Colors and RtValues of Standard Dyes
Color
Dye
RI
Fluorescein
Yellow
0.77
Bromocresol Green
Yellow-Green
0.67
Methyl Red
Red
0.58
Basic Fuchsin
Purple-Red
0.41
Rhodamine B
Pink
0.27
New Methvlene Blue
Blue
0.12
20-30 min, the dyes should be well-separated, and the plate can be removed from t h e chamber. At this point, t h e solvent front should be clearly marked with a pencil. The plate is then placed in a wellventilated place, ifpossible, to allow removal of any acetic acid fumes. Determining Composition and Rf Once the plate is dry, the students can determine the composition of their unknown. In addition, either in class or later, they can determine the Rr for each dye standard and unknown for a defmitive comparison. The Rr i s determined bv measuriLg the distance from the marked origin to the center of the dye spot. Divide this value by the distance from the point of origin to the solvent fmnt. See the table for a list of measured Rrvalues for the experimental dyes under the conditions of this experiment. Column Chromatography Making the Chromatographic Column A chromatoera~hiccolumn is made by i&erkng a small Figure 3. Diagram of the chro plug of glass wool into the matographic column. neck of a 9-in. disposable pipet. The glass wool is gently, but firmly, packed in the bottom end of the pipet and then covered with a thin layer of sand. Next approximately 850 mg of silica gel is firmly packed in the column by gently tapping the outside wall of the pipet with a pencil. The column is then prepared for development by washing the silica gel with one volume of eluting solvent (waterll-propanoUacetone1:l:l). Filling and Eluting the Column The dye mixture contains fluorescein, new methylene blue, and rhodamine B in ethanol (1mg of each dye1mL ethanol). Two dmps of the dye solution are applied carefully to the top ofthe prewashed silica gel. When the liquid level has dropped to the top of the silica gel, a thin layer of sand is applied to the top of the column (see Fig. 3).
The column is then fdled to the top with the eluting solvent. The rate of elution can be increased by slipping a 4-cm section of latex tubing (114-in.-0.d. with a 3132-in.thick wall) over the top of the column and then filling the tubing with eluting solvent. The Separation The components of the mixture separate quickly as they move down the column. Fluorescein elutes from the column within 1 P 1 8 min, and rhodamine B is completely off the column within 2 5 3 2 min. The new methylene blue remains a t the top of the column, but can be removed fmm the column after the other two dves have been collected bv acid (1:l:l:l;. eluting with water~l-~ro~anoUa~:eton~acetic ? h e seuarated d v e ~ can be collected in individual vials for later TLC evaluation to confirm complete separation, if desired. Literature Cited 1. Freese,J.M.; Olemn,B.;tinniek, H. R., Jr;Useted, J.T.J. Cbm.Edue lLW7,54,684. 2. Olemn, B.; Hopson, D. J. Cbm. Educ. IS=, 60,232. 3. MeKone, H. T:Nelson, G.J. J. Cbm. Edue. 1916.53.722. 4.Madsen.B. C. J. Chem. Educ. 1973,50,85%853. 5. Ruppel, I. R.,Jr.: Cuneo, F L.; Ifmuse, J. G. J. Ckm. Edue. 1971,48,635. 6. Scisrn,A. J. J Cbm. Educ 1985,62,361. 7. Mmre, J. A;Dalrymple, D. L. Elpsrimnfal Mothads in Ogonic Chemistry;W B. Saundera: Philadelphia, 1971; pp 51-61.
Separation Science and Chromatography A Colorful Introduction Larry S. Wigman Mobil Research and Development Corporation Paulsboro Research Laboratory Paulsboro, NJ 08066 Catherine T. Kelsch Triton Regional High School Runnemede, NJ 08078
Tours of the chromatography laboratory a t Mobile Research and Development often are used to introduce students to industrial research. Explaining highly automated gas chromatography (GC)and liquid chromatography (LC) equipment to students unfamiliar with chromatography is difficult. Thus, two-dye separation experiments (extraction and column chromatography) were devised to intmduce the fundamental concepts of phases, partitioning, equilibrium, plate height, and resolution in a colorful and memorable way The entire demonstration takes only 5 min. The dye separation served a s an excellent introduction to separation science and chromatography and will be evaluated by secondary school level students. Experimental Material and Supplies 'Caution. Rhodamine B Base is a suspected carcinogen.
