Paper chromatogram:
Many times, it happens which all the components of a mixture cannot be separated using a single solvent system; some components separate better in one solvent and some in another. Within such cases, two-dimensional paper chromatography might be employed. In this method, a square paper of 30 cm x 30 cm is taken and the sample is spotted at one corner of the sheet. Chromatogram is established using one solvent whence solute migrates parallel to one edge of the paper. Further time, the paper is turned at 90o and developed in a second solvent system which carries the solutes into the unused portion of the paper. A classical chromatogram of a protein hydrolysate using ninhydrin-stained spots is shown in Figure.
Figure: Illustration of two-dimensional paper chromatogram of a protein hydrolysate where solute is placed in corner at H and it is first run towards the right with an acetic acid-butanol solvent and then at perpendicular using phenol + resol/water solvent