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Structure of water Water is a molecular compound containing H2O molecules in which two Hydrogen atoms are bound to the oxygen atom forming an angle of 104.5°. The OH distance (bond length) is 9.57 1011 metres. Because an oxygen atom has a greater electronegativity than a hydrogen atom, the OH bonds in the water molecule are polar, with the oxygen bearing a partial negative charge 1 () and the hydrogens having a partial positive charge (+). The electron arrangement in the water molecule can be represented as follows. Each pair of dots represents a pair of unshared electrons (i.e., the electrons reside on only the oxygen atom). 2 This electronic structure leads to an interaction between water molecules that is called hydrogen bonding, in which a positive hydrogen atom on one molecule is attracted to the electron density of one of the unshared electron pairs on the oxygen atom of another molecule. 3 4 Concentration based on volume The majority of reaction studied by biochemists occur in solution. Concentration based on the amount of dissolved solute per unit volume are the most widely used in biochemistry laboratories. Solution concentration: Concentration is the amount of a solute in a given amount of solution. The molarity of a solute is the moles of solute per liter of solution (the number of moles of solute per liter of solution). Molarity = Moles of solute/ Liters of solution Units: 1 Molar (M) = 1mole/L 5 Molar con are usually given in square brackets. Ex, [H]= molarity of H ion. To calculate M , we need to know the weight of dissolved solute and its molecular weight, MW. Wtg/ MW = moles. Dilute solutions are often expressed in terms of millimolarity, Micromolarity, and so on , where: 1 mmole = 10-3 moles 1μmole = 10-6 moles 1nmole = 10-9 moles Therefore: 1 mM= 10-3 M = 1mmole/ Liter = 1μmole/ml 1 μM = 10-6 M = 1μmole/Liter = 1 nmole/ ml 6 Normality (N)= the number of equivalents of solute per liter of solution. To calculate N we need to know the weight of dissolved solute and its equivalent weight, EW. Wtg/ EW= equivalents One equivalent (EW) of an acid or base is the weight that contains 1g-atom (1mole) of replaceable hydrogen or 1g-atom (1mole) of replaceable hydroxyl. Ew= Mw/n N= the number of replaceable H or OH per molecule( for acids and bases) 7 The molarity and normality are related by: N=nM Ex, a 0.01M solution of H2SO4 is 0.02N. Q: How many moles of NH3 are dissolved in 75 ml of 6.0 M NH3? Q: How do you prepare 250 ml of 1.00 M CuSo4 solution? Mw of CuSo4= 249.7 8 Introduction The branch of chemistry that deals with the separation, identification and determination of components in a sample. Thus, it is answers to the basic questions: What is in this sample? and How much of it is there? The first question is qualitative and it is related to the detection and identification of the components of a sample. The second question is quantitative. 9 Applications of chemical analysis: Applications of chemical analysis are found everywhere in industry, universities, hospitals…etc The chemical analysis is playing an important role in the following area: 1- Environmental: Chemical analysis of air and water is required for measuring pollutants or contamination. 2- Medical: Measuring the levels of critical substances in the blood stream of hospital patient. For example, the presence of high amount of bilirubin and concentration of transaminases enzymes in patient s blood stream is assign of an impaired liver function. 10 3-Nutrition: Determination of nitrogen content of breakfast cereals and other foods can be directly related to their protein content and thus their nutritional qualities. 3- Agricultural: Chemical analysis of soil and plants is used to determine the nutrients or fertilizers which must be add to the soil to increase productivity. 4-Research Quantitative analysis data are the backbone of research activity in chemistry, biochemistry, biology and other braches of sciences. 11 There are other areas in which chemical analysis is playing an important role are pharmacological and industry. Analytical chemistry can be broken down in to general areas of analysis: 1- Qualitative analysis deal with finding what materials are present in an analytical sample. It is not a method for determining the concentration. If the chemical species (materials) are known the concentration of that species in the sample can be determined by quantitative analysis. A variety of tools are used for qualitative analysis such as spectroscopic methods are quick and simple 12 ways to detect metals and organic compounds. Microscopic examination is also a powerful qualitative tool. It may be necessary to do a complete qualitative analysis on a sample before discovering how to determine just one or two components quantitatively. This is because many elements are sufficiently similar in their behavior that they interfere in quantitative determination. 