Vitamins A and D are fat-soluble substances and are thus concentrated in the cream. The fats in milk are primarily triglycerides, which are esters of saturated and unsaturated carboxylic acids with glycerol, a tri-alcohol. About two thirds of the fatty acids in milk are saturated, and consist primarily of C12, C14, and C16 acids. Milk is unusual in that about 12% of the fatty acids are short-chain fatty acids (C2-C10) like butyric, caproic, and caprylic acids. Additional lipids (fats and oils) in milk include small amounts of cholesterol, phospholipids, and lecithins. The phospholipids help to stabilize the whole milk emulsion, as the phosphate groups help to achieve partial water solubility for the fat globules. All the fat can be removed from milk by extraction with petroleum ether or a similar organic solvent.

There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All three are globular proteins, which tend to fold back on themselves into compact, nearly spheroidal units and are more easily solubilized in water as colloidal suspensions than fibrous proteins are. They are "complete proteins", so-called because they contain all the amino acids essential for building blood and tissue, and they can sustain life and provide normal growth even if they are the only proteins in the diet. These proteins not only contain more amino acids than plant proteins, but they contain greater amounts of amino acids than the proteins in eggs and meats.

Casein, the main protein in milk, is a phosphoprotein, meaning that phosphate groups are attached to the hydroxyl groups of some of the amino acid side-chains. Casein exists in milk as the calcium salt, calcium caseinate. It is actually a mixture of at least three similar proteins which differ primarily in molecular weight and the amount of phosphorus groups they contain. Alpha- and beta-casein have molecular weights in the 25,000 range and possess about 9 and 4-5 phosphate groups per molecule, respectively. They are both insoluble in water. Kappa-casein has a molecular weight of about 8,000 and possesses 1-2 phosphate groups per molecule. It is responsible for solubilizing the other two caseins in water by promoting the formation of micelles.

Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore, casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized (by protonation of the phosphate groups) and the neutral protein precipitates, with the calcium ions remaining in solution:

Ca-caseinate + 2H+ ---> casein + Ca2+

A natural example of this process occurs when milk sours. The souring of milk is an intricate process started by the action of microorganisms on the principal carbohydrate in milk, lactose. The microorganisms hydrolyse the lactose into glucose and galactose. Once galactose has been formed, lactobacilli, a strain of bacteria present in milk, convert it to the sour-tasting lactic acid. Since the production of the lactic acid also lowers the pH of the milk, the milk clots when it sours due to the precipitation of casein.

When the fats and proteins have been removed from milk, the carbohydrates remain in the whey, as they are soluble in aqueous solution. The main carbohydrate in milk is lactose. Lactose (4-O-( -D-galactopyranosyl)-D-glucopyranose) is the only carbohydrate that mammals synthesize. It is a dissacharide consisting of one molecule of D-glucose and one molecule of D-galactose joined in 1,4'-fashion, and is synthesized in the mammary glands. In this process, one molecule of glucose is converted to galactose and joined to another of glucose. Galactose is thought to be needed by developing infants to build brain and nervous tissue. It is more stable to metabolic oxidation than glucose and affords a better material for forming structural units in cells. The digestion of lactose involves the enzyme lactase, which hydrolyzes the disaccharide into its two component sugars.

In the first part of this experiment, we will isolate casein and lactose from cows' milk and carry out a few chemical tests on the isolated casein and lactose. As implied above, these are rather simple operations to carry out. Casein is precipitated by simply adjusting the pH of the milk to be sufficiently acidic that the protein is insoluble, taking care not to acidify too much so that the lactose does not hydrolyze. The other proteins remain water-soluble in acidic solution, but they can also be precipitated and isolated by merely heating the acidic solution and filtering. The isolated casein is insoluble in water, alcohol, and ether, but dissolves in alkaline and some acidic solutions. Once the casein is removed, lactose can be isolated as the alpha-anomer by addition of ethanol and crystallization from the resulting water-ethanol mixture at room temperature.

Casein is isolated from milk commercially and is industrially important because after dissolving in alkaline solutions and drying, it becomes a sticky substance that can be used in glues, the coating of paper, and the binding of colours in paints and wallpaper. It is also used as a coating for fine leather, and is cured with rennet to produce a plastic material used for buttons. When isolated under sanitary conditions and dissolved in alkaline solutions, casein is also employed in the manufacture of pharmaceutical and nutritional products.

