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BIOL 130L Study Notes on every lab.doc

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Dragana Miskovic

Summary for Quiz Monday, May 30, 2005 12:13 AM Title Identification of Some Macromolecules Gist of Experiment Use different tests to check for the existence of macromolecules in various substances • o Iodine test checks for starch and/or glycogen o Benedict's test checks for reducing sugars o Biuret test checks for protein Notes on Underlying Theory Introduction • The most abundant elements in living material are: o Carbon o Hydrogen o Oxygen o Nitrogen o Sulfur o Phosphorus • There are 4 major types of biological macromolecules: o Carbohydrates Monosaccharides (i.e. glucose, fructose) • • Disaccharides (i.e. sucrose) • Polysaccharides (i.e. starch, glycogen) o Lipids o Proteins o Nucleic acids Tests Iodine test • o Information on starch: • It is a polysaccharide used by plants to store glucose Glucose is held together with glycosidic bonds • • It is a mixture of 2 different polymers: amylose and amylopectin  Amylose • It is unbranched and helical molecule • The glucose is joined by alpha 1 -> 4 linkages  Amylopectin • It is straight and highly branched • The glucose is joined by alpha 1 -> 6 linkages o Information on glycogen: • It is a polysaccharide used by animals to store glucose • Glucose is held together with glycosidic bonds It is heavier than starch • • It is similar to amylopectin in overall structure, but is more highly branched o How does the test work? Iodine solution is usually pale yellow • • It turns blue-black in the presence of starch because of the amylose • It turns red-brown in the presence of glycogen because of the multi-branched components • Benedict's test o Information on sugar: • All sugars can exist as straight chains or in ring form The straight-chain forms are called aldose sugars •  They have a terminal aldehyde group (C single-bonded to H, double- bonded to O) o How does the test work? • Blue cupric ions (Cu ) in Benedict's solution are reduced to cuprous ions (Cu )+ by the free aldehyde groups, and we get a precipitate of cuprous oxide (Cu O): + - - + - 2  4Cu + 2OH + 2e -> 2Cu O + 2H2+ 2e • The amount of cuprous oxide formed is proportional to the concentration of free aldehyde groups  The color of the precipitate varies depending on this as well (blue -> green -> orange -> red -> brown) • Ketose sugars (i.e. non-aldose, which means non-straight chain, which means no free aldehyde group) can ALSO reduce Benedict's solution  This happens because the basic conditions of the experiment isomerize a ketose to an aldose, and then the reduction happens with an aldose • Biuret test o Information on proteins: • They are composed of amino acids, which are connected by peptide bonds A peptide bond is the carboxyl group of one amino acid covalently linked to the • alpha-amino group of the next amino acid o How does the test work? • Biuret solution is a solution of sodium hydroxide (NaOH) and copper sulfate (CuSO4) • Under alkaline conditions (caused by the NaOH), the peptide bonds within proteins react with the Cu++ ions to form a purple complex • So we identify the presence of protein by looking for this purple color Summary for Quiz Monday, May 30, 2005 12:13 AM Title Isolation of Some Macromolecules Gist of Experiment • Use a variety of techniques to isolate macromolecules from a starting mixture Experiment Procedure and Justification Thereof 1. We start with a yeast-sand mixture a. Yeast cells have: i. Glucan (a polysaccharide) in the cell walls ii. Glycogen, proteins, and nucleic acids in the cytoplasm b. Grind the yeast to rupture the cell walls and release all this stuff 1. Add TCA (trichloroacetic acid) and continue grinding a. Polysaccharides (in this case glucan) are soluble in TCA, so they will go into solution a. But the proteins and nucleic acids will stay suspended! 1. Centrifuge the suspension (so just the non-sand part) a. When you do this on a liquid (remember the polysaccharides are suspended in the liquid) with particles suspended in it (remember these are the nucleic acids and proteins), all the suspended stuff goes to the bottom (it "sediments") and the liquid remains on top i. The sediment is known as the precipitate, also known as "pellet" ii. And the top liquid stuff is the "supernatant" 1. Now we focus just on the pellet (i.e. the nucleic acids/proteins) a. Add NaCl to the pellet i. Nucleic acids are soluble in strong NaCl, so they go into solution! ii. But the proteins remain in suspension 1. Again we centrifuge this, and the proteins become the pellet, and the nucleic acids are the supernatant 1. Now we are going to SPLIT the nucleic acids and the proteins, and do stuff with each Nucleic Acid Portion 1. Alright, first remember that we are dealing with a liquid here, because the nucleic acids were in solution! 1. But the first thing we'll do is to add chilled ethanol, which will cause the acids to precipitate out of solution to form a suspension 1. As before, we centrifuge to isolate the acids (which are in the pellet, of course) 1. But then we take the pellet and we add sulfuric acid, which makes the nucleic acids go into solution again 1. Then we boil the stuff - but only ONE of the test tubes! (We have 2 test tubes' worth of nucleic acid) a. Boiling in acid is a "hydrolyzing process" - it breaks up the nucleic acid into the nucleotide subunits, and then even FURTHER into the base and sugar and phosphoric acid subunits! 1. So now we have one test tube of "hydrolyzed nucleic acid" and another of "unhydrolyzed nucleic acid" Good times! 