Testing for biochemicals

Add a drop of Iodine solution (iodine in potassium iodide solution)

'STARCH

IODINE

cr
o
CD
OJ
(j)

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:::r

Aqueous solution, suspension or extract of test substance

Positive result (blue-black coloration)

= starch

present

Principle: iodine binds to the centre of the starch helix, forming a starch-iodine complex which is intense blueblack in colour

Perform emulsion test (add 5 ern" of ethanol to sample - mix - add to 5 em? water - shake). Positive result (milky-white suspension = emulsion)

Negative result (solution remains clear blue) = absence of reducing

= fat or oil present

Positive result (green to orange-red precipitate) Principle: the fat or oil dissolves in the ethanol, which is miscible with water. When the oil-laden ethanol is added to the water the oil is left as minute drops suspended in the water - these drops scatter light rays in many directions and give the suspension a milky-white appearance. Positive result (mauvepurple coloration) = protein present
L- __ '"

sugar
Hydrolyse with dil. HCI and neutralize with NaHCOJ

= reducing sugar present

N.B. In order to make these tests comparative, e.g. to compare the amount of protein present in several different. test solutions, it is essential that as many factors as possible are maintained constant. For example, Equal volumes of reagents should be used in each test; Results should be compared after equal time intervals; Any necessary heating should be carried out in a water bath for a fixed period of time; A control blank (a volume of water equal to the volume of text solution) should be run alongside the tests; any colour change in the blank would suggest contamination or unreliable technique.

Positive result (green to orange-red precipitate)'

More intensely-coloured precipitate

= non-reducing sugar present

Principle: Copper (II) sulphate, which forms a blue solution under alkaline conditions, is reduced to copper (I) oxide, which forms a brick-red precipitate under the same conditions

= both

reducing and nonreducing sugars present

CONCENTRATION OF REDUCING SUGAR

COLOUR OF SOLUTION AND PRECIPITATE BLUE GREEN

Principle: the alkali (sodium hydroxide) hydrolyses the protein into fragments containing the peptide linkage -CO-NHwhich then forms coloured complexes with copper (II) from the copper sulphate.

---....,~ BLUE

. Cu"
RED
INCREASING

YELLOW PALE BROWN ORANGE RED

O=C-NH

The test is semi-quantitative - the greater the concentration oi reducing sugar, the darker and heavier the precipitate.

li
.

/ "'-.1 \ HN Cu NH \ /\ NH-C=O / O=C-NH

NH-C=O

REMEMBER SAFETY PROCEDURES 1. Wear eye protection when heating solutions 2. Use a safety pipette - no pipetting by mouth

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