In the test section of the experiment, we will carry out a few chemical tests on the isolated casein and lactose, as well as on test samples of other representative amino-acids and carbohydrates. Historically, these tests were designed for the purpose of structure elucidation. Since we already know the structures of these substances, we will use the chemical tests to demonstrate various aspects of the chemical reactivity of the protein casein. Of course, these tests depend on the specific structural features present in the molecules.


Isolation of Casein and Lactose from Milk

A. Isolation of Casein and Lactose from Milk 

Isolation of Casein


Weigh out 5 grams of powdered non-fat dry milk and dissolve it in 20 mL of warm water in a 100 mL beaker. Bring the temperature of the solution to 55oC (do not exceed) on a hot plate, remove the thermometer, and then add dropwise a solution of 10% acetic acid while stirring with a stirring rod. Do not add the acid too rapidly. Continue the acid addition (slightly less than 2 mL will be required), keeping the beaker on the hot plate, until the liquid changes from milky to almost clear and the casein no longer separates. It is important not to add too much acid, because it may hydrolyze some of the lactose in the milk and reduce your yield in Experiment 11B. Stir the precipitated casein until it forms a large amophous mass; then remove it with a stirring rod or tongs and place it in another beaker.

Immediately add 0.75 grams calcium carbonate to the original beaker containing the remaining liquid, stir for a few minutes, and save the resulting mixture for the later separation of lactose below. The separation of lactose should be done as soon as possible during the same laboratory period.

Collect the casein by suction filtration to remove as much water as possible. Press the solid with a spatula. Place the casein in a 100 mL beaker and add 5 mL of a mixture of 1:1 ethyl ether and ethanol (CAUTION: HIGHLY FLAMMABLE - NO FLAMES). Stir the casein in the ether for a few minutes, decant the ether, and repeat the process with a second 5 mL portion of ether. After the second washing with ether, suction filter the product. The ether washings remove any small quantities of fat that may have precipitated with the casein. Place the casein between several layers of paper towels to help dry the product, and let it stand in the air for 10-15 minutes. Divide the wet product in half, and weigh the two portions. Place one portion in a 125 mL Erlenmeyer flask with 35 mL of water and 0.5 mL of 1M NaOH, stopper the mixture, shake it to ensure solution of as much of the casein as possible, and save it for use in the chemical tests below. (You may carry out the chemical tests for the protein during this lab period if you have time, or in your next lab period.) Allow the second portion to dry in your locker over the following two weeks. When dry, weigh this portion and calculate the total yield of casein from the powdered milk. Show your calculations.

Isolation of Lactose


Gently boil the original liquid to which the calcium carbonate was added after isolation of casein. Bumping will not be a problem so long as you stir the solution constantly and vigorously with a glass rod. The solution will foam somewhat as it refluxes. This procedure precipitates the remaining proteins lactalbumin and lactoglobulin. Suction filter the hot mixture to remove the proteins and calcium carbonate, and transfer the hot, slightly yellow filtrate to a 125 mL Erlenmeyer flask. Concentrate the filtrate to a volume of about 5 mL by heating with constant swirling, again being careful to avoid bumping. Foaming can be controlled by heating the liquid less vigorously and gently blowing onto it.

To the hot, concentrated solution, add 25 mL of hot 95% ethanol and 0.2 gram of decolourizing carbon. Put this mixture aside and prepare a slurry of about 1 gram of Celite and 7.5 mL of 95% ethanol. Suction filter the slurry into a Hirsch funnel containing a correct sized filter paper to obtain a filter pad of Celite, and discard the alcohol in the filter flask. [The Celite filter pad helps collect the very fine particles of carbon and prevents the normal filter paper from becoming clogged.]

To the slightly cooled ethanol mixture containing the lactose, add 1 mL water. Suction filter the mixture through the Celite filter pad, making sure the filtrate is clear. If the filtrate is cloudy, heat it up and add another 0.5 mL of water. Transfer the filtrate to a 125 mL Erlenmeyer flask, heat it until it clears, then allow to cool slowly. Stopper the flask and allow it to stand in your locker until your next lab period.

Collect the crystals of lactose by suction filtration, and wash the product with a small amount of cold 95% ethanol. Thoroughly dry the lactose and determine its weight and melting point. Determine the percentage yield of lactose from the powdered milk, and show your calculations.

