144 APPENDIX C 



by reducing the tendency of the molecules to 

 move around. 



Specific tests enable us to characterize the iso- 

 lated components. The reagent in the biuret 

 test gives a color with substances composed of 

 linked amino acids. Similarly, Benedict solution 

 reacts with aldehyde groups ( — HC=0) found 

 in sugars. 



Differentiation between large and small mole- 

 cules can be accomplished by placing the milk 

 in a sac which has pores so fine that only small 

 molecules may pass through. The wall of such 

 a sac is thus a semipermeable membrane, and 

 the process of ultrafiltering mixtures of large 

 and small molecules through such a semiperme- 

 able membrane is called dialysis. 



The amino acid composition of casein will be 

 examined by allowing a proteolytic enzyme to 

 break it down. The proteolytic enzyme is a 

 protein isolated from the pancreas gland, which 

 forms important enzymes for the digestive sys- 

 tem. It is capable of splitting the links between 

 the amino acids in a protein. The breakdown 

 products of casein will be analyzed by paper 

 chromatography. 



ON THE SEPARATION OF 

 COMPOUNDS 



Organic chemistry began as the chemistry of 

 carbon compounds that occur in, or are pro- 

 duced by, living organisms. So it remained for 

 a time until it was discovered that it was pos- 

 sible to synthesize innumerable unnatural com- 

 pounds of carbon, and organic chemistry went 

 its own way to leave the naturally occurring 

 compounds in the realm of biological chemistry. 



The major effort in biochemistry was devoted 

 for many years to its medical aspects, and 

 methods were developed for the qualitative and 

 quantitative determination of constituents of 

 milk, saliva, blood, urine, feces, and gastric 

 juices. These are materials easily obtainable for 

 analysis, and valuable for the clinical diagnosis 

 of human diseases. 



Many of the methods developed in this con- 

 nection, however, are not exact, since chemical 



tests which may be satisfactory for the detection 

 of a substance in one biological fluid or extract 

 are unsatisfactory in other preparations. The 

 difficulty is that such complicated mixtures vary 

 greatly in chemical composition (even within 

 the different tissues of the same organism), and 

 interfering compounds occur in some prepara- 

 tions though not in others. For this reason it 

 has been necessary to devise procedures to 

 separate mixtures into simpler fractions, in the 

 best case containing pure or nearly pure com- 

 pounds. 



One of the most rapid and convenient methods 

 for doing this is paper chromatography. This 

 method in combination with the use of relatively 

 specific chemical tests provides a general scheme 

 for evaluating the chemical composition of all 

 kinds of complex mixtures of biological origin. 



You will use this technique, not only to reveal 

 the complexity of a large natural molecule, the 

 protein casein, but also to identify a single sub- 

 stance among a variety of possibilities. 



The movement of substances on filter paper 

 depends on their solubility in the developing 

 solvent, adsorption on the paper, and often on 

 partition between two solvents. Some sub- 

 stances can be separated fairly well in distilled 

 water, but mixtures of water with various organic 

 solvents are usually more satisfactory. The 

 aqueous portion usually contains acid or base 

 to minimize the existence of more than one 

 ionized form of any dissociable substance in the 

 sample. This is not always necessary, and neu- 

 tral or buffered solvents are frequently used. 

 The volatile alcohols (methanol, propanol, 

 butanol) and acids (formic, acetic, hydrochloric) 

 are convenient, and ammonia is the most 

 generally satisfactory base. 



EXPERIMENTAL PROCEDURES 



Measure 200 ml of skimmed milk into a 

 400-ml beaker. Add hydrochloric acid drop 

 by drop until a precipitate of casein appears. 

 About 50 drops will be needed. Stir with a 

 glass rod while adding the acid. Add 40 drops, 



