180 
cules, by hyroloysis either with an acid or ferment into peptones, etc. Then 
we would expect the formol number to increase, double, if each protein 
molecule were split into two simpler ones. This is true, so formol titration 
gives a measure of the hydrolytic cleavage. We know that the proteids of 
milk are neutral to indicators, but on the addition of the formaldehyde 
become decidedly acid to these indicators. 
Now if we can determine a factor or equivalent of the acidity produced 
on the addition of the formaldehyde to milk proteids, we can at once deter- 
mine the percent of proteids in milk by titrating the acidity with a standard 
alkali. 
In 1912, E. Holl Miller, of England, worked out a method for estimating 
the proteids in butter, and the same method is used in determining the 
proteids in milk. 
Directions for estimating the proteids in butter. Weigh into a tared beaker 
exactly 10 grams of butter, which is placed in a water bath at 60° to 70° C. - 
until the butter is completely melted. Twenty-five cc. of carbon dioxide 
free water is then added at about 60° C. and 1 ce. of phenolpthalein solution. 
The contents are well agitated. Run in te NaOH until a faint permanent 
pink color is formed. It is found that the end point is masked by the yellow 
color of butter fat, the contents of the beaker should be allowed to settle 
and the bottom aqueous layer observed, and the addition of alkali continued 
until the pink tint is obtained. Five ec. of formaldehyde (40 per cent.) is 
added. The formaldehyde must either be neutralized before addition or its 
5 ce. obtained and afterwards deducted. After the 
N NaOH 
run in until a permanent faint pink color is produced in the aqueous layer. 
N 
at 
equivalent to the acidity of the formaldehyde. No deduction is necessary 
acidity equivalent for 
formaldehyde has been added the beaker is well shaken and again 
The number of ce alkali used in the second titration less the amount 
if the formaldehyde was neutralized before being added to the butter. Now 
the number of ce. aa alkali used to neutralize the acidity produced on the 
addition of the formaldehyde is proportional to the protein present. One 
ee. of N alkali is equivalent to .01355 grams of protein nitrogen or .0864 
grams milk protein, assuming a definite proportion of casein and albumen. 
0864 X 100 Xce. 
Then to calculate the percent of protein we have 10 
= per- 
cent protein if 10 grams of butter were taken. 