B. Chemical Tests for Proteins and Carbohydrates

Chemical Tests for Proteins and Amino Acids

In this experiment, you will perform chemical tests on the sample of casein which you isolated from milk, in order to determine the presence of specific amino acids in this type of protein. The tests will also be carried out on the amino acids, to help you identify a positive test with your sample, and on egg albumin, which is the main protein present in egg whites and is similar to the lactalbumin found in milk. While there are literally dozens of tests that are characteristic for only certain amino acids, we will carry out only three.

You will use the aqueous solution of casein which you prepared above (suction filter it if it is cloudy), along with stock solutions of egg albumin, tyrosine, glycine, and cysteine which have been prepared for you. Since some of the reagents used in these tests are toxic and/or corrosive, wear gloves, carry out the FIRST TWO tests in the fume hoods, and dispose of your waste in the proper labelled containers. Since we have more students than fume hoods, you will have to apportion your time carefully and stagger the amino acid tests with the carbohydrate tests, which you can carry out at the bench.


1. Millon's Test

Millon's test is given by any compound containing a phenolic hydroxy group. Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red colour. The Millon reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids (CAUTION: MILLON'S REAGENT IS HIGHLY TOXIC AND HIGHLY CORROSIVE). The red colour is probably due to a mercury salt of nitrated tyrosine.

Procedure: Place 1 mL of casein, 2% egg albumin, and 0.1 M tyrosine into separate, labelled, 12 x 75 mm test tubes. Add 3 drops of Millon's reagent and immerse the tubes in a boiling water bath for 5 minutes. Cool the tubes and record the colours formed.

2. Ninhydrin Test

The ninhydrin reaction is used to detect the presence of -amino acids and proteins containing free amino groups. When heated with ninhydrin, these molecules give characteristic deep blue colours (or occasionally pale yellow). The reactions involved in this test are shown below


Place 1 mL of of casein, 2% egg albumin, and 0.1 M glycine into separate, labelled, 12 x 75 mm test tubes. Add 4 drops of 0.1% ninhydrin solution. (CAUTION: NINHYDRIN IS A CARCINOGEN - AVOID DIRECT CONTACT) Add a boiling chip to each test tube and heat to boiling in a hot-water bath. Record the results.

3. Sulfur Test

The presence of sulfur-containing amino acids such as cysteine can be determined by converting the sulfur to an inorganic sulfide through cleavage by base. When the resulting solution is combined with lead acetate, a black precipitate of lead sulfide results.
Sulfur-containing protein ----NaOH----> S2- ----Pb2+----> PbS

Procedure: Place 1 mL of casein, 2% egg albumin, and 0.1 M cysteine into separate, labelled 16 x 150 mm test tubes. Add 2 mL of 10% aqueous sodium hydroxide. Add 5 drops of 10% lead acetate solution. Stopper the tubes and shake them, then remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results.

Chemical Tests for Carbohydrates

In this experiment, you will perform tests and reactions on the sample of lactose which you isolated from milk and on samples of selected other mono- and disaccharides.

1. Benedict's Test

Benedict's test determines whether a monosaccharide or disaccharide is a reducing sugar, and is hence similar in purpose to the Tollens test. To give a positive test, the carbohydrate must contain a hemiacetal which will hydrolyse in aqueous solution to the aldehyde form. Benedict's reagent is an alkaline solution containing cupric ions, which oxidize the aldehyde to a carboxylic acid. In turn, the cupric ions are reduced to cuprous oxide, which forms a red precipitate.
RCHO + 2Cu2+ + 4OH- -----> RCOOH + Cu2O + 2H2O
Procedure Place 15 drops of the following 1% carbohydrate solutions in separate, labelled 12X75 mL test tubes: glucose, fructose, sucrose, lactose, and maltose. Also place 1 mL of distilled water in another tube to serve as a control. To each tube, add 1 mL of Benedict's reagent and heat the tubes in a boiling water bath for 5 minutes. Remove the tubes and note and record the results.

2. Barfoed's Test

Barfoed's test is similar to Benedict's test, but determines if a carbohydrate is a monosaccharide or a disaccharide. Barfoed's reagent reacts with monosaccharides to produce cuprous oxide at a faster rate than disaccharides do:
RCHO + 2Cu2+ + 2H2O -----> RCOOH + Cu2O + 4H+
Procedure: Place 15 drops of the following 1% carbohydrate solutions in separate, labelled 12X75 mL test tubes: glucose, fructose, sucrose, lactose, and maltose. To each tube, add 1 mL of Barfoed's reagent and heat the tubes in a boiling water bath for 10 minutes. Remove the tubes and note and record the results.

