INTRODUCTIONform orstructure of a molecule plays a significant role in the function of thatmolecule. Since weare interested in the function of molecules. it helps to study theirstructure. One of themajor classes of organic compounds found in cells are carbohydrates.
Thesecarbohydrateare made of carbon, hydrogen, and oxygen in a ratio of 1:2:1 respectivelywith a generalformula of X(CH2O)n. When the carbohydrates consists of one unit of sugar,X=1, it iscalled monosaccharide. If it consists of 2 units, X=2, the carbohydrate iscalleddisaccharide. Carbohydrates made up of more than two units, X>2, are calledpolysaccharides. Carbohydrates can also be branched or unbranched dependingon thetype of linkage.
Those with alpha 1:4 linkages are linear or unbranched,while those withalpha 1:6 linkages are branched. Carbohydrates are necessary biomoleculesbecause theyplay a role in energy metabolism as a source of potential chemical energy,also they areimportant building blocks for other biomolecules. The word carbohydrate is very general, so in order to understand thesemoleculesmore precisely, we need to be able to identify more specificclassifications. Ourexperiments try to accomplish this using three common bioassay tests.Order now
Thefirst, theBenedict test, will test various compound for reducing sugars. Allsix-carbon hexosesugars are reducing carbohydrates, as are most disaccharide. Sucrose is theexception. Most polsaccharides are not reducing. Secondly, we have the Barfoed testwhich isdesigned to test for monosaccharides. The third and final Iodine test isusedto test forpolysaccharides that are either branched or unbranched.
By combining thesetests wewere able to make accurate predictions about the carbohydrate contents of agiven sample. Now, let’s take a closer look at how these bioassays do work. The Benedictandthe Barfoed tests are based on the reaction of cupric ions with aldehyde orketone groups. In the presence of a reactive group, the blue cupric ions are reduced toredcuprous ions. The Benedict test is a basic solution and upon heating turns green, yellow,orange or brickred which indicates a positive reaction. The final color is dependent onthenumber ofreactive sites available; green indicates few sites, yellow more, and reddenotes many sites.
The Barfoed solution is acidic and only free aldehyde or ketone groups ofmonosaccharides can reduce the blue ions to red ions. The color change tored will occurimmediately. The lack of a change indicates only that the solution is not amonosaccharide. The iodine test is used for polysaccharides. Iodinecombines with anyexisting alpha helices.
The more coiled the sample the darker the iodinewill turn. Thecolor change can range from deep black-blue with a sample of many coils toarust redviolet with fewer coils and more branchings. When there are no coils, thereis no colorchange. Mono and disaccharides give negative results. In summary, this lab attempts to investigate several different samples bymeans ofseries of tests, and based on the combined results of all three tests wecanattempt tounderstand the carbohydrate composition of unknown samples.
We hope to beable topredict the results of three bioassays for an unknown solution if given itssaccharide typeand reducing property. We should also be able to predict the saccharidetypeand reducingcapability of an unknown solution if given the results of the threebioassays. MATERIALS AND METHODS*Like any other experiment, this experiment needs some specific materialsincluding, beaker, graduated cylinder, hot plate, 11 test tubes, test tubeholder, wax pencil,liquid soap, and test tube brush. Also, we used the Barfoed reagent,Benedict reagent,and iodine reagent.
Our eleven samples were distilled water (control), glucose, fructose,maltose,lactose, sucrose, glycogen, starch, potato soup, and dilute honey. First, we marked our test tubes with the wax pencil to keep track on thesubtances,then we place the eleven samples in the corresponding tubes. The first testthat weperformed was Benedict, followed by Barfoed, ending with iodine test. Whenneeded thesamples were heated and our results were immediately recorded in thefollowing tables. Inall three cases distilled water was used as a control.
