Chem 40.1 Post-Lab Finals PDF

CHEM 40.1 POST-LAB Engr. MGAT BILE Bile is golden greenish-yellow colored viscous liquid which is inte rmitte ntly discharged into duodenum. Daily output ranges from 700-1200 ml. Bile helps with digestion. It breaks down fats into fatty acids, which can be taken into the body...

CHEM 40.1 POST-LAB Engr. MGAT BILE Bile is golden greenish-yellow colored viscous liquid which is inte rmitte ntly discharged into duodenum. Daily output ranges from 700-1200 ml. Bile helps with digestion. It breaks down fats into fatty acids, which can be taken into the body by the digestive tract. Organic Constituents of Bile 1 3 Bile Salts (Sodium & Potassium Salts) Lecithin Bile salts are the major organic solutes in bile and the third major lipid constituent in bile. It is the normally function to emulsify dietary fats and major endogenous source of choline in the body. facilitate their intestinal absorption. Bile salts appear in the urine when there is an obstruction to the biliary tract. 2 4 Cholesterol Bile Pigments (Bilirubin and Diglucuronide) In bile, it is either converted into bile acids for biliary the third major lipid constituent in bile. It is the secretion or transported by ABCG5/G8 into the bile. major endogenous source of choline in the body. Inorganic Constituents of Bile 1 Sodium Ions 3 Chloride Ions 2 Potassium Ions 4 Bicarbonate Ions Test for Bile Salts & Pigments POSITIVE REACTION Pettenkofer’s Test Red color indicates the presence of bile salt Smith’s Test A green ring is formed at the junction of two layers indicating the presence of bile salt Hay's test Bile salts when present lower the surface tension of urine. Activity of bile acid can help human body to transfer and When sulfur powder is added to the urine, sulfur particles absorb fat from food. It is possible to observe this by sink to the bottom of the tube. In the case of normal urine, it colloidal sulfur (sulfuric powder). will float on the surface. Fouchet’s test When barium chloride is added to urine it combines with sulfate radicals in urine and precipitate of barium phosphate is formed. If bile pigments are present in urine, Oxidation of yellow bilirubin to green bilverdin. they will adhere to these large molecules. Ferric chloride present in Fouchet reagent then oxidizes yellow bilirubin in presence of trichloroacetic acid to green bilverdin.  Color Reactions of Proteins & Amino Acids; Detection of Proteins, Lipids, and Carbohydrates Proteins are polymers of amino acids joined together by peptide bonds. When proteins are hydrolyzed, amino acids are produced. The protein in your diet provides the nitrogenous material needed to build protoplasm. All enzymes and many hormones are proteins. Different functional groups present in certain amino acids and proteins react with the variety of reagent forming colored products.  Color Reactions for Proteins & Amino Acids Ninhydrin Reaction Biuret Test Xanthoproteic Test Compounds containing peptide The ninhydrin reaction is widely used bonds react with alkaline copper Xantho means yellow. When reaction of the alpha amino group that sulfate to give violet colored treated with concentrated nitric can be used to estimate amino acids complex products. This is called acid, the aromatic rings on the quantitatively in very small amounts. On “biuret test” because it is given side chains of proteins become heating, an alpha amino acid and by the substance biuret obtained substituted with nitro groups. The peptides having free alpha amino group upon heating urea to 180 C. The nitrated products are yellow, so reacts with ninhydrin to yield purple color may vary from pink to violet the solutions becomes yellow or a colored products. Proline and blue, the color depending as the yellow precipitate forms. When hydroxyproline do not have free alpha number of peptides bonds the solution is made basic with amino group yield products with a increases. Thus, it is widely used sodium hydroxide, the color characteristics yellow color. as a qualitative general test; becomes more intense and however, the substances ranges from yellow to orange to present in biological material brown. Thus the positive reaction only protein will give a positive indicates the presence of tyrosine reaction. and or tryptophan in proteins.  Color Reactions for Proteins & Amino Acids Millon-Nasse Test Hopkins-Cole Bromine Water Test This is due to the reaction of the The indole group such as is The addition of bromine water hydroxyphenyl group with the present in the tryptophan and n-amyl alcohol to a solution Millon-Nasse reagent (HgSO4) condenses with a number of containing free tryptophan forming colored (old rose) complex aldehydes like glyoxylic acid in the results in the formation of a which is probably the mercury salt presence of concentrated H2SO4 pinkish lavender or violet color in of the phenolic compound. forming a violet ring appearing in the alcohol layer. Tyrosine, phenol and thymol will the junction of 2 liquids. give positive test. The indole group such as is present in the tryptophan condenses with a number of aldehydes like glyoxylic acid in the presence of concentrated H2SO4 forming a violet ring appearing in the junction of 2 liquids.  