3. Hydrolysis Test for Glucose

Disaccharides and polysaccharides can be hydrolyzed in acidic solution into their component monosaccharides, and then submitted to chemical tests like Benedict's test. In this experiment, several disaccharides and a sample of starch will be hydrolyzed, and tested for the presence of glucose. The glucose test will be carried out using a commercially available product called Tes-Tape. Available at most drug stores, the tape contains the enzymes glucose oxidase and peroxidase, as well as ortho-toluidine. The glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide. Once formed, the hydrogen peroxide reacts with peroxidase to produce oxygen, which oxidizes the ortho-toluidine to give green-coloured products.


Place 5 mL of the following 1% carbohydrate solutions in separate, labelled 16 x 150mm test tubes: sucrose, lactose, maltose, and starch. Add 3 drops of concentrated hydrochloric acid to each of the tubes, and heat them in a boiling water bath (400 mL beaker) for 10 minutes. Cool the tubes in an ice bath. Carefully neutralize each of the four solutions with 10% sodium hydroxide, using litmus or pH paper. The pH MUST be neutral or very slightly alkaline in order for the Tes-Tape to work. If necessary, make final pH adjustments with 0.1M HCl and/or 0.1M NaOH solutions. Test each solution with Tes-Tape (by placing a drop on the tape and recording the colour change - use plain distilled water as a control) and if time permits, with Benedict's reagent. Record the results and compare them with those obtained earlier with the Benedict's tests on the unhydrolysed carbohydrates.


Chemistry of carbohydrates

Simple sugars, starches and cellulose are organic compounds that have the approximate formula C(H2O)n, which accounts for the name carbohydrate (or hydrate of carbon) that is usually applied to this group of compounds. They are not truly hydrates of carbon but are polyhydroxy (alcohol) compounds that contain an aldehyde or ketone functional group. These functional groups give the carbohydrates some of their chemical properties that will be studied in this lab.

Simple sugars are called monosaccharides (one sugar), or disaccharides (2 sugars). Some monosaccharides are glucose, fructose, galactose, and xylose. Note that xylose is a pentose and fructose is a ketose.

Two common disaccharides are sucrose (table sugar) and lactose (milk sugar); sucrose is a combination of glucose and fructose linked together by their anomeric carbons to produce a nonreducing sugar (it does not reduce Cu2+), whereas lactose is a combination of galactose and glucose linked together by a β-1,4-glycosidic bond to produce a reducing disaccharide. When many sugar molecules are linked together into a polymer, the resulting compound is called a polysaccharide. Starches and celluloses are polysaccharides. Amylose is a linear chain polymer of glucose, whereas amylopectin (a plant starch) and glycogen (an animal starch) , starches and cellulose are organic compounds that have the approximate formula C(H2O)n, which accounts for the name carbohydrate (or hydrate of carbon) that is usually applied to this group of compounds. They are not truly hydrates of carbon but are polyhydroxy (alcohol) compounds that contain an aldehyde or ketone functional group. These functional groups give the carbohydrates some of their chemical properties that will be studied in this lab.


Qualitative Tests for Carbohydrates

Carbohydrates are a class of organic molecules with the general chemical formula Cn(H2O)n. These compounds are literally carbon hydrates. Only the monomeric form of these compounds, the monosaccharides, fit this description precisely. Two monosaccharides can be polymerized together through a glycosidic linkage to form a disaccharide. When a few monosaccharide molecules are polymerized together, the result is an oligosaccharide. A polysaccharide is an extensive polymer of carbohydrate monomers.

The monosaccharide glucose is our primary energy source. The function of the polysaccharides starch (plants) and glycogen (animals) is to store glucose in a readily accessible form, as well as to lower the osmotic potential of internal fluids. Some polysaccharides serve a structural role in living organisms. The glucose polymer cellulose is a major component of plant cell walls. Chitin, a polymer of N-acetylglucosamine, is a major structural component of the exoskeleton of insects and crustaceans. Hyaluronic acid and chondroitin sulfate occurs in the connective tissues of animals, especially in cartilage. Oligosaccharide side chains of glycoproteins may also serve as signals for intracellular sorting of the protein (i.e. mannose-6-phosphate signal designating lysosomal enzymes).

