Ii8  Standardization  of  Preparations.      { AmMJ£ch*<m?m' 
The  physiological  method  of  testing  was  described  in  the  first  por- 
tion of  the  paper.  The  chemical  method  for  determination  of 
digitoxin  employed  is  essentially  that  of  Keller,  as  outlined  in 
Lyon's  little  book  on  the  "Assay  of  Drugs,"  and  is  based  upon  the 
relative  solubility  of  the  digitalis  glucosides  in  chloroform  and 
water,  depending  for  the  final  purification  of  the  digitoxin  from  other 
principles  on  its  complete  insolubility  in  light  petroleum  benzine. 
The  method,  as  used  in  our  laboratory,  is  as  follows  : 
Twenty  grammes  of  the  powdered  leaves  are  exhausted  by  per- 
colation with  70  per  cent,  alcohol,  and  the  percolate  is  evaporated 
at  low  temperature  on  the  water-bath  until  all  alcohol  has  been  dis- 
sipated (or  if  a  tincture  or  fluidextract  is  to  be  tested,  200  c.c.  of 
the  former  or  20  c.c.  of  the  latter  are  taken  and  evaporated).  The 
residue  is  diluted  with  or  dissolved  in  sufficient  water  to  make  150 
c.c.  Fifteen  cubic  centimeters  of  solution  of  lead  subacetate  (25  per 
cent.)  is  then  added,  and  the  mixture  diluted  to  200  c.c.  The  pre- 
cipitate is  filtered  out  and  allowed  to  drain  thoroughly,  after  which 
sufficient  water  is  passed  through  the  precipitate  on  the  filter  to  insure 
saving  all  retained  mother  liquor.  The  united  filtrates  are  again 
diluted  to  2CO  c.c.  and  the  excess  of  lead  is  precipitated  by  means 
of  dried  and  powdered  sodium  sulphate  or  sodium  phosphate. 
After  standing  for  twenty-four  hours  the  precipitate  is  filtered 
out,  allowed  to  drain  well,  and  is  then  rinsed  with  water,  in  order  to 
save  all  of  the  solution.  (The  taking  of  aliquot  parts,  thereby 
avoiding  the  necessity  for  completed  filtrations  and  the  washing  of 
precipitates,  cannot  be  recommended  in  digitalis  assays  on  account 
of  the  vast  bulk  of  the  lead  subacetate,  sodium  sulphate,  or  sodium 
phosphate  precipitates.)  The  solution  is  transferred  to  a  separatory 
funnel,  2  c.c.  of  10  per  cent,  ammonia  water  added,  and  the  mix- 
ture shaken  out  with  five  portions  of  chloroform  of  30  c.c.  each. 
The  united  chloroform  solutions  are  evaporated  to  dryness  on  the 
water-bath  in  a  tared  flask  and  the  residue  of  crude  digitoxin  redis- 
solved  in  3  c.c.  of  fresh  chloroform.  Ten  cubic  centimeters  of  ether 
and  70  c.c.  of  light  petroleum  benzine  (the  so-called  86°,  which  must 
leave  no  trace  of  residue  on  evaporation)  are  added  and  the  flask 
allowed  to  stand  in  a  cool  place,  covered  by  an  inverted  beaker  for 
twenty-four  hours.  (In  hot  summer  weather  the  flask  is  placed  in 
a  refrigerator.) 
The  digitoxin  in  micro-crystalline  form  will  be  found  adhering  to 