1. OK, remember we had sulfuric acid in there? Now we have to neutralize the solution! Details below… a. We're going to use barium hydroxide, a base, to neutralize this solution a. We're essentially going to perform a titration, where we use litmus paper to figure out when the solution is acidic, when it is basic, and when it is neutral a. The chemical formula is this: H2SO4 + Ba(OH)2 -> BaSO4 + 2H2O i. Note that the precipitate (salt) which forms is barium sulfate - we will filter this out later! Protein Portion 1. OK, so remember that back in the day, we had protein and nucleic acid resulting from a centrifugation…Well, now we're dealing with the protein portion, which is solid 1. We take half the protein and add pancreatic enzyme a. This enyzme will hydrolyze the protein into its amino acid subunits a. This simulates how the hydrolytic process is carried out naturally, because in real life it is done with enyzmes! 1. And the other half of the protein we add phosphate buffer, which will not hydrolyze it at all! 1. To both we add thymol crystals, which prevent the growth of bacteria Summary for Quiz Monday, June 06, 2005 10:28 AM Title Characterization of Some Macromolecules Gist of Experiment • Use the method of chromatography to separate the proteins and nucleic acids earlier into their individual components Experiment Theory • Chromatography is a technique that separates mixtures into their individual components o For example: • If we put black washable ink onto a tissue, the ink will spread outwards from the place where we blotted it However, the various components of the ink can't all move at the same speed • as it spreads out - so the components will visibly separate • The pigment which moves the slowest will "stop" first, followed by the next slowest, and so on… • The stationary phase in any chromatogram is the "matrix" - it is the substance onto which we place the stuff to be measured (i.e. in the above example, it was the tissue - or more specifically, the cellulose in the tissue which was reacting with the ink in the fibers) o Note that the "matrix" has to be INERT - meaning that when we place the mixture we want to examine onto it, they can't react! Or else the whole point of it will be ruined! • And the mobile phase is the solvent - meaning that the mixture we want to study will DISSOLVE in this solvent, and then the solvent will move up the "matrix" (paper, in the above case), and like with the paper example provided above, certain parts of the mixture /solvent will stop based on how much the matrix slows them down o Or as the lab manual says, "separation depends on the relative tendencies of molecules in a mixture to associate more strongly with one or the other phase." • Here are some factors which affect how far a given substance (within a mixture) will travel: o How soluble is it in the solvent? • If it is COMPLETELY soluble, it'll just travel as far as the solvent does, and we won't see any separation • If it is NOT SOLUBLE at all…it won't travel anywhere at all! o How heavy is it? (What is the molecular weight?) o What is the overall polarity of the compound? • The thing we measure in chromatography is the difference between how far a substance (from the mixture) travels compared to how far the solvent travels o Rf = (distance traveled by a substance) / (distance traveled by the solvent) Experiment Procedure and Justification Thereof • Well, this is a very general overview, but… • Put the mixtures on the chromatography paper (just a spot of each) • Sew the paper so that it forms a cylinder (but the ends of the paper should NOT overlap) • Put the "cylinder" into the solvent, making sure that the spots are just over the level that the solvent comes up to o Solvent: For proteins, it is FORMIC ACID (10% formic acid, 70% isopropanol, 20% • water) • For nucleic acids, it is ACETIC ACID (15% acetic acid, 60% butanol, 25% water) Summary for Quiz Sunday, June 12, 2005 11:19 PM Title Spectroscopy Gist of Experiment Measure the concentrations of unknown solutions using the concentration curves derived from the measurement of solutions of known concentration (all this using spectrophotometers). Notes on Underlying Theory • The energy content of light depends on its wavelength (because light moves as waves) o The human eye can recognize light between 400 nm (violet) and 750 nm (red) • A spectrophotometer has a white light source which focuses on a prism that splits it up into the different portions of the spectrum…after this, we can focus each different "incident beam" on a sample specimen o The sample specimen is dissolved in a solvent, and it is housed in a tube called a cuvette When the incident beam hits the sample, one of 3 things will happen: • o It gets absorbed o It gets transmitted o It gets reflected • The part that gets transmitted goes through and hits the photoelectric cell, which generates an electric current - and the current tells us what the intensity of the transmitted beam is! Or in other words, it tells us how much got through… • The current is measured in 2 ways: o Percent transmittance - this is an arithmetic scale with equidistant units from 0% to 100% which tells us what percentage of the light was transmitted (i.e. how much got through) o Absorbance - this is a logarithmic scale with unequal divisions from 0.0 to 2.0 which tell us how much light was absorbed • Beer's Law says that the concentration of a light-absorbing solute is directly proportional to the absorbance over a given range of concentrations  So this means that as we vary the amount of solute we put in the solvent, obviously the absorbance readings we get from the spectrophotometer will change…but they will be LINEAR! On the other hand, the relationship between the solute concentration and the • percentage transmitted is NOT linear • Random note: Know that the photocolorimeter we use can measure the entire visible spectrum and slightly overlaps into the U.V. and infrared ranges, but other spectrophotometers