Judith Faye Rubinson and Kenneth A. Rubinson University of Cincinnati Cincinnati. OH 45221
II
A Laboratory Introduction to Quantitative Column Chromatography
One of the first problems a chemist faces when attempting t o investigate the properties of some substance is obtaining a pure sample. Liquid chromatography, with its multitude of combinations of eluents and column materials available, provides a powerful tool in the separation of from each other. Several experiments involving separation by liquid chromatography have been described for use in undergraduate laboratories (1-7). With t h e excention of a n excellent exnerimrnt illu\trating sepilrutim hy ion c,xd~ilnget;,, thcsi, i l l volvr tmreulistir rontliti~msor extensive schemes ior elutiun or visualization. I n t h e procedure outlined below, the chromatographic separation alone can be done easily and provides a n introduction t o column chromatography. However, t h e entire quantitative experiment described demonstrates several other concepts important in analytical chemistry. T h e first a n d most important concept is found in preparing t h e Beer's Law plot calibration a n d comparing with i t t h e ahsorhance of a n unknown. All conditions except t h e concentration of t h e comnound t o be measured must remain t h e same. T h e second is the elutropic series, illustrated by t h e elution of the less tiehtlv bound c o m ~ o n e nwith t ethvl acetate and rhr more right'l! l k n d unr afkmvards with &hatwl. Third. m i c r o \ ~ ~ h m ~ tterhniuuc.; ris are tlsrd, nlth~,urh - larrrr samples and columns can he employed if desired. In t h e laboratory, t h e students receive small volumes of liquid unknown. his is a mixture of bromocresol green (3', 3", 5', 5"-tetrabromo-m-cresolsulfonephthalein)and methyl orange (p-[[p-(dimethylamino)pheny1]azo]benzenesulfonic acid) in their base forms. An aliquot is placed on a small silica gel column. Ethyl acetate containing a small amount of hydrochloric acid is used t o elute t h e hromocresol green into a volumetric flask containing a few drops of triethylamine, producing a purple solution of t h e base form. Methanol with added triethvlamine is then used t o elute t h e methvl oranee intoa contninrr t o u,hich has iwvn added n ira tlrc~psui H('1, p r d u r i n g thc acidic fmm ahich is red. l'hr. sample< are then diluted to a known volume and quantitated by iisible spectrophotometry. The entire experiment-column preparation, separation, standard curves, and analysis of unknown-were completed in one 3-hr laboratory period by students who already had learned how t o d o quantitative serial dilution, t o operate t h e spectrophotometers, and t o find the assay wavelengths for t h e dyes. Directions for the Instructor Equipment and Materials. All spectrophotometric measurements were made using a Bsusch and Lomh Speetronic 20 Spectmphotometer. Simple Pyreurn test tubes (13 X 100 mm) were used instead of the usual c&et
