going beyond. going further
edited by N. H. ET~INGER George Washington High Schwl Bustleton Ave. and Verree Road Philadelphia. PA 19116
An Inexpensive Experiment for the Introduction of High Performance Liquid Chromatography Brian A. Bidlingmeyer and F. Vincent Warren, Jr. Waters Associates, 34 Maple Street, Milford, MA 01757 Column liquid chromatography (LC) is one of the fastest growing segments uf analyjical instrumentation. This phenomenal aruwth is in large part due to the excellent qualitaand preparative capabilities which are oftive, fered by LC. Furthermore, LC can be applied to a greater variety of samples than any other separation technique. Analyses of the extremely complex mixtures which chemists encounter in chemical and biological systems promise to challenge separation scientists for many years. Despite the continuous advances in selective chemical reagents - and improvements in physical techniques for measurement, LC separations will continue to play a complementary and vital role in all areas of chemical analysis. Technoloeical develo~mentsduring the past decade have brought signkcant impr&ements to tKe instrumentation and column packings used in these LC separations. As a result, high performance liquid chromatography (HPLC) has emerged as the preferred method for the separation and quantitative nnaiysis of a wide range of samples. Modern HPLC analyses are imt and efficient, and detection of as little as 100 pg of material is routinely achievable. Opportunities for applying HPLC are almost unlimited, with the result that HPLC instruments have become indispensable tools for a variety of scientists and manufacturers. This widespread popularity indicates the importance of introducing future scientists to the principles of LC a t an early stage in their trainine. her; is a need for a basic LC experiment which demonstrates the fundamentals in a vivid manner, yet is safe (uses no "hazardous" chemicals), uses relatively common and familiar materials, and above all, is inexpensive. The experiment should illustrate the mechanisms of LC and should provide data for the calculation of important chromatographic parameters such as resolution. Additionally, it would be helpful if the demonstration could he expanded to a quantitative analysis experiment for students wishing t o pursue their interest in the subject. Unfortunately, most previously described
Normally, it is unwise to use adjecti5,es like m m t or rofert mdescribe experiments. Hut the words me appropriate when desurilrine" thc HP1.C r x o r r i m c n t ~outlmed helw. These are the must compact experiments this teacher has ever tried and, definitely, they are the safest! What's more, they are intriguing and educational. They can be done anywhere a small amount of water is available. No special cautions are called for. Miriam C. Nagel Avon High School 125 Juniper Drive Avon, CT 06001
716
Journal of Chemical Education
introductory LC experiments (1-3) have relied upon expensive and sophisticated HPLC instruments and have used mobile phases containing harsh additives. We believe that a safe and affordable alternative should be available. Ideally, an introductory LC experiment should mirror the processes which occur in a typical HPLC analysis. The same mechanisms of separation should be operative, and the apparatus used should correspond directly to the components of an HPLC instrument. Experiments in paper chromatog-
"Going Beyond, Going Further" should answer a need long felt by many who teach or supervise high school science programs. While students' interest often are piqued by possible involvement in Science Fairs and Competitions, they will just as often be ata loss as to what lo do--particularly in the area of chemistry. Not surprisingly. the teacher will also find it difficult to choose a topicthat will: (a) beat a proper intellectual level. (b) be reasonably safe. (c) not require a great expense, or Id\ , ~m.e n avenues far further research. We hooe to address each of tneSe concerns Some 01 3s nave 'pel" cnemistry pralecls whom we m y share wtth O J swdenlS. ~ Iyod feel that the move criterla are met by your project, please submit it ~
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Neli Ettinger is a teacher at George Washington High Schwi in Philadelphia where he serves as head of the Science Department. He received his BA and his MA from Temple University in Pennsylvania in 1954 and 1955, respectively. He completed a second MA in science education in 1966 at the University of Pennsylvania. Along with his 29 years of teaching experience. Ettinger is actively involvedwith several professionalorganizations including the American Association for the Advancement of Science, the National Science Teachers Association, the American Chemical Society, and the American Society of Microbiologists.
