Practical Biochemistry Flashcards
Testing for carbohydrates- Starch:
To test for starch, add iodine solution (in potassium iodide) to a sample. If starch is present, you will see a colour change of yellow-brown to blue-black. When dissolved in potassium iodide, to iodide forms a triiodide ion I3-, which slips into the middle of the amylose helix. This causes the colour change.
Testing for carbohydrates- reducing sugars:
These include all monosaccharides and some disaccharides. They are known as reducing sugars because they can reduce, or give electrons to, other molecule. If you heat a reducing sugar with Benedicts solution (alkaline copper (II) sulfate), there is a colour change from blue to green to yellow to orange-red.
Benedicts solutions contains Cu2+ ions, which are reduced to to Cu+ ions, forming orange- red copper (I) oxide (CU2O). This is called a precipitate because it comes out of solution and forms a solid, suspended in the reaction mixture.
Testing for carbohydrates- non-reducing sugars:
To test for a non-reducing sugar, we have to hydrolyses the bond first, to ‘free up’ these ‘reducing group’, and then test for reducing sugars as normal.
- First, test a sample for reducing sugars to check there are none there in the first place.
- Take a separate sample and boil it with hydrochloric acid to hydrolyze the sucrose into glucose and fructose.
- Cool the solution and use sodium Hydrogencarbonate solution to neutralize it.
- Test for reducing sugars again
A positive result (green-yellow-orange-red) indicates that non-reducing sugar (e.g. sucrose) was present in the original sample.
Testing for Lipids:
The emulsion test is used to test for the presence of lipids:
- Take a sample and mix it thoroughly with ethanol. Any lipid will go into solution in the ethanol
- Filter
- Pour the solution into water in a clean test tube
- A cloudy white emulsion indicates the presence of lipids. This is made up of tiny lipid droplets that come out of solution when mixed with water.
Testing for Proteins:
Biuret Test:
If the protein is present, the colour changes from light blue to lilac.
You may find that the reagents are supplied to you separately as biuret A (sodium hydroxide), which you add first, and biuret B (copper sulfate), which you add next.
The colour is formed by a complex between the nitrogen atoms in a peptide chain and Cu2+ ions, which is why this test really detects the presence of peptide bonds.
Quantitave testing for reducing sugars:
Benedict’s reagent the presence of reducing sugars. If there is more sugar present:
- the amount of precipitate will increase.
- the amount of copper (II) ions remaining in solution will decrease
We can try to quantify the concentration of sugar in the original sample by assessing how these variables change, using a technique called colorimetry.
Colorimeters:
A colorimeter works by shining light through a sample. In this case, we would use a centrifuge to separate the precipitate and any excess Benedict’s solution (the supernatant).
Using a pipette, we can take the supernatant and place it in a cuvette (a small vial), which is then placed into the colorimeter. The cuvette is commonly made of glass or plastic.
Colour filters are often used for greater accuracy. By using a red filter in this case, we can shine red light through the solution, and detect how much passes through (percentage transmission). The solution reflects blue light but absorbs red light.
Creating a Calibration Curve:
Using a colorimeter gives us a Semi-Quantitave test for sugar, as we can compare how much sugar is contained in different samples. To find the exact amounts, we need to create a calibration curve:
- First, take a series of known concentrations of reducing sugars
- Using a sample of each, carry out Benedict’s test
- Use a colorimeter to record the percentage transmission of light through each supernatant.
- Plot a graph to show ‘transmission of light against the concentration reducing sugar. This provides a calibration curve, which you can use with other ‘unknown’ sample to determine the concentration of sugar in the original sample.
Use of Biosensors:
Biosensors take a biological or chemical variable which cannot easily be measured, and convert it to an electrical signal.
Biosensors have many other applications, For example, they can be used to detect contaminants in water, and pathogens and toxins in food. They can even be used to detect airborne bacteria, for example in counter-bioterrorism programmes.
What are the principles of chromatography?
The aim of chromatography is to separate a mixture into its constituents; in this case biological molecules. There are two key components, known as the stationary phase and the mobile phase.
What is the Stationary Phase?
This is either the chromatography paper or a thin-layer chromatography (TLC) plate. The paper is made of cellulose. The TLC plate is often a sheet of plastic, coated with a thin layer of silica gel or aluminum hydroxide. In each case, there are free -OH groups pointing outwards in contact with the mobile phase.
What is the mobile phase?
This is the solvent for the biological molecules. We can use water (for polar molecules) or ethanol ( for non-polar molecules). The mobile phase flows through and across the stationary phase, carrying the biological molecules with it.
Chromatography Method:
- Draw a line in pencil and put a tiny dot on the line to show where you place your solution mixture, If you draw it in ink, the pigments in the ink will also separate.
- Spot the solution mixture onto the pencil dot several times using a capillary tubing. Wait for the spot to dry before putting on the next spot, and try to make the spot as thin as possible. When it is completely dry, lower it into the solvent. Ensure the level of the solvent at the start is below the pencil line.
- Cover the beaker with a watch glass, or glass plate.
- Let the apparatus ‘run’ until the solvent has reached a point just underneath the top of the paper/ TLC plate. Then remove it from the solvent, and layer it on a white tile to dry