Several qualitative tests have been devised to detect members of this biologically significant class of compounds. These tests will utilize a test reagent that will yield a color change after reacting with specific functional groups of the compounds being tested. The following exercises are reactions that can detect the presence or absence of carbohydrates in test solutions. They range in specificity to the very general (i.e. Molisch test for carbohydrates) to the very specific (i.e. mucic acid test for galactose).


Molisch Test for Carbohydrates

The Molisch test is a general test for the presence of carbohydrates. Molisch reagent is a solution of alpha-naphthol in 95% ethanol. This test is useful for identifying any compound which can be dehydrated to furfural or hydroxymethylfurfural in the presence of H2SO4. Furfural is derived from the dehydration of pentoses and pentosans, while hydroxymethylfurfural is produced from hexoses and hexosans. Oligosaccharides and polysaccharides are hydrolyzed to yield their repeating monomers by the acid. The alpha-naphthol reacts with the cyclic aldehydes to form purple colored condensation products. Although this test will detect compounds other than carbohydrates (i.e. glycoproteins), a negative result indicates the ABSENCE of carbohydrates.


Add 2 drops of Molisch reagent to 2 ml of the sugar solution and mix thoroughly. Incline the tube, and GENTLY pour 5 ml of concentrated H2SO4 down the side of the test tube. A purple color at the interface of the sugar and acid indicates a positive test. Disregard a green color if it appears.


Benedicts's Test for Reducing Sugars

Alkaline solutions of copper are reduced by sugars having a free aldehyde or ketone group, with the formation of colored cuprous oxide. Benedict's solution is composed of copper sulfate, sodium carbonate, and sodium citrate (pH 10.5). The citrate will form soluble complex ions with Cu++, preventing the precipitation of CuCO3 in alkaline solutions.


Add 1 ml of the solution to be tested to 5 ml of Benedict's solution, and shake each tube. Place the tube in a boiling water bath and heat for 3 minutes. Remove the tubes from the heat and allow them to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing sugars.


Barfoed's Test for Monosaccharides

This reaction will detect reducing monosaccharides in the presence of disaccharides. This reagent uses copper ions to detect reducing sugars in an acidic solution. Barfoed's reagent is copper acetate in dilute acetic acid (pH 4.6). Look for the same color changes as in Benedict's test.


Add 1 ml of the solution to be tested to 3 ml of freshly prepared Barfoed's reagent. Place test tubes into a boiling water bath and heat for 3 minutes. Remove the tubes from the bath and allow to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing monosaccharides. Do not heat the tubes longer than 3 minutes, as a positive test can be obtained with disaccharides if they are heated long enough.


Lasker and Enkelwitz Test for Ketoses

The Lasker and Enkelwitz test utilizes Benedict's solution, although the reaction is carried out at a much lower temperature. The color changes that are seen during this test are the same as with Benedict's solution. Use DILUTE sugar solutions with this test (0.02 M).


Add 1 ml of the solution to be tested to 5 ml of Benedict's solution to a test tube and mix well. The test tube is heated in a 55oC water bath for 10-20 minutes. Ketopentoses demonstrate a positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react. Aldoses do not react positively with this test.


Bial's Test for Pentoses

Bial's reagent uses orcinol, HCl, and FeCl3. Orcinol forms colored condensation products with furfural generated by the dehydration of pentoses and pentosans. It is necessary to use DILUTE sugar solutions with this test (0.02 M).


Add 2 ml of the solution to be tested to 5 ml of Bial's reagent. Gently heat the tube to boiling. Allow the tube to cool. Formation of a green colored solution or precipitate denotes a positive reaction.


Mucic Acid Test for Galactose

Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids. However, oxidation of galactose yields an insoluble mucic acid. Lactose will also yield a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose subunits.


Add 1 ml of concentrated nitric acid to 5 ml of the solution to be tested and mix well. Heat on a boiling water bath until the volume of the solution is reduced to about 1 ml. Remove the mixture from the water bath and let it cool at room temperature overnight. The presence of insoluble crystals in the bottom of the tube indicates the presence of mucic acid.  