*The details of the materials and the methods can be obtained from the labmanual:Experiments in Biology, From chemistry to sex by Linda Van Thiel, page 13. RESULTSThe actual results of the Benedict test are as follows: distilled waterremainedblue, glucose turned a dark green, fructose blue-green, galactose was red,maltose wasslightly red, lactose blue-green on the top of the test tube and red on thebottom, sucrose,glycogen, starch, and potato soup were all negative(blue). Finally, thedilute honey samplewas dark orange. The actual results of the Barfoed test are as follows: distilled waterformed noprecipitate, glucose, fructose and galactose did form red precipitate,maltose, lactose,sucrose, glycogen, starch, and potato soup did not form a precipitate,dilutehoney didform a precipitate. The actual results of the iodine test are as follows: distilled water,glucose,fructose, maltose, lactose, and sucrose all remained yellow or negative.
Glycogen turneda rust color, starch was black-blue, potato soup was rust colored, andfinalsample dilutehoney remained yellow. DISCUSSIONCombining the three tests we have the over all results as follows: for ourcontroldistilled water we can conclude that it is non-reducing, non-monosaccharide,and non-polysaccharide; glucose, fructose, and galactose were all reducing,monosaccharides,non-polysaccharides. Maltose and lactose were both reducing,non-monosaccharides,non-polysaccharides. Sucrose was non-reducing, non-monosaccharide, non-polysaccharide.
Glycogen was a non-reducing, non-monosaccharide, and abranchedpolysaccharide. Starch was a non-reducing, non-monosaccharide, and aunbranchedcoiled polysaccharide. Potato soup was non-reducing, non-monosaccharide,andabranched polysaccharide. Dilute honey was reducing, monosaccharide, and anon-polysaccharide. Let’s continue the discussion of this lab with a closer look at ourmonosaccharides.
Based on our results we can conclude that glucose, fructose, galactose anddilute honeyare the monosaccharides since they all formed a precipitate in the Barfoedtest. Thesample of dilute honey was of greatest interest to me since we did not knowprior to thetest whether it were a monosaccharide or not. I suspected that it wasreducing since thehoney was diluted. A non-reducing carbohydrate I do not believe we coulddilute since itwill not dissolve.
Based on the precipitate formation of dilute honey intheBarfoed, it canbe concluded that it is comprised of monosaccharides. Looking at our results I can reasonably conclude that the disaccharidesamples aremaltose, lactose, and sucrose since they all were negative for both theBarfoed and iodinetests. If we also look at the probable disaccharides, we see that none ofour tests usedwere designed to specifically test positively for them. Since we know thatdisaccharidesare comprised of two monosaccharides by way of dehydration reaction, wecouldtest fordisaccharides by adding water to the possible disaccharide samples and maybeheat themso they will undergo a hydrolysis process, then run them through theBarfoedtest again. If the sample which before adding water was negative in the Barfoed test,butwas positiveafter adding water then we could conclude that the original sample was adisaccharide.
Our tested samples that we believe to be polysaccharides are glycogen,starch, andpotato soup since they all had some color change in the presence of Lugol’siodine. Polysaccharides can be further classified by their overall structure, inparticular, whetherthey are highly branched, highly coiled and unbranched, or both slightlycoiled andbranched. We learned that the starches can be coiled profusely or coiledwith no branches. The iodine test will result in a different degree of color change based onthe amount ofcoiling present.
Namely, a highly coiled carbohydrate will turn a darkblue-black color. The particular highly coiled polysaccharide that we discussed in the classisamylose whichis an unbranched storage starch found in plants. Since our starch sampleturned black, itmay be compromised of amylose starch. The potato soup sample did not turnasdark, acolor indicating to me that the starch in this sample probably consisted ofsmaller starchunits called dextrin.
Dextrin have very short terminal ends that coil onlysightly so thecolor change would not be so dramatic as in the presence of highly coiledstarch likeamylose. The potato soup was made from dehydrated buds. This dehydrationprocess ofthe fresh potatoes does cause structural change in the starches. A freshpotato sample Ipredict to turn a dark black since its starches would be intact.
Glycogenturned a rustcolor as we should expect since we know that glycogen is a slightly coiledpolysaccahride. I did predict prior to the experiment that the color change in the presenceof the iodinewould be different for starch and glycogen since they have differentcoilingcharacteristics. The data, in my opinion, did not conflict with our expected results. Thesetestswhen used together allow us to make predictions about unknown samples withconfidence. I believe that the data provide sufficient information tobetterunderstandcarbohydrates and how we can more precisely describe carbohydrates.