Exercise 



CHEMICAL COMPONENTS OF CELLS (1) 19 



neighbors need to have a flame at the bench, 

 either delay the alcohol precipitation or do it at 

 a distance. Locate the fire extinguisher and fire 

 blanket early in the period, in case they may be 

 needed. 



Neutralization of acids and bases 



You have four neutralizations of acids to per- 

 form, two on the hydrolysates of glycogen and 

 nucleic acid, the other two on the controls for 

 these reactions. The glycogen had been taken 

 up in normal (i.e., 1 A') HCl, and is neutralized 

 with 1 N NaOH. A normal solution contains 

 1 gram molecular weight of hydrogen or hy- 

 droxy! ions per liter. The molecular weight of 

 HCl is 36.5 grams, so that a normal solution 

 contains this amount of HCl per liter; similarly 

 a normal solution of NaOH contains 40 grams 

 per liter. (How do we obtain these numbers?) 

 A given volume of 1 A'^ HCl should be very 

 nearly neutralized by the addition of the same 

 volume of 1 N NaOH. What are the products 

 of this neutralization? 



To neutralize the glycogen solution in HCl, 

 add about 30 drops of NaOH solution (about 

 1.5 ml), stir with a glass rod, and then touch the 

 end of the wet rod to a piece of red litmus paper. 

 Such paper is red when acid, but turns blue on 

 being made alkaline. You will find that the wet 

 spot you have made remains red, as it should at 

 the beginning of such a titration. Now add 

 more NaOH drop by drop, stirring well each 

 time with the glass rod, and touching a fresh bit 

 of the litmus paper each time with the wet rod. 

 Do this until the addition of a final drop just 

 turns the paper blue. That marks the end of 

 your titration. The faint blueness of the paper 

 shows that your solution is now slightly alkaline, 

 which is how we want it. 



The same essential procedure is used to neu- 

 tralize the H2SO4 in which nucleic acid was 

 hydrolyzed. The sulfuric acid solution also is 



1 N. Since each molecule of H2SO4 contains 



2 atoms of hydrogen, to make a normal solution 

 we dissolve half the gram molecular weight of 

 H2SO4 (i.e., ^ of 98 = 49 grams) in one liter. 



In this case we use barium hydroxide to neu- 

 tralize the acid. The reason is that the salt 

 formed by the neutralization, BaS04, is insolu- 

 ble and precipitates, leaving a salt-free solution 

 in which the paper chromatography to be done 

 next week will go better than if the salt were 

 present. 



Fractionation and isolation of molecules 



One of the principal tasks in biochemistry is 

 to divide a complicated mixture of molecules 

 into its components, ultimately separating out 

 single molecular species. This is the enterprise 

 in which you are now engaged, and we should 

 like to say a little more about the procedures 

 you are using. 



The crudest of them involves separating a 

 soluble from an insoluble fraction. That could 

 be done by filtration, the filter paper holding 

 back the insoluble material and permitting the 

 clear solution to run through. We accomplish 

 the same thing more rapidly and cleanly by 

 centrifugation, which among other things avoids 

 losing any material such as inevitably would 

 have stuck to filter paper. This is one of the 

 commonest procedures in biochemistry and can 

 be used to throw down any particles which are 

 denser than the liquid in which they are sus- 

 pended. 



A second method of fractionation in these 

 experiments is dialysis. This is a refined kind 

 of filtration, in which the dialysis sac is the 

 filter. It is composed of a cellulose membrane 

 that contains tiny pores which allow water and 

 small molecules to pass through but block the 

 passage of macromolecules. In other words, 

 this process divides small from large molecules. 

 In your dialysis setup, the large molecules 

 should stay inside the sac, and the small mole- 

 cules distribute themselves evenly inside and 

 outside. If, however, you had chosen to replace 

 the outside solution repeatedly with fresh dis- 

 tilled water, you would eventually have removed 

 all the small molecules, and only the large mole- 

 cules would have remained inside the sac. In 

 this way you could have washed the macro- 