Figure 1. Block diagram showlng the component3 of an isonstic HPLC Instrument. Figure 2. Students performing the LC experiments in Miriam Nagel's d a s . (Photograph by Barry Rydberg.)
raphy or thin-layer chromatography only partially fulf~U these goals. In this report we describe an inexpensive experiment in column LC which seoarates the food dves foundin a nooThe first process which governs an LC separation is the ular powdered drink mix, grape-flavorei ~ i o l - ~ i d @ . ~ i i h differential migration of solutes, which can be explained to a first approximation in terms of the competing attractions isocratic and eradient elution methods are used. The data collection from this experiment allow calculation of resolution of the mobile and stationary phases for each solute. The forces res~onsiblefor interactions between the solute and the two tR). selectivitv In) and column effiriencv- IN1 . . usine the ;ame phases are the same forces which explain solubility: electroeq"ations which would be applied to HPLC data.static, dipole, and dispersive (Van der Waals) forces. In reFimre 1shows the com~onentsof a simole HPLC instrumentwhich is capahle of i&ratic (single el"ant) separations. versed-phase LC, polar solutes will be more strongly attracted Each component of Figure 1 is re~resentedin this introducto the mobile phase than to the stationarv phase and will tory expe&nent. A disposable syiinge will serve as the comtherefore eluterather quickly from the coiukn. Less polar bined eluant reservoir and solvent delivery system (Fig. 2). solutes will wend more time in interactions with the staThe injector is a smaller disposable syringe. The miniature tionary phase-and will be retained longer by the column. LC column (Sep-Pak" cartridge or equivalent) is packed with By adjusting the composition of the mobile phase, a wide Silica Gel@or octadecyl-modified silica. The eluant consists variety of solutes of differing polarity can be easily separated of isopropanol mixed with water or with household, distilled by reversed-phase LC. Even closelv related molecules such white vinegar (dilute acetic acid). Since the samples are highly as homologs-can be resolved on efficient HPLC columns. colored dyes, the human eye provides an effective detector. Mobile phases can be more complex than those used in this Finally, recording of the experiment is done manually with experiment. Depending on the nature of the sample compopen and laboratory notebook. nents, it may be necessary to use additional solvents or additives such as acids, bases, salts or surfactants. The other Background variable which can be adjusted is the stationaw phase, but this Two processes characterize the chromatomaohic seoaration necessitates owning an-assortment of packed LC columns, of a mixture: differential migration of the various components which is expensive. In addition, the diversitv of mobile uhases (solutes) and the subsequent spreading along the column of which can be prepared is far greater than the variety ofcomthe molecules of each solute. When a sample is first injected mercially available stationary phases. The technique of graonto an LC column. it forms a narrow hand a t the head of the dient elution LC, in which the combination of two or more column. As mo\.ingnolvent (mubile phase) is passed through eluants can be varied with time, lends additional mobile phase the column, the initial band senarntes into individual bands control of separations. Gradient elution is an approach which for each solute, each of which migrates a t a rate governed hy eliminates excessive resolution and is often used for samples the equilibrium distribution of solute between the mobile containing solutes of widely differing polarities for which phase and the surface of the column packing (stationary isocratic separations are not appropriate. The interested phase). T o control the separation i t is therefore necessary to reader is directed to several introductorv texts (4-8) . . and remanipulate the experimental variables which have the view arti(:Im (%I21 for additional information. greatest influence on the equilibrium distribution: the comThe secund characteristic of LC srparhons is the spreading position of the mobile phase, the nature of the stationary of each solute hand during dutiun through the column. For phase and, much less importantly, the temperature. For the cartridre columns used in this exnerin~cnt.an initial band maximum versatility, the mobile and stationary phases are of solutes ;hi& occupies less t h a n 1 ml may generate two usually chosen to have contrasting polarities. In normal-phase separated hands of dves having volumes greater than 1 ml LC. the stationarv each. This band-broadening is tKe result of physical processes - ohase . is oolar while the mobile ohase is composed of nonpolar solvents. An example of normal-phase which relate to the efficiencv (see the appendix) of the LC LC would be the separation of steroids on an alkvlaminecolumn. The cartridges used here are inefficient due to the use modified silica stationary phase using a mixture of isopropanol of large (50-100 pm), irregular-shaped particles. Highly effiand hexane as the mobile phase. The situation is onuusite tor cient HPLC columns contain small particles of 5 or 10 pm reversed-phase LC, in which the mobile phase is more polar diameter tightly packed into steel or polyethylene tubes. High than the stationary phase. This is the type of chromatography performance solvent delivery systems are required to move used for all the separations performed in the experiments mobile phase across these columns at pressures up to 6000 psi. listed below. The isopropanollwater and isopropanollvinegar A more complete discussion of column efficiency and bandmobile phases are typical of reversed phase eluants, which broadening may be found in general LC texts. generally are composed of water mixed with polar organic Materials &difie Slowlv i n i m I mlofthrsample. (.V