Color Reactions for Proteins & Amino Acids Pauly Reaction Lead Acetate Sakaguchi Reaction This test is positive if histidine or If protein is boiled with KOH and Substances that contain the tyrosine is present. The reagent then treated with Pb(OAc)2 guanidino group react with is made by mixing NaNO2 solution, a black precipitate is alpha-naphthol and an oxidizing solution with sulfanilic acid fo r m e d i f s u l p h u r c o n t a i n i n g agent such as sodium dissolved in HCl solution. Upon amino acid such as cysteine is hypobromite to give a red or adding to a protein solution and present. The strong alkali orange color. Arginine, either free making alkaline with NH4OH a removes sulphur from this amino or combined in proteins, red or orange results. Histidine acid forming K2S that react with responds positively to the test. gives red to orange color. Pb(OAc)2 to form PbS, a black Tyrosine gives lighter orange compound. The sulfur of color. methionine is not readily removed by alkali and is not affected by the reaction. Nucleic Acids Nucleic acids are biopolymers in which the repeating unit is mononucleotide. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are the two structural classes of nucleic acids that occur in living cells. Nucleic acids are major components of all cells making up 5-15% of their dry weight. The highly cellular organs such as spleen, liver, thymus and pancreas are rich in nucleic acids, whereas brain and muscle tissues have a much lower concentration. In tissues with high nuclear/cytoplasmic ratio, the DNA concentration is high. Thus, a lymphoid tissue is very good source of DNA, with thymus as the best source and spleen as good substitute. Tissues with a high cytoplasmic volume have a high RNA value. The unicellular fungus, Saccharomytes cerevisiae (yeast) contains 4% RNA by weight. Nucleic acids have the following properties: 1. Sparingly soluble in cold water 2. Insoluble in alcohol 3. Readily soluble in weak alkali with the formation of alkali metal salts 4. They are precipitated from alkaline solutions by the addition of acids  Isolation of Nucleic Acids Step 1 Step 2 Step 3 Disruptions of the cell Treatment with a reagent that Purification of isolated nucleic membranes to release the causes the dissociation of acid. The methods used are nucleic acids. This involves the nucleoproteins and gradient centrifugation, gel denaturation of the enzymes denaturation of electrophoresis and (deoxyribonucleases or the proteins. The following are chromatography ribonucleases) therefore usually used: enzymatic degradation of the a. Detergents with phenol - nucleic acids in avoided. used to release and denature proteins b. S o l v e n t m i x t u r e s l i k e chloroform - isoamyl alcohol or chloroform - octanol  Isolation of RNA from Yeast Step 1 Step 2 Step 3 Heating with alkali like NaOH. Acid extraction at pH 4-5. This Precipitation of the isolated This is used to extract the is used to separate the nucleic RNA. This is accomplished by nucleic acids and water-soluble acid from associated proteins treatment with alcohol, ether or proteins. At the same time this and other interfering other organic solvents to inactivates the nucleases so substances. remove lipids. that the RNA will not be degraded. Enzymatic Browning Enzymatic activity causes the rapid darkening (usually browning) of fruits and vegetables after they have been peeled, cut and sliced. The mechanism of brown color development involves oxygen from the air or from the intercellular spaces within the tissue in contact with the cut surface that contains enzyme polyphenoloxidases (PPO) and phenolic compounds as suitable substrates. Brushing or ether injury to the tissue allows the contents to make contact with oxygen and causes browning.. Enzymatic Browning Enzymatic browning produces undesirable changes such as: 1. Darkening of the cut surfaces of fruits and vegetables 2. Deterioration of the flavour, odor and nutritive value. The methods of prevention of enzymatic browning most commonly practiced concentrates on the enzyme and oxygen because the elimination of the substrate (phenolic compounds) but this is not practical. The principal approaches are: A. Inactivation of the enzyme polyphenoloxidases (PPO) 1. Blanching 2. Use of inhibitors 3. Lowering of pH A. Minimize contact with oxygen 1. Use of syrup solution 2. Use of antioxidants such as ascorbic acid and sulfur dioxide  Enzymatic Browning When polyphenols mix with PPO and oxygen, they create a compound called 1,2- Benzoquinone. This can also be called ortho-quinone or o-quinone. The individual molecules of o-quinone connect together to make larger molecules. This process is called polymerization. It creates a compound called melanin, which causes the apple to look brown.