Preparation of Glass Columns for Visual Demonstration of Reversed-phase Liquid Chromatography Lane C. Sander Organic Analytical Research Division, Center For Analytical Chemistry, National Bureau of Standards, Gaithersburg, MD 20899 The principles of reversed-phase liquid chromatography1 can he vividly demonstrated by preparing a glass column for the separation of dyes. Used with aconventional liquid chromatographic pump, the glass column can separate various dyes within a period of about 2 minutes. Background Conventional stainless steel columns employed in high performance liauid chromatoeraohv - . . are usuallv orenared . kith silica substrates -3-10 pm in diameter. The use of small particles increases column efficiencv but also increases columi hack-pressure compared to colu&s with larger diameter particles. Commercial columns are prepared by pumping a slurry of the packing material into a column of stainless steel tuhing under high pressure (often as high as 15,000 psi). This slurry packing process is required to prepare efficient columns with suhstrates smaller than 20 pm diameter. Because of the small article size. column hackpressure during normal operation can exceed 2000 p s i . ~ o r these reasons, standard liquid chromatographic packing materials are unsuitable for the preparation of glass columns. Back-pressure can be reduced (with a subsequent loss in column efficiency) by using larger diameter silica particles. Particles -50-100 pm in diameter or larger will reduce backpressure to a level compatible with glass tuhing, and columns can he prepared from such suhstrates by a dry packing process. Large-diameter chromatographic packings are available for the preparation of guard columns, and are also suitable for making glass chromatographic columns. A similar type of packing material is used in commercial products for solid phase extraction. Designed for sample cleanup and preconcentration, these products are actually small (-1 cm 0.d. X 2 cm length) plastic chromatographic columns containing about 0.35 g of a large diameter particle size packing material. Material of this tvpe can he used in the nrenaration of low-resolution glass c&mns. A series of introductory HPLC experiments utilizing CIEand silica Sep-Paks2 (Waters Associates, Milford, MA) has been previously described for the separation of grape-flavored Kool-Aid.3 "
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Column Preparatlon and Use
A 40-cm length of '!-in. 0.d. X 2-mm i.d. Pyrex tuhing was cut using a diamond-tip pencil to insure that both ends of the tubing were flat and square. A conventional HPLC end
Contribution of the National Bureau of Standards. Not subject to CODVriaht. .. Snyder. L. R.; Kirkland, J. J. Introduction to Mcdern Liquid Chrcmatography; Wiiey: New York, 1979. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experlmentai procedure. Such identificationdoes not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Bidiingmeyer, B. A.; Warren F. V., Jr. J. Chem. Educ. 1984, 61,
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fitting and frit was attached to the tuhing using a'I4-in. Vespel ferrule (see the figure). CIS packing material was reclaimed from C18 Sep-Paks (about three required) by cutting open the plastic casing. Additionally, a small quantity (less than 1g) of 60-80 mesh size glass beads was used in C,, bonded column preparation. Dry CIE silica packing material was added stepwise to the tuhing while the tubing was gently tapped and rotated. Each addition of the packing material was sufficient to fill about 1-2-cm length of the tubing. The last 5 cm of the column was carefully filled with hare (unmodified) glass beads without tapping, su that a sharp interfare between the two types of packVespel ing materials was~iaingined. ferrule Thecolumn was completed by attaching a second end fitting to the top of the tuhing. e n d fitting The liquid chromatograph Detail of gias column showing in- was assembled using a conterface between glass beads and ventional HPLC pump and Cq8bonded silica. fixed loop injector with a 1mL loop. Various pumps may he suitable for demonstration purposes; however, the pump should be capable of delivering a flow rate of at least 2 mL1 min a t 500 psi. Lower capacity pumps may be applicable if lower flow rates and longer separation time can he tolerated. Proper safety precautions should he taken when using the glass column.
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Dve Seoarations Sewral common food coloring dyes can he separated isocratically (i.e., with a constant solvent environment) using a mohile phasecomnosition of 8OC: water 200: ~reronirrileat a flow rate of 1m ~ j m i nAs . an example, prepare a solution of ~ food coloring to 10 green food colorina using about 1d r o of mL water. Fill the;njectbr loop with this mixture, and-inject i t into the column. The green dye mixture will ranidlv concentrate a t the interface hetwien the glass beads and C18 suhstrates and then will migrate somewhat more slowly along the length of the C18 portion of the column. Two components will separate in this process, FD&C #1 blue and FD&C #6 yellow dyes. The concentration effect results from the injection of a large volume of water (the green dye mixture) onto the column. In the presence of this high water environment, the nonpolar dyes are unretained on the glass beads but are strongly retained on the nonpolar C18 surface. Once the water has moved away from the interface between the glass Volume 65 Number 4
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beads and CIB substrate, the mobile phase environment becomes more nonpolar and the dye mixture begins to elute. The yellow dye is more polar than the blue dye, and it elutes first. Other mixtures of dyes can be separated. A particularly dramatic separation of five components occurs for the mixture of red, yellow, and blue food coloring dyes. Also of interest is the separation of the artificial colors in grape soda soft drinks. Using the same conditions as above, red and blue dyes can be resolved from certain brands of grape drinks. The effect that changes in mobile phase polarity have on solute retention can be easily demonstrated by altering slightly the composition of the mobile phase. Solutes are
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retained longer with polar (high water composition) mobile phases than with nonpolar (high organic composition) mobile phases. Dye mixtures will be less retained and less well separated using a higher percentage of acetontrile