13 2- Quantitative analysis deals with the determination of how much of a material is present in a sample. The goal is to determine the amount of each component in a sample. Quantitative analysis may be classified based on the size of sample which is available for analysis. When a sample weighing more than 0.1g is available the analysis called as macro analysis. Micro analysis deal with samples weighing from 1 to 10mg. 14 Sampling: The type of sample that must be taken for analysis will depend on the information desired. In the case of biological fluids, the conditions under which the sample is collected can be important. For example, whether a patient has just eaten. Because the composition of blood varies before and after meals and for many analyses a sample is collected after the patient has fasted for a number of hours. Preservatives such as sodium fluoride for glucose preservation and anti coagulants may be added to blood samples when they are collected these may affect a particular analysis. Blood samples may be analyzed as whole blood or they may be 15 separated to yield plasma or serum according to the requirement of the particular analysis. If whole blood is collected and allowed to stand for several minutes, the soluble protein fibrinogen will be converted by a complex series of chemical reactions (involving calcium ion) into the insoluble protein fibrin, which form the basis of a clot. The clotting process can be prevented by adding a small amount of an anticoagulant, such as heparin or a citrate salt. Handling the sample: Precautions should be taken in handling and storing samples to prevent or minimize contamination, loss. The sample may have to protect from the atmosphere or from light. 16 Examples 1- Alkaline substance will react with carbon dioxide in the air. 2-Blood samples analyzed for carbon dioxide should be protected from the atmosphere. 3- Glucose is unstable and a preservative such as sodium fluoride is added to blood samples. 4- Protein and enzymes tend to denature on standing and should be analyzed without delay. 5- Addition 1 or 2 ml of acetic acid per 100 ml of urine prevent precipitation (pH 4.5). Store under refrigeration. Because urine samples are unstable. 6-Whole blood, serum, plasma and tissue samples can be frozen for prolonged storage. 17 Types of Methods 1- Volumetric analysis or titrimetric analysis. Methods based on a measured volume 2- Gravimetry Methods based on a measured weight 3- Electrochemical Methods based on a measured of current, charge and resistance. 4- Chromatography Methods based on the interaction with two different phases. 5- Chemometrics 18 The statistical treatment of data. Q: What type of information do you need? Complete analysis which is mean determines the amount of each component in a sample. Partial analysis: Determining one or a limited number of species in a sample. Ex, electrolyte levels in blood and presence of lead in a water sample. 19 Titrimetric analysis (Volumetric analysis) Titration slow addition of a standard solution to a solution of analyte until the reaction between the two is complete. It is used for determining the concentration of a solution. A known volume of a solution of unknown concentration is reacting with a known volume of a solution of known concentration (standard). The standard solution is delivered from a burette so the volume added is known. 20 For example of titrimetric analysis is the titration of a solution of an unknown con of hydrochloric acid (HCl) with sodium hydroxide (NaOH) solution of known conc. The end point of titration is located with an acid-base indicator. OH from NaOH combines with Hydrogen ion HCl as the following: H + OH H2O Generally, the burette is filled with the titrant, while the titrate is in the conical flask. On every titrant addition, the reaction consumes part of the titrate. If all the titrate is consumed by the 21 last titrant addition, the equivalence point is reached. Since both the concentration and the volume of NaOH solution are known the number of moles of OH which react with the HCl can be calculated. Equivalence point –reached when the amount of added titrant is chemically equivalent to the amount of analyte. Standard Solution Standard solution is a reagent whose concentration is known exactly. Standard solutions are used to perform titrations in which the quantity of analyte in a solution is 22 determined from the volume of standard solution that is consumes. Prepared of standard solution By dissolving a carefully weighed quantity of the pure reagent and diluting to an exactly known volume. It is essential for the chemical standards to meet the following: 1- High purity 2- Stability in air (not be attacked by atmosphere) 3- Reasonable solubility in solvent 4- Availability at modest price 23 Classification of Volumetric methods The are major general classes of volumetric methods 1- Neutralization titrations 2- Oxidation- Reduction titration: These redox titrations involve the titration of an oxidizing agent with reducing agent. An oxidizing agent gains electrons in a reaction between them. 3- Precipitation titration: In the case of precipitation, the titrant forms an insoluble product with analyte. Ex, the titration 24 of chloride ion with silver nitrate solution. Also, indicators can be used to detect the end-point. Chemical Reactions in Solution: A solution is a homogenous mixture. The substance present in the greatest amount is the solvent. The other substances are solutes. In many reactions, one or more of the reactants are in solution. Solution concentration: Concentration is the amount of a solute in a given amount of solution. 