Standard curve
Standard curves are used to determine the concentration of substances. They are
obtained by relating a measured quantity to the concentration of the substance of
interest in "known" samples, i.e. Standards of known concentration. These standards
provide a reference to determine unknown concentrations. Thus amounts chosen of
standards need to span the range of concentrations expected to be found in the
"unknown" sample concentration.
The quantity (assay measurements as i.e. luminescence, radioactivity, fluorescence,
and optical density of various known concentrations of a substance) graphed on y-axis
and standard concentrations on x-axis. Data analyzed by fitting a line on curve (see
Figure below).
To determine the unknown concentration of a substance in a sample (with same assay
as for standards used), intersect across the assay measurement on y with standard
concentration, and down to x. The concentration of substance in unknown sample is
the value on x.
Quantitative estimation of the amount of proteins present in an unknown
solution
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To determine protein content of an unknown requires:
1- an assay in which measurable quantity is related to concentration
2- standards for comparison
Biuret test: is the method that forms a blue-violet color on reaction of protein with
copper ion in alkaline solution. Is a good protein assay.
Spectrophotometer: is an optical instrument that measures the light energy
transmitted throughout the continuous band of wavelength in spectromagnetic
spectrum. A beam of light is focused by a lens onto an entrance slit, where it is
collected by a second lens and refocused on the exit slit after being reflected and
dispersed by a diffraction grating (used to select ). After passing the slit , the light
goes through the sample being measured and picked up by a phototube. The
amount of light absorbed by the sample is read on the dial.
Colorimetry: white light passed through a solution containing colored compounds .
Standard solutions: known concentrations of samples
Procedure:
1. Use the stock solution of concentrated bovine serum albumin. Check
concentration
2. Number 10 tubes, and place in each the following volumes of the bovine serum
albumin: 0.0, 0.1, 0.2, 0.3, up to 1.0 ml
3. Bring total to 1 ml volume with distilled water.
These solutions of varying concentrations of protein will provide the data that will
give the standard curve.
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Application of Standard curve
Procedure:
1. To all the standard solutions and unknown of the same total volume, add 4 ml
of biuret reagent, mix by vortex
2. incubate for 20 min at 37 C
3. Prepare the spectrophotometer:
a.
Place solution from tube 1 into matched tubes or cuvettes. wipe outside
of tube and put in spec. and mark ref at the absorbance wavelength of
540 nm. Reading should be Zero.
b. pour back solution into tube 1. Now place solution from tube 2 in to
tube or cuvette, and measure the Absorbance. Do not reset reading to
Ref.
c. Similarly read O.D. of all standard solutions and unknown.
4. Plot standard curve, make sure amount of protein in (mg/ml). And determine
concentration of unknown.
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Acids, Bases and Buffers
Acid is a substance that yields hydrogen ions (H+) when dissolved in water. As
hydrogen ion is a proton, an acid is defined as any substance that can donate a
proton to some other substance.
A base is a substance that yields hydroxide ions (OH) when dissolved in water, or
any substance accepting a proton from some other substance.
To measure acidity, the amount of H+ in a solution is expressed, for convenience,
by a logarithmic pH scale, which ranges from 0 to 14. The lower the pH, the more
acidic the solution, the higher the pH, the more basic the solution.
Acid - base balance maintained by buffers. A buffer solution is a solution of a
weak acid and its conjugate base or weak base and its conjugate acid. It resists
changes in its pH despite the addition of small quantities of strong acid or strong
base.
i.e. a buffered solution of pH 4.0, on addition of small quantities of NaOH, keep the
pH from changing drastically, and goes to 4.2. If same amount of NaOH added to
the same volume of an un-buffered solution of pH 4, the pH may change drastically
to 12.
The buffer solution works by shifting equilibrium system, if pH too low (addition
of H+) one chemical will bind some of the H+ and raise the pH. While if pH too
high the other chemical will donate some H+ to lower the pH.
i.e. Phosphate buffer: mixture of HPO4 and H2PO4 where H2PO4 is weakly acidic,
and HPO4 is lightly basic, are in an equimolar solution.
If a limited amount of strong acid is added, part of the basic is converted to the
weakly acidic dihydrogen phosphate: HPO4 + H+
H2PO4
If, however, a strong base is added, the opposite conversion occurs
H2PO4 + OH
HPO4 + H2O
Maintaining the balance of acids and bases fairly constant is essential to keep
organisms healthy. i.e. Blood pH must be kept close to 7.4 in mammals. Addition
of 0.2 to either direction is serious.
Determination of buffering capacity of solutions
The buffering capacity of solutions is its ability to react with acids or bases and
neutralize their effect. For example, if the same weak acid solution added to 2 vials
of solutions both with a pH 7, but one with no buffering power and the other with
buffering power, the pH of the first one will immediately drop while the pH of the
other will change very little or not at all. Once buffering capacity used up by too
much acid or base addition, the pH drops rapidly.
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To follow the pH change, pH indicators can be used. Indicators are compounds
whose color is affected by acid and base. i.e. Phenol red indicator, is an acid base
indicator with a color change from orange red to yellow in the presence of acids,
and to red in base (alkaline) conditions.
Experimental procedure:
1. In a test tube rack, set up 4 empty test tubes and number them from 1 – 4.
Pipette 5 ml distilled water and place into tube 1 to serve as a control. Similarly in
tube 5, put 5 ml of phosphate buffer.
In tubes 2 and 3, pipette 4 ml distilled water, plus 1 ml of H2PO4 in tube 2, while in
tube 3, 1 ml of HPO4 solution.
To all test tubes add 10 drops of Phenol red. Mix well.
2. Observe color in each tube.
3. Start adding 1 drop of HCl at a time to tube 1 and mixing. Observe color
Change, and drops added.
4. Now add 1 drop of base at a time to tube 1. mix and observe color and drops
added.
5. repeat procedure with tube 2 starting by adding base, count the drop added till
color change.
6. Similarly with tube 3 this time starting with the acid instead of base. Count the
drops.
7. Finally add acid or base to tube 5, count the drops added, did the color change?
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