Chapter 2 Flashcards
Testing for Key Biological Molecules
-There are a number of tests that can be carried out quickly and easily in a lab to determine if a sample contains one of the key biological molecules (carbohydrates, proteins and lipids) -The following tests are qualitative – they do not give a quantitative value as to how much of each type of molecule may be present in a sample
The Benedict’s test for reducing sugars
-Add Benedict’s reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution in a test tube -Heat the test tube in a water bath or beaker of water that has been brought to a boil for a few minutes -If a reducing sugar is present, a coloured precipitate will form as copper (II) sulfate is reduced to copper (I) oxide which is insoluble in water -A positive test result is, therefore, a colour change somewhere along a colour scale from blue (no reducing sugar) to brown/brick-red (a high concentration of reducing sugar) —-This test is semi-quantitative as the degree of the colour change can give an indication of how much (the concentration of) reducing sugar present
The iodine test for starch
-To test for the presence of starch in a sample, add a few drops of orange/brown iodine in potassium iodide solution to the sample —-The iodine is in potassium iodide solution as iodine is insoluble in water -If starch is present, iodide ions in the solution interact with the centre of starch molecules, producing a complex with a distinctive blue-black colour -This test is useful in experiments for showing that starch in a sample has been digested by enzymes
The emulsion test for lipids
-Lipids are nonpolar molecules that do not dissolve in water but will dissolve in organic solvents such as ethanol -Add ethanol to the sample to be tested, shake to mix and then add the mixture to a test tube of water -If lipids are present, a milky emulsion will form (the solution appears ‘cloudy’); the more lipid present, the more obvious the milky colour of the solution -If no lipid is present, the solution remains clear
The biuret test for proteins
-A liquid solution of a sample is treated with sodium or potassium hydroxide to make the solution alkaline -A few drops of copper (II) sulfate solution (which is blue) is added to the sample —–Biuret ‘reagent’ contains an alkali and copper (II) sulfate -If a colour change is observed from blue to lilac/purple, then protein is present. —–The colour change can be very subtle, it’s wise to hold the test tubes up against a white tile when making observations) -If no colour change is observed, no protein is present —–For this test to work, there must be at least two peptide bonds present in any protein molecules, so if the sample contains amino acids or dipeptides, the result will be negative
The Benedict’s Test for Reducing Sugars
-Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar solution to determine the concentration of reducing sugar present in the sample —-It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present -The intensity of any colour change seen relates to the concentration of reducing sugar present in the sample —-A positive test is indicated along a spectrum of colour from green (low concentration) to brick-red (high concentration of reducing sugar present)
semi-quantitative test
a result that gives a broad range/estimation for the substance being tested
Benedict’s Test for Reducing Sugars when concentration is unknown
-can be carried out by setting up standard solutions with known concentrations of a reducing sugar (such as glucose) -These solutions should be set up using serial dilution of an existing stock solution -Each solution is then treated in the same way: add the same volume of Benedict’s solution to each sample and heat in a water bath that has been boiled (ideally at the same temperature each time) for a set time (5 minutes or so) to allow colour changes to occur —-Remember it is really important thing is to ensure that an excess of Benedict’s solution is used -Any colour change observed for each solution of a known concentration in that time can be attributed to the concentration of reducing sugar present in that solution -The same procedure is carried out on a sample with an unknown concentration of reducing sugar which is then compared to the stock solution colours to estimate the concentration of reducing sugar present
Alterations
-It is also possible to standardise this test but instead of waiting a fixed amount of time for a range of colours to be observed, time how long it takes for the first colour change to occur (blue to green) —-The higher the concentration of reducing sugar in a sample, the less time it would take for a colour change to be observed -To avoid issues with human interpretation of colour, a colorimeter could be used to measure the absorbance or transmission of light through the sugar solutions of known concentration to establish a range of values that an unknown sample can be compared against a calibration curve
Serial dilutions
-Serial dilutions are created by taking a series of dilutions of a stock solution. -The concentration decreases by the same quantity between each test tube -They can either be ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm-3) -Serial dilutions are completed to create a standard to compare unknown concentrations against
Making serial dilutions
Serial dilutions comparisons can be
-Visual -Measured through a calibration/standard curve -Measured using a colorimeter
Serial dilutions: can be used when:
-Counting bacteria or yeast populations -Determining unknown glucose, starch, protein concentrations
Colorimeter
-A colorimeter is an instrument that beams a specific wavelength (colour) of light through a sample and measures how much of this light is absorbed (arbitrary units) -They provide a quantitative measurement
Colorimeter features
-They contain different wavelengths or colour filters (depends on the model of colorimeter), so that a suitable colour can be shone through the sample and will not get absorbed. This colour will be the contrasting colour (eg. a red sample should have green light shone through) —-Remember that a sample will look red as that wavelength of light is being reflected but the other wavelengths will be absorbed