25 The molarity of a solute is the moles of solute per liter of solution (the number of moles of solute per liter of solution). Molarity = Moles of solute/ Liters of solution Units: 1 Molar (M) = 1mole/L Molarity is the conversion factor between moles of solute and volume of solution. Molar con are usually given in square brackets. Ex, [H]= molarity of H ion. To calculate M , we 26 need to know the weight of dissolved solute and its molecular weight, MW. Wtg/ MW = moles. Normality (N)= the number of equivalents of solute per liter of solution. To calculate N we need to know the weight of dissolved solute and its equivalent weight, EW. Wtg/ EW= equivalents One equivalent (EW) of an acid or base is the weight that contains 1g-atom (1mole) of replaceable hydrogen or 1g-atom (1mole) of replaceable hydroxyl. 27 Ew= Mw/n N= the number of replaceable H or OH per molecule( for acids and bases) The molarity and normality are related by: N=nM Ex, a 0.01M solution of H2SO4 is 0.02N. Q: How many moles of NH3 are dissolved in 75 ml of 6.0 M NH3? 28 Q: How do you prepare 250 ml of 1.00 M CuSo4 solution? Mw of CuSo4= 249.7 Atomic weight An atom consists of protons, neutrons and electrons. The protons and neutrons are in the nucleus and electrons are somewhere around the nucleus but at great distance in relation to the size of nucleus. Most of the weight of an atom is concentrated in the nucleus. The mass of individual atoms is around 10-24 gram. For example: one hydrogen atom weighs 1.6738 × 10-24 g. Because atoms 29 have such small weights, scientists have agreed a set of relative masses of atoms called atomic weights Because atomic weights represent relative masses, one element must be selected as the standard. Carbon isotope C12 was assigned arbitrary value of 12 atomic weight units and the weight of all other elements were related to carbon- 12. Note: Isotopes are atoms of the same element which differ only in atomic weight (as all atoms of an element have the same number of protons, 30 isotopes differ in neutrons number) e.g. Carbon has two isotopes 12 to 13. Gram Atomic Weight: The gram atomic weight of an element is the atomic weight in grams. Atomic number is the number of protons in the nucleus. Ex; H = 1.0079 31 Molecular Weight The molecular weight for a compound is the sum of the atomic weights of each atom that makes up the compound. Example: the molecular (or formula) weight of sulfuric acid H2SO4 is : 2× 1.01 = 2.02 1 × 32.06 = 32.06 4 × 64.00 = 64.00 Molecular weight = 98.08 Equivalent Weight 32 If a substance can react in more than one way, it will have more than one equivalent weight. Thus the definition of equivalent weight for a substance is always based on its behavior in a specific chemical reaction. In neutralization reaction: the equivalent weight of a substance is that weight which either contributes or reacts with one mole of (H) ion in the reaction. For example the equivalent weight for potassium hydroxide KOH and HCl must be equal to their molecular weight because each has only a single reactive hydrogen ion (H) or Hydroxide ion. Barium hydroxide , Ba (OH)2 is a strong base containing two reactive hydroxide ions. This base will necessarily react with two hydrogen ions (H) in any acid-base reaction: thus its 33 equivalent weight will be one-have its molecular weigh. We define an equivalent of any substance as its equivalent weight (aq. Wt) expressed in gram. Thus, 1 equivalent of H2NO3 In an acid-base reaction, one equivalent of any acid exactly neutralize one equivalent of any base. EW= MW/n N= number of H or OH (per molecule for acids and bases) 34 Electrolytes: An electrolyte dissolves in water to form ions. A non electrolyte does not form ions. Strong Electrolytes: Strong electrolytes are completely dissociated into their ions, such as strong acids and strong alkalis, also most inorganic salts are strong electrolytes. HCl is a good example as strong electrolytes. Thus if 100 molecules of HCl are dissolved in water, 100 H ions and 100 Cl ions are produced, no HCl molecules exist in aqueous solution. 