Iodine Test for Starch and Glycogen

The use of Lugol's iodine reagent (IKI) is useful to distinguish starch and glycogen from other polysaccharides. Lugol's iodine yields a blue-black color in the presence of starch. Glycogen reacts with Lugol's reagent to give a brown-blue color. Other polysaccharides and monosaccharides yield no color change; the test solution remains the characteristic brown-yellow of the reagent. It is thought that starch and glycogen form helical coils. Iodine atoms can then fit into the helices to form a starch-iodine or glycogen-iodine complex. Starch in the form of amylose and amylopectin has less branches than glycogen. This means that the helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine complex.


Add 2-3 drops of Lugol's iodine solution to 5 ml of solution to be tested. Starch gives a blue-black color. A positive test for glycogen is a brown-blue color. A negative test is the brown-yellow color of the test reagent.


Seliwanoff's Test

Seliwanoff's reagent contains resorcinol in 6 M hydrochloric acid. Hexoses undergo dehydration when heated in this reagent to form hydroxymethylfurfural, that condenses with resorcinol to give a red product. Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherry-red condensation product. Other sugars may produce yellow to faint pink colors.

Empty, wash and rinse the test tubes from part B and make sure you have sufficient water in your boiling water bath.

Caution: Seliwanoff’s reagent is caustic, rinse thoroughly with water if you get this solution on your skin or clothing.

Add about 3 mL of Seliwanoff's reagent to each labeled test tube.

Add 1 drop of the respective sugar solution and 1 drop of water blank to the appropriate test tubes as described in part A-1 above and mix well.

Place all the test tubes in the boiling water bath at the same time and heat for 3 min after the water begins to boil again.

Record observations on the report sheet.

What conclusions can you make about your unknown now?

Discard these solutions in the sink and rinse with plenty of water.


Benedict's reagent

Contains Cu2+ ions in alkaline solution with sodium citrate added to keep the cupric ions in solution. The alkaline conditions of this test causes isomeric transformation of ketoses to aldoses, resulting in all monosaccharides and most disaccharides reducing the blue Cu2+ ion to cuprous oxide (Cu2O), a brick red-orange precipitate. This solution has been used in clinical laboratories for testing


Barfoed's reagent

Barfoed's solution contains cupric ions in an acidic medium. The milder condition allows oxidation of monosaccharides but does not oxidize disaccharides. If the time of heating is carefully controlled, disaccharides do not react while reducing monosaccharides give the positive result (red Cu2O precipitate). Ketoses do not isomerize with this reagent. Carbohydrates are dehydrated in the presence of nonoxidizing acids to form furfural and hydroxymethylfurfural.


Bial's reagent

Bial's reagent contains orcinol (5-methylresorcinol) in concentrated HCl with a small amount of FeCl3 catalyst. Pentoses are converted to furfural by this reagent, which form a bluegreen color with orcinol. This test is used to distinguish pentoses from hexoses


Enzymatic Digestion of Starch

  1. Collect about 2 mL of saliva in a clean test tube.
  2. Add 6 mL of neutral (pH 7) buffer solution to the saliva and mix well.
  3. Pour half of the saliva mixture into another clean test tube and label these test tubes S1
    and S2 (for saliva)
  4. Add about 4 mL of neutral buffer (no saliva) to each of 2 other clean test tubes and label
    these B1 and B2 (for buffer).
  5. Add about 10 drops of starch solution to each of the 4 tubes (2 with saliva and 2 with
    buffer only) and mix well. Allow the mixtures to stand while you proceed with parts A
    thru E.
  6. After allowing the solutions to stand for at least 30 min, test for glucose with Benedict's
    reagent and starch with iodine as described below.
  7. Add 3 drops of Benedict's reagent to one tube containing saliva (S1) and to one tube
    containing buffer (B1), mix well and place both tubes in the hot water bath for several
  8. Add 1 drop of iodine solution to the other tube containing saliva (S2) and the other tube
    containing buffer (B2) and mix well. It is not necessary to heat these 2 tubes.
  9. Record your observations for these 4 tubes on the report sheet and answer the questions
    for this part.



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Any substance or mixture of substances intended for preventing, destroying or controlling any pest, including vectors of human or animal disease, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs, or substances which may be administered to animals for the control of insects, arachnids or other pests in or on their bodies. The term includes substances intended for use as a plant growth regulator, defoliant, desiccant or agent for thining fruit or preventing the premature fall of fruit, and substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport.


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Classification of pesticides (Algicides, Bactericides,Insecticides, Herbicides, etc..)