can use the whole U.V. range (180 to 350 nm) and infrared range (780 to 300,000 nm) • So how do we analyze a substance? o First we dissolve the substance in a suitable solvent o Then we insert a cuvette containing ONLY the solvent into the electrophotometer (this is called the "blank" cuvette), and we zero the scale at this level so it acts as our baseline o Then we replace the solvent-only cuvette with a cuvette containing a solution with some solvent in it…And obviously the solute in this solvent will absorb some light so the reading will be different • Graphical analysis of a substance o Firstly, we have to plot an absorption spectrum for the substance - this means that you read the absorbance of that substance at many different wavelengths at one constant concentration (remember, this would mean that when you use the spectrophotometer and split it up into different wavelengths of light, you don't just use one of those…you use many!) • If you draw a curve that relates absorbance to wavelength, look for the highest point on that curve - it will tell you what the wavelength of maximum absorption is o Then, you make a concentration curve - so you set the spectrophotometer to use the wavelength of maximum absorption which you just figured out, and then take multiple measurements with different concentrations of the solute • Note that you only have to measure 2 or 3 different concentrations and then plot them - the rest of the points can be deduced by drawing a straight line, because according to Beer's Law, it is a directly proportional relationship! The plot should go from 0.025 to 1.0 • • This entire process allows us to determine the concentration of an unknown sample! Because once we have the concentration curve, we can just take any other sample of that substance (even if we don't know its concentration) and figure out what the absorbance level is! And from there, we can check the curve to find what the associated concentration is… Notes on Experimental Procedure • Experiment 1 - Congo Red o Basically, we are creating solutions of different concentrations of Congo Red first… o Then we get the maximum absorbance by measuring some of the cuvettes at varying wavelengths (from 400 nm through 600 nm, going up by 20 nm each time) • But when we narrowed it down to a wavelengths, re-measure the area going up by 5 nm each time to be even more precise o Then we use this wavelength and try all the different concentrations so that we can get our concentration curve • Experiment 2 - Chloroplast pigments o Information: • Within chloroplasts, there are different pigments, and they all absorb different parts of the visible spectrum…but the parts of the spectrum they can't absorb are reflected! And these are the colors we see! • The major pigments in a chloroplast:  Chlorophylls • Chlorophyll A • Chlorophyll B  Carotenoids • Carotenes • Xanthophylls o Procedure: • We get the chloroplast extract from spinach leaves and we dab it along the line of a paper which we will perform chromatography on (the procedure is much the same as last week)  Note that the solvent in this case is 90% petroleum ether, 10% acetone • When the chromatogram is finished it should look like so (starting from the distance the solvent traveled and going backwards): A thin orange band (carotene)   2 distinct yellow bands (xanthophylls)  Green band (chlorophyll A)  Green band (chlorophyll B) • Then cut up the paper into these different strips and put them into test tubes of acetone, which will facilitate elution of the pigment into the solvent - we only want Chlorophyll A and Chlorophyll B! • Now we have different test tubes with different pigments in them! We now use these with the spectrophotometer and we find a maximum absorbancy wavelength for each of them Summary for Quiz Monday, June 20, 2005 2:28 AM Title Enzymes Gist of Experiment See the effect that enzyme concentration has on reaction time and the effect that substrate concentration has on enzyme reaction. Notes on Underlying Theory General • Enzymes are: o Biological catalysts (remember from CHEM 123 what a catalyst does) o Specific in their action (specificity is determined not only by amino acid order, but also by 3-D conformation) o Proteins (except for a small subset of enzymes which are called ribozymes) • Enzymes combine with a substrate to form a substrate-enzyme complex, which then breaks down into the enzyme again (which is UNALTERED!) and the product: o Substrate + enzyme -> substrate-enzyme complex -> product + enzyme • Here are some factors which affect the rate at which the enzyme converts the substrate into the product: o Temperature o pH o Enzyme concentration o Substrate concentration o Product concentration o Energy of activation • As for the direction of the reaction, all enzyme-mediated reactions are theoretically reversible, but the direction which the reaction actually goes in depends on the conditions under which the reaction is taking place Experiment #1: Salivary amylase • Salivary amylase is a digestive enzyme found in saliva • It acts on starch molecules by breaking off maltose molecules from the end of the starch chain Each time the chain is broken, a water molecule is consumed - thus this reaction needs water • and is called a hydrolytic reaction o The general term for bond-breaking with water is hydrolysis Experiment #2: Phosphorylase • Phosphorylase is an enzyme that acts on starch by breaking off glucose molecules • Instead of using water to do this, we consume phosphoric acid - and so the general term for bond-breaking with phosphoric acid is phosphorolysis 2- • Here is the general reaction: (Glucosn) + HPO4 (Glucose)n-1+ Glucose-1-phosphate o When the reaction goes -->, it is phosphorolysis o When the reaction goes
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