35 Another example of strong electrolyte is sodium hydroxide NaOH which contain hydroxide ions (OH) that completely dissociate when dissolved in water. Weak electrolytes: They are substances that produce relatively few ions when dissolved in water or other ionizing solvents (poor conductors of electric current). The most common weak electrolytes are weak base and weak acids. Ex of weak electrolyte is acetic acid when 100 molecules of CH3 COOH are dissolved in water approx 99 molecules of CH3 COOH remain intact and only H ion and one CH3 COO- ion are produced (1%). Non electrolyte: 36 Non electrolytes are substances that dissolve in water but do not produce any ions. Nonelectrolyt solution does not conduct electric current. Ex, ethanol and sucrose. Sucrose is stable sugar which is very soluble in water but produces no ions when it dissolves. Acids and bases Acid is a substance that donates protons (hydrogen ions). Base is a substance that accepts protons. A strong acid is a substance that ionizes almost 100% in aqueous solution. Ex HCl in solution is essentially 100% to H+ and Cl-. Other examples of acids are H2 SO4 , HNO3, A strong base is a substance that ionizes extensively in solution to yield OH ions. Potassium hydroxides are ex of strong inorganic bases. 37 KOH in a solution is essentially 100% ionized to K+ and -OH. Other ex. NaOH, Buffers; A buffer is a solution that resists change in pH following the addition of acid or base. A buffer solution consists of a mixture of a weak acid (HA) and its salt (A-) or of a weak base and its salt. A buffer can be created by mixing equal concentration of a weak acid (HA) and its conjugate base (A-). The quantitative relation between the concentration of a weak acid (HA) and its conjugate base (A-) is described by the Henderson equation. 38 The Henderson equation. Weak acids are only slightly ionized in solution and a equilibrium is established between the acid and the conjugate base. If HA represents a weak acid, then : HA H + + A- Weak acid Proton salt form or conjugate base The "salt" or conjugate base A- is the ionized form of a weak acid. 39 The dissociation constant of the acid, Ka is defined as: [H+] Ka = [HA] 40 [A-] What is gel electrophoresis? Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode. Electrophoresis The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means "to carry across." 41 A GEL A gel is a net. The suspended particles are single large molecules or aggregates of molecules or ions ranging in size from 1 to 1000 nanometers. Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium. Agarose There are two basic types of materials used to make gels: agarose and polyacrylamide. Agarose is very fragile and easily destroyed by handling. Agarose gels have very large "pore" size and are used primarily to separate very large molecules with a molecular mass greater than 200 kdal. Agarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior. That is, the bands formed in the agarose gels 42 are fuzzy and spread far apart. This is a result of pore size and it cannot be controlled. Agarose is a linear polysaccharide made up of the basic repeat unit agarobiose. Agarose is usually used at concentrations between 1% and 3%. Preparation of Agarose Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel. Polyacrylamide polyacrylamide gel electrophoresis (PAGE) involves separation of protein on the basis of charge and molecular size. The pore size of the gel may be varied to produce different molecular sieving effects for separating proteins of different sizes. In this way, the 43 percentage of polyacrylmide can be controlled in a given gel. By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2,000 Kdal. This is the ideal range for gene sequencing, protein, polypeptide, and enzyme analysis. Gradient gels It provides continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels. Proteins Proteins are important in the structure and function of all living organisms. Some proteins serve as structural components while others function, defense, and cell regulation. Some proteins serve as enzymes that act as biological catalysts which control the biochemical events. 44 Amino Acids The fundamental unit of protiens is the amino acid. Each amino acid contains an amino group (-NH2) and a carboxylic group (-COOH) attached to a central carbon called the alpha carbon. A R-group (or side chain) is also attached to the alpha carbon. The R-group, or side chain, determines the nature of different amino acids. Twenty amino acids have been identified as constituents of most proteins. These amino acids differ from each other in the nature of the R-group attached to the alpha carbon. The identity of the particular amino acid depends on the nature of the R group. 45 Classification of Amino Acids Amino acids are classified according to properties of the R groups. The first of these depends on the polar or nonpolar nature of the side chain. The second depends on the presence of an acidic or basic group in the side chain. Another criteria that is taken into account is the presence of functional groups in the side chains and the nature of those groups. Electrophoresis of Proteins Proteins can be separated and purified by electrophoresis. Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. The source of a protein is generally 46 tissue or microbial cells. The cell must be broken open and the protein must be released into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles. Once the extract or organelle preparation is ready, a variety are available for separation of proteins. Ionexchange chromatography can be used to separate proteins with different charges (similar to the way amino acids are separated). Other chromatographic methods take advantage of differences in size, binding affinity, and solubility. Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures. In addition to chromatography, another important set of methods is available for the separation of proteins, based on the migration of charged proteins in an electric field, a 47 process called (gel) electrophoresis. Gel electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Amino acids differ not only in R-group characteristics but also in molecular weight. Different amino acids are linked together in a linear chain by peptide bonds in various combinations and sequences to form specific proteins. The net charge of a protein will depend on its amino acid composition. If it has more positively charged amino acids such that the sum of the positive charges exceeds the sum of the negative charges, the protein will have an overall positive charge and migrate to 48 the cathode (negatively charged electrode) in an electrical field. Proteins even with a variation of one amino acids will have a different overall charge, and thus are electrophoretically distinguishable. polypeptides that make up complex proteins. The break up of complex proteins into their respective polypeptides allows us to study the structure of proteins that result from the interaction of several genes. A gene is a discrete unit of hereditary information that usually specifies a protein. A single gene provides the genetic code for only one polypeptide. Thus, a protein consisting of four polypeptides requires the interaction of four genes to synthesize that specific protein. 49 A molecular weight protein marker is used to prepare a standard separation curve with which various unknown proteins or polypeptide fractions can be identified. Nucleic Acids The double helix structure originally proposed by Watson and Crick is the most striking feature of DNA structure. The two coiled strands run in antiparallel directions and are held together by hydrogen binds between complentary bases. Adenine pairs with thymine and guanine with cytosine. Eukaryotic DNA is complexed with histones and other basic proteins, while prokaryotic DNA occurs in "naked" form not complexed to proteins. 50 Nucleic acids transmit hereditary information and determine which proteins a cell manufactures. There are two classes of nucleic acids found in cells: ribonucleic acids (RNA) and deoxyribonucleic acids (DNA). DNA comprises the genes, the hereditary material of the cell and contains instructions for making all the proteins needed by the organism. RNA functions in the process of protein synthesis. Nucleic acids are large, complex molecules. Their name --nucleic acid-- reflects that they are acidic and were first identified in nuclei. Nucleic acids are made of only four nucleotides in a regular and an interesting arrangement. Beadle et al experiments had shown that genes control the production of enzymes, which are proteins. 51 Nucleic acids are polymers of nucleotides, molecular units that consist of the following: 1. a five-carbon sugar, either ribose or deoxyribose, 2. a phosphate group, and 3. a nitrogenous base, a ring compound containing nitrogen. The nitrogenous base may be either a double-ringed purine or a single-ringed pyrimidine. DNA commonly consists of Purines o adenine (A) o guanine (G) Pyrimidines o cytosine (C) o thymine (T) with the sugar deoxyribose and phosphate. RNA commonly consists of 52 Purines o adenine (A) o guanine (G) Pyrimidines o cytosine (C) o uracil (U) with the sugar ribose and phosphate. The removal of the phosphate group from a nucleotide yields a compound termed a nucleoside, composed of a base and a sugar. Nucleic acid molecules are made of linear chains of nucleotides. The nucleotides are linked together by covalent bonds to each other. The specific information of the nucleic acid is coded in the unique sequence of the four kinds of nucleotides present in the chain. DNA is composed of two nucleotide chains entwined around each other in a double helix. The base sequence of nucleic acids can be determined in a manner similar to determination of the amino acid sequence of protein, but it is more efficient, particularly for DNA, to use specialized techniques. Restriction endonucleases can be used to cleave DNA molecules into fragments of suitable size. In a direct chemical method, four 53 samples of a given restriction fragment can each be treated with a selective reagent, causing cleavage at a given base. The resulting mixtures can be analyzed by gel electrophoresis, which separates, on the basis of size, the oligonucleotides produced by this treatment. The base sequence of the oligonucleotide can be "read" directly from the sequencing gel. 54 55 56