Biochem Lab

• pH


  A convenient way of expressing the concentration of hydronium ions [H3O+] present by means of the logarithmic function
• The hydronium ion concentrations encountered in biological materials vary from 0.1 M to 10-8 M or 0.00000001 M
• Henderson-Hasselbach Equation


  A rearrangement of the equilibrium constant for the dissociation of a weak acid, Ka into a useful expression
• Buffer solution


  A solution that resists changes in hydronium ion concentration upon addition of small amounts of acid or alkali
• Buffer solution


  • Consists of a mixture of weak acid (or weak base) and its conjugate base (or conjugate acid)
  • Resists large changes in pH most effectively when the pH is close or equal to the pKa of the weak acid
  • The choice of buffer pair should be such that the pH to be stabilized falls within the range equal to the pKa ± 1
• Buffering region


  The region where a buffer system resists changes in pH most effectively, starting from pKa -1 to pKa +1
• pH meter


  An instrument used for the precise measurement of pH, composed of a reference electrode, a glass electrode, and an electrometer
• pH meter


  • The response of the glass electrode must be calibrated accurately against standard buffers of known pH
  • Errors can arise if the unknown and the standard solution differ substantially in ionic strength or if the calibration is carried out at different temperatures
  • Standardization with two buffer standards is preferable to the use of one standard only
• Biochemical reactions are greatly influenced by hydronium ion concentration
• Organisms have evolved certain methods to maintain the solution in their cells within narrow limits of hydronium ion concentration
• Acetate buffer


  0.1 M, pH 4.7
• Record the pH of each of the buffer solutions
• Record the pH of each buffer solution after the addition of alkali
• Ratio of conjugate base to weak acid

  Affects the pH of a buffer solution
• Make 25 mL of each of the buffer solutions assigned
• Measure and record the pH of each of the buffer solutions
• Account for the magnitude of pH shift in each with reference to the direction of pH shift
• Choosing the proper buffer solution

  For protein precipitation, a 5mM buffer solution with pH 5.2 is needed
• Buffer solutions: Acetate buffer (pKa 4.73), Tris- (hydroxymethy) aminomethane (pKa 8.08), Phosphate buffer (pKa 7.20)
• Plot pH vs. mL NaOH added and determine the pKa's of the sample
• Draw the structure of the amino acid at each pKa value
• Blood pH must be kept within narrow limits for life and health
• Blood contains compounds whose concentrations fluctuate and those whose concentrations are constant
• Mineral ions in blood act as buffers to maintain pH balance
• Compounds that could act as buffers at physiological pH (around 7.4)

  • Carbonic acid
  • Phosphoric acid
  • Amino acids
• Carbohydrates are the major compounds in our diet that provide energy to run chemical reactions in the body and make us move
• Carbohydrates


  Polyhydroxy compounds that have either a keto group or an aldehyde group
• Glucose


  A typical monosaccharide
• Maltose


  A typical disaccharide
• Glucose exists almost entirely in the closed-chain hemiacetal form
• In maltose, two glucose units react to form an acetal
• Benedict's test for reducing sugars

  1. Monosaccharides and most disaccharides are easily oxidized

  2. Aldoses and ketoses react with Benedict's reagent

  3. Cupric ion, Cu2+, reacts with reducing sugar to form a red precipitate of cuprous oxide, Cu2O (s)
• Sucrose, a disaccharide, does not react to Benedict's reagent
• Sucrose


  The monosaccharides glucose and fructose are bonded through the oxygen atoms of the hemiacetal parts of the molecules, so there is no hemiacetal that can revert to the open-chain form to cause reduction of the cupric ion
• Barfoed's test


  • Used to distinguish between monosaccharides and disaccharides
  • Cupric ion in the acidic reagent is reduced much more rapidly by monosaccharides to produce the red cuprous oxide (Cu2O)
• Seliwanoff's test


  • Used to distinguish between ketohexoses and aldohexoses
  • Ketohexoses form a deep red color rapidly, aldohexoses form a light pink color that develops more slowly
• Iodine test for polysaccharides

  • Starch, a polysaccharide, contains amylose (straight-chain polymer) and amylopectin (branched-chain polymer)
  • Amylose gives a deep blue-black color with iodine, other polysaccharides give red to brown colors
• Hydrolysis of disaccharides and polysaccharides

  1. Disaccharides hydrolyze in the presence of an acid to give their individual monosaccharides

  2. Polysaccharides like amylose in starch can be hydrolyzed to smaller polysaccharides (dextrins) and to maltose, producing many glucose molecules

  3. This hydrolysis is also catalyzed by salivary and pancreatic enzymes during digestion
• Fermentation


  1. Some sugars will ferment in the presence of yeast, which contains the enzyme zymase

  2. The products are ethyl alcohol and carbon dioxide
• The formation of bubbles of carbon dioxide is used as confirmation of the fermentation process