Pesticides can be classified by target organism, chemical structure, and physical state. Pesticides can also be classed as inorganic, synthetic, or biologicals (biopesticides), although the distinction can sometimes blur. Biopesticides include microbial pesticides and biochemical pesticides. Plant-derived pesticides, or "botanicals", have been developing quickly. These include the pyrethroids, rotenoids, nicotinoids, and a fourth group that includes strychnine and scilliroside.

Many pesticides can be grouped into chemical families. Prominent insecticide families include organochlorines, organophosphates, and carbamates. Organochlorine hydrocarbons (e.g. DDT) could be separated into dichlorodiphenylethanes, cyclodiene compounds, and other related compounds. They operate by disrupting the sodium/potassium balance of the never fiber, forcing the nerve to transmit continuously. Their toxicities vary greatly, but they have been phased out because of their persistence and potential to bioaccumulate. Organophosphate and carbamates largely replaced organochlorines. Both operate through inhibiting the enzyme acetylcholinesterase, allowing acetylcholine to transfer nerve impulses indefinitely and causing a variety of symptoms such as weakness or paralysis. 

Organophosphates are quite toxic to vertebrates, and have in some cases been replaced by less toxic carbamates. Thiocarbamate and dithiocarbamates are subclasses of carbamates. Prominent families of herbicides include pheoxy and benzoic acid herbicides (e.g. 2,4-D), triazines (e.g. atrazine), ureas (e.g. diuron), and Chloroacetanilides (e.g. alachlor). Phenoxy compounds tend to selectively kill broadleaved weeds rather than grasses. The phenoxy and benzoic acid herbicides function similar to plant growth hormones, and grow cells without normal cell division, crushing the plants nutrient transport system. Triazines interfere with photsynthesis. Many commonly used pesticides are not included in these families, including glyphosate.
  • Algicides or algaecides for the control of algae
  • Avicides for the control of birds
  • Bactericides for the control of bacteria
  • Fungicides for the control of fungi and oomycetes is a very bad
  • Herbicides (e.g. glyphosate) for the control of weeds
  • Insecticides (e.g. organochlorines, organophosphates, carbamates, and pyrethroids) for the control of insects - these can be ovicides (substances that kill eggs), larvicides (substances that kill larvae) or adulticides (substances that kill adults)
  • Miticides or acaricides for the control of mites
  • Molluscicides for the control of slugs and snails
  • Nematicides for the control of nematodes
  • Rodenticides for the control of rodents
  • Virucides for the control of viruses
Pesticides can be classified based upon their biological mechanism function or application method. Most pesticides work by poisoning pests. A systemic pesticide moves inside a plant following absorption by the plant. With insecticides and most fungicides, this movement is usually upward (through the xylem) and outward. Increased efficiency may be a result. Systemic insecticides, which poison pollen and nectar in the flowers, may kill bees and other needed pollinators.

In 2009, the development of a new class of fungicides called paldoxins was announced. These work by taking advantage of natural defense chemicals released by plants called phytoalexins, which fungi then detoxify using enzymes. The paldoxins inhibit the fungi's detoxification enzymes. They are believed to be safer and greener.


Particular uses of titrations and titration curves and Acid number, Iodine number, Saponification value, Karl Fischer titration

  • As applied to biodiesel, titration is the act of determining the acidity of a sample of WVO by the dropwise addition of a known base to the sample while testing with pH paper for the desired pH=8.5 reading. By knowing how much base neutralizes an amount of WVO, we discern how much base to add to the entire batch.

  • Titrations are a very common procedure held in secondary education, to assess a chemistry student's practical aptitude.
  • Titrations in the petrochemical or food industry to define oils, fats or biodiesel and similar substances. An example procedure for all three can be found here:
o Acid number: an acid-base titration with colour indicator is used to determine the free fatty acid content. See also: pH of fatty acids.

o Iodine number: a redox titration with colour indication, which indicates the amount of unsaturated fatty acids.

o Saponification value: an acid-base back titration with colour indicator or potentiometric to get a hint about the average chain length of fatty acids in a fat.

o Karl Fischer titration a method to analyse trace amounts of water in a substance



Identification of the constituents of a given sample and the nature of the constituents are mainly done in the qualitative analysis. But the determination of how much of each component present in a sample is obtained by quantitative analysis.

