PAPER CHROMATOGRAPHY 3()7 



Application of Sample. The applioation of the sample to the paper is 

 quite simple and yet may be tedious when large numl)ers of chromatograms 

 are involved. Usually 5-m1 pipettes are employed. If it is necessary to 

 use larger amounts of solution, the paper should be dried after each 5-m1 

 application. Larger pipettes may be used if the sample can be contin- 

 uously dried in a current of warm air. If the initial spot is too large 

 (greater than about 1-cm diameter), the chromatogram tends to be diffuse 

 and indefinite. Williams (9) has described a method for the stacking of 

 filter-paper sheets between supports in echelon which facihtates the appli- 

 cation of the sample. 



Solvents. Numerous solvent mixtures have been employed for specific 

 separations, and an indication of usage is presented later in the tabular 

 summaries. In most solvent mixtures the water is used to saturate the 

 organic solvent, and only the saturated organic phase is used on the chro- 

 matogram. The following is a description of some of the more widely 

 used solvents, as reported by Wilhams (9) : 



Phenol solvent: 100 g phenol (Mallinckrodt, analytical grade) saturated with an 

 aqueous solution containing 6.3 per cent sodium citrate and 3.7 per cent sodium (or 

 potassium) dihydrogen phosphate. About 20 ml of the aqueous soUition is refiuued 

 to saturate the phenol. The salts serve a dual purpose: they inhibit the diffusion oi 

 the spots on urine chromatograms, and they prevent the migration of a contaminant 

 in the paper which otherwise obscures the lower half of the chromatogram. 



Butanol-acetic acid solvent: 80 ml n-butanol, 20 ml glacial acetic acid, and 20 ml 

 water This solvent must be freshly prepared for each determination. 



Butanol-ethanol solvent: 80 ml n-butanol, 20 ml of 95 per cent ethanol, and 20 ml 



water. 



Isobutijric acid solvent: 80 ml isobutyric acid and 20 ml water. 



Lutidine solvent: 65 ml 2,6-lutidine and 35 ml water. Lutidine is preferred to col- 

 lidine because of the greater uniformity of the commercially available product. 



Collidine-lutidine solvent: 33 ml of the fraction of mixed coUidines boiling between 

 158 and 165°C, 50 ml 2,6-lutidine, and 17 ml water. 



Pyridine-butanol solvent: 80 ml pyridine, 20 ml /i-butanol, and 20 ml water. 



Butanol-pyridine solvent: 50 ml n-butanol, 50 ml pyridine, and 20 ml water. 



Butanol-ethanol-ammonia solvent: 80 ml n-butanol, 10 ml of 95 per cent ethanol, 

 and 30 ml concentrated ammonium hydroxide. 



Ethanol solvent: 100 ml of 95 per cent ethanol. 



Methanol solvent: 95 ml absolute methanol and 5 ml water. 



Pyridine solvent: 65 ml pyridine and 35 ml water. 



Etkanol-acetic acid solvent: 95 ml of 95 per cent ethanol and 5 ml glacial acetic acid. 

 This solvent must be freshly prepared before each determination. 



Ethanol-ammonia solvent: 95 ml of 95 per cent ethanol a..d 5 ml conc-entrated 



ammonium hydroxide. , , , 



Butanol-ethylene gly col-hydrochloric acid solvent: 80 ml «-l.ut:.n.>l, 20 n:l ctliyl.Tic 



glycol, and 20 ml of 0.1 N hydrochloric acid. 



Phenol-acetic acid solvent: Same as phenol solvent except that ghicial a<-etic acid is 

 employed in a separate container within the chromatograph chamber to maintain an 

 acid atmosphere. This solvent is useful in separating acidic substances. 



Phenol-ammonia solvent: Same as phenol solvent except that concentrated ammo- 



