PAPER CHROMATOGRAPHY 373 



is adilt'd to the warm solution, and tlic roascMt allowed to stand lor 21 hr. It is then 

 filtered and is ready for use. This reagent is stable indefinitely. It is used exclusively 

 for the determination of sodium. 



Quantitative Aspects. Numerous methods have been suggested for the 

 quantitative estimation of substances separated on 1 he paper chromato- 

 gram. Again, where radioactive materials are iinoKcd, the estimation of 

 amounts of substance present is greatly simplified. Advantage may be 

 taken of isotope-dilution procedures (see Chap. 1) to a^•oi(l tlic time and 

 trouble necessary for complete separations. 



Colo7- Co)npariso7i. Since the intensity of the color varies with the 

 c}uantity of material present, it is possible to make estimations by visual 

 comparison with known samples chromatographed in exactly the same 

 way as the unknown. Difficulties arise on account of (a) lack of contrast 

 between spot and background, (h) necessity of using an amount in the 

 concentration range in which the visible gradation of color will be a func- 

 tion of concentration, and (c) interfering substances that may be present 

 in the sample and not in the standard. The success of this method will be 

 primarily dependent upon standardization of experimental conditions. 



Various modifications have been employed using densitometers or 

 colorimeters in an effort to determine the total color of the spot or the 

 maximum color density and to relate these empirically to standard solu- 

 tions (8). Commercial equipment is now available specifically designed 

 for this purpose. 



Determination of Spot Area. It has been shown that usually the log- 

 arithm of the area of a spot is a linear function of the concentration (8). 

 The area can be experimentally determined by use of a planimeter or by 

 cutting out the spot and weighing the paper. The area method is usually 

 less sensitive than the color-comparison method but is less affected by 

 experimental variables. It can be used only where the boundaries are 

 distinct, and in any event standards are required. 



Elution. Of most general application, perhaps, is the procedure of 

 cutting out the spot of the chromatogram containing the separated mate- 

 rial, and analyzing it by the appropriate method. The limiting factor 

 will usually be the sensitivity of the analytical method. 



Use of an Electrical Field. The differential migration of solutes due to 

 application of an electrical current also provides a means for resolution of 

 mixtures (10b). Haugaard and Kroner (28) combined the chromato- 

 graphic and electrophoretic procedures to increase the degree of sepa- 

 ration of basic and acidic amino acids. The chiomatogram was devel- 

 oped with phenol in the usual way, the electrical potential being applied 

 at right angles to the direction of solvent fiow. 'I'his procedure allows 

 separation of substances under conditions in wliich either the migrations 

 due to electrical current or the R( values are different. An excellent 