When a sample is given for quantitative analysis there can be several techniques to be applied. But the chemist should follow the most suitable technique in order to get the best result. The function of the chemist is to obtain a result as near to the true value as possible by the correct application of the procedure employed. For that purpose he/she must be familiar not only with the practical details of various techniques, but also with theoretical principles involved, the possibilities of interferences from other ions, elements and compounds as well as with the accuracy and precision he/she uses.


Techniques employed analysis -Classical,Spectroscopics,Electroanalytical,Separational,Radio Chemical,Thermoanalytical,Kinetic Methods

Some of the techniques employed in such analysis is given below.

1. Classical Methods
Titrimetry and Gravimetry

2. Spectroscopic Methods
Colorimetry, Atomic Adsorption and Atomic Emission Spectroscopy, Flame Photometry & X-ray Fluorescence

3. Electroanalytical Methods

Coulometry, Potentiometry, Polarography & Striping Voltammetry.

4. Separational Methods
Solvent Extraction, Gas Chromatography (GC), HPLC, Ion Exchange methods, Ion Chromatography

5. Radio Chemical Methods
Neutron Activation Analysis & Gamma Spectroscopy

6. Thermoanalytical Techniques
Thermometric Titrimetry

7. Kinetic Methods

Some of the above mentioned techniques require quite expensive instruments, therefore classical methods are still in wide use.


Analysis techniques in detail- CLASSICAL METHODS- Titrimetric Analysis, Standard solution, ‘end point’ of the titration, indicator, Free Energy Change

Titrimetric analysis refers the quantitative chemical analysis carried out by determining the volume of a solution of accurately known concentration which is required to react quantitatively with the solution of the substance to be determined. The solution of accurately known strength, the Standard solution, is called the titrant and the substance being titrated is termed the titrand. A titration is a reaction between the titrant in the burette and the titrand in the flask. The process of adding the standard solution until the reaction is just complete is termed a titration, and the substance to be determined is titrated.

All chemical reactions can not be considered as titrations. A reaction can serve as a basis of a titration procedure, only if the following conditions are satisfied.

a) The reaction must be a fast one
b) It must proceed stoichiometrically
c) The change in free energy (ΔG) during the reaction must be sufficiently large for spontaneity of the reaction.
d) There should be a way to detect the completion of the reaction.

The point at which an equivalent amount of the titrant has been added is called the ‘equivalence point’ or ‘theoretical or stoichiometric end point’. This is detected by some physical change, produced by the solution by it self or more usually by the addition of an auxillary reagent known as an ‘indicator’. After the reaction is practically complete, the indicator should give a clear visual change (either a colour change or a formation of turbidity) in the solution being titrated. The point at which this occurs is called the ‘end point’ of the titration. In the idea titration the visible end point will coincide with the stoichiometric or theoretical end point. In practice, however a very small difference usually occurs; this represents the titration error. Therefore the indicator and the experimental conditions should be selected so that this difference becomes as small as possible.

When a titration is carried out the free energy change for the reaction is always negative.
e.g. During the initial stages of the reaction between A & B, when the titrant A is added to B the following reaction takes place.

a = activity

Large values of the equilibrium constant K implies that the equilibrium concentration of A & B are very small at the equivalence point. It also indicates that the reverse reaction is negligible and the product C & D are very much more stable than the reactants A & B. Greater the value of K the larger the magnitude of the negative free energy change for the reaction between A & B. Since

Free Energy Change = ΔG = -RT ln K

Where R = Universal gas Constant = 8.314 JK-1mol-1

T = Absolute Temperature.

The reaction of the concentration of A & B leads to the reduction of the total free energy change. If the concentrations of A & B are too low the magnitude of the total free energy change becomes so small and the use of the reaction for titration will not be feasible.


Monday, January 9, 2012

Analysis tecniques in detail- Classical methods -Titration curves

The titration curve can be plotted using experimental results or can be calculated theoretically. It gives an indication of the completion of the reaction. The size and the steepness of the vertical portion of the titration curve around the experimental point governs the feasibility of the titration by accurate and precise location of the experimental end point.

 A generalized titration curve

The derivative curve

pB = - log [B]
When the magnitude of the free energy during a particular titration is larger the vertical portion of the slope of the titration curve is long and steep and the maximum value of the derivative curve is also large. The titration curve is useful :

a) In establishing the feasibility of a particular reaction to be used in titration,
b) In the prediction of the sharpness of the end point,
c) In the selection of the suitable indicator.


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