Practical Skills

• Chromatography involves passing a mixture dissolved in a mobile phase (a solvent) through a stationary phase (e.g. chromatography paper), which separates the molecules according to their specific characteristics (e.g. size/charge).
• In thin-layer chromatography, such as the separation of photosynthetic pigments, the stationary phase is a thin layer of absorbent material (e.g. silica gel or cellulose) attached to a solid plate. A sample is placed near the bottom of the plate which is placed in an appropriate mobile phase (solvent).
• Rf value equation:

  Distance Travelled By The Spot (x) ÷ Distance Travelled By The Solvent (y)
• Rf values are useful because they can be compared with Rf values from known samples or standards to provide objective and accurate results.
• Quantitative test:

  identifies the amount of a substance that is present.

  e.g. testing for the concentration of an acid
• Qualitative test:

  shows whether a particular substance is present but does not give an indication of how much is present.

  e.g. testing for a colour change
• Semi-quantitative test:

  a systematic procedure using specific chemical reactions to confirm whether certain ions/other electrons are present and approximately in what concentrations.

  e.g. serial dilution test
• Identifying Reducing Sugars:

  1. add 1/4 spatula of sugar to a test tube
  2. add 2cm³ of Benedict's solution
  3. heat in a water bath and observe colour changes

  Results:

  brick red colour if reducing sugars are present
• Identifying Non-Reducing Sugars:

  1. add 1/4 spatula of sugar to a test tube
  2. add 2cm³ HCl and heat in a water bath
  3. cool in a beaker and add sodium bicarbonate until it stops effervescing
  4. add 2cm³ of Benedict's solution
  5. heat in a water bath and observe colour changes

  Results:

  brick red colour if non-reducing sugars are present
• In serial dilution tests, the 'neat' stock solution contains a 100% concentration. Each new solution made contains 10% less of the neat solution than the previous (0.1, 0.01, 0.001, etc).
• The purpose of serial dilution tests is to create a set of known standards with known concentrations which can be used for comparison to find the concentration of an unknown solution.
• Serial dilution tests are semi-quantitative.
• Systematic errors affect all the data in the same way.
• Random errors don't affect all the data in the same way.
• Paper chromatography method:

  1. a pencil line is drawn and a spot of mixture is placed here and left to dry
  2. the paper is suspended in a solvent (mobile phase) just below the pencil line
  3. as the solvent travels upwards the components of the mixture move with it at different speeds
  4. produces a chromatogram
• The rate of change on a graph can be measured at any point on a curve by measuring the gradient of that point. The point's gradient is equal to the tangent's gradient to the curve.

  To accurately draw a tangent line you use the normal line (a line passing through a point at a 90° angle and is perpendicular to the tangent).

  To find the gradient you use the equation y/x.
• Control variables in enzyme reactions:

  • volume and concentration of substrate
  • volume and concentration of enzymes
• Colourimeters remove subjectivity from a conclusion as qualitative data can be converted into objective quantitative data.
• Colourimetry is measured in % absorption (how much light is absorbed) or % transmission (how much light is transmitted through).
• A colourimeter is based on the photometric technique, which states that when a beam of incident light of intensity passes through a solution, a part of the incident light is reflected, a part is absorbed, and the rest is transmitted through.
• Colourimeters must be calibrated first using the standard solutions of the known concentration of the solute that has to be determined in the test solution (e.g. in the Benedict's test you calibrate when it is blue).
• There is a ray of light with a certain wavelength that is specific for the assay (what you're measuring). Before reaching the solution the ray of light passes through a series of filters and lenses used for the navigation of the coloured light in the colourimeter. The filter splits the beam of light into different wavelengths, allowing the required wavelength to pass through it and reach the cuvette containing the standard solutions. It analyses the reflected light and compares it with a predetermined solution.
• cm -> x10 -> mm

  mm <- /10 <- cm

  mm -> x1000 -> um

  um <- /1000 <- mm

  um -> x1000 -> nm

  nm <- /1000 <- um

  cm -> x10,000 -> um

  cm -> x10,000,000 -> nm
• A student investigated the difference in the reducing sugar content of two fruit juices. He performed a biochemical test on each fruit juice using Benedict's solution. He then used a colorimeter with each test result.

  How can the results from the colorimeter identify the fruit juice containing the higher sugar content?

  The fruit juice with a higher concentration of reducing sugar will have a higher absorbance (produces a darker red colour).
• Describe a biochemical test you could use with a solution to confirm that amylase affects starch in agar?

  Heat in Benedict's solution to test for the presence of a reducing sugar; should produce a red precipitate.
• What is the problem of using a ruler to measure the diameter in mm?

  Human error / uncertainty
• Describe how you could use aseptic techniques to transfer a small amount of bacteria in liquid culture from a bottle onto an agar plate.

  • use a sterilised pipette
  • work close to upward air movement
  • disinfect surfaces regularly
  • disinfect equipment immediately after use
• The scientists incubated the flasks containing the leaf discs at 26 °C and gently shook the flasks. Suggest one reason why the scientists ensured the temperature remained constant and one reason why the leaf discs were shaken.

  1. Maintain temperature so no change in shape of membrane proteins;
  2. Shaking so all surfaces of the leaf discs are submerged in water;
• What is a positive control?

  A control that produces the expected result (e.g. using glucose in a reducing sugars test).
• What is a negative control?

  A control that involves the absence of a reagent/the substance expected to cause a change (e.g. using water in a reducing sugars test).