Rhodamine B Base, diphenylthiocarbazone (dithizone), and heptane were obtained from Aldrich (P. 0. Box 355, Milwaukee, WI 53201). Flash chromatography grade Silica Gel (32-63 pm) was purchased from Fisher (50 Valley Stream Pkw., Great Valley Corporate Center, Malvern, PA 19355) and used without drying. All other chemicals were reagent grade. Distilled water was used for the extraction. A plug of glass wool was added to a 5-U4 in. disposable glass Pasteur pipet and was used as the chromatography column. The chemicals used for this experiment are available in smaller amounts frpm Flinn (P. 0. Box 219, 131 Volume 69 Number 12 December 1992
991
Flinn St., Batavia, IL 60510) but have not been evaluated for performance. Dispose of all chemicals used for this experiment aceording to regulations at your local level. Preparation of Dye Mixture
Weigh approximately 100 mg each rhodamine B base and diphenylthiocarbazoneinto a 5-mL vial. Add 2 mL acetone to dissolve the mixture. 'Coulion. Permanent stains to fabrics result from eontact with this dye mixture. Mix well before using. Prepare the mixture within one week of t h e demonstration, because the green dye (diphenylthiocarbazone)will fade. Extraction Exoeriment
To a 15-mlbeaker, add 25 ml, water and25 m1. heptane. Add a few drops of the dye mixture and swirl slightly. Stir if necessary to achieve a bright pink lower phase (water) and a rich green upper pbase (beptane). Column Preparation
Place a small glass wool plug into the bottom of a disposable glass Pasteur pipet. Fill the pipet approximately 213full with silica gel. Add a small amount of sand to the top of the bed. Column Chromatography Experiment
Wet the bed with approximately 114 mL (-5 drops) of heptane. Add two drops of the dye mixture directly to the too center of the bed. Allow the dve mixture to mierate into thk bed. Add heptane with a dropper to the remaining 1,3 of the oioet. Keeo addine heotane until the meen dve migratesaway from the die. Do not allow ;be bedto run dry until a suitable separation is achieved. Aschematic of this experiment is shown in the figure. Results and Discussion
The fundamental concepts of separation science include: equilibria phases partitioning plate height resolution A logical way to introduce these concepts is to discuss extraction, then chromatography. Extraction
Extraction involves the concept of equilibrium, two phases, and partitioning between the phases. It is a single plate or a one-step experiment. The dyes were selected because of their high solubility in one phase and low solubility in the other phase. The blue dye mixture rapidly equilibrates, partitioning completely between water and heotane. A brieht ink aoueous ~ h a s econtrasts the rich green organic {ha&. partition coefficient, hydrophilic and hydrophobic wmpounds and Craig Counter-Current chromatography may be discussed at this point (1,2). The partition wefficient (K,,,= [solutelorganic[solutelaqueous) defines the molar distnbution of a solute between an aqueous
992
Journal of Chemical Education
1 Sand Pink Dye
1
phase. Craig Counter-Current Chromatography is a series of extractions t h a t can separate compounds that are not completely separated by one extraction. Chromatography
Green Dye
Chromatography involves a solid stationary phase and a liquid (LC) or gaseous (GC) mobile phase. Continuous partitioning, caused by mobile pbase, silica Gel flow, results in multiple plates and higher resolution. While the column is developing, the concepts of theoretical plates, see lectivity, detection, capacity factor, normal phase and reGlass Wool versed phase may be discussed (13). I Using the same dye mixture for both extraction and chromaDiagram of experimental set-up. tography links a single plate experiment to a multi-plate experiment. Atheoretical plate can be conceptualized as one complete equilibrium such as one extraction. The chromatography column can be conceptualized as a series of extractions, each of one theoretical plate, thus leading to multiple theoretical plates. Capacity Factor (k'= Kp * Volume,,bil~olume,ati~)is simply the partition wefficient corrected for phase ratios. Selectivity is the ability of a wlumn to separate one compound class from another. This experiment is classified as normal phase chromatography, because a polar stationary phase and a nonpolar mobile phase is used. Normal phase cbromatography is generally selective for bydrophilic compounds (the pink dye). Reversed-phase chromatography uses a polar mobile phase and a nonpolar stationary pbase. Reversed-phase chromatography is generally selective for hydrophobic compounds (the green dye). This introduction the necessary fundamentals allowine for a meanineful and memorable oresentation of
i
Literature Cited 1. Beuer, H. H.; Christian, G.D.: O'tkilly, J.E.;lnstrum~nfalAdy~is,AUynandBamn, he.: Boston,1978: Chaptera 2-22. 2. Giddidigs, J.C. lketiseAnol. Cham. 2nd ed. 1882, I,@); 63-184 3. Horuath, C;Lin, H. J. J. Chmmamgr. 1978,149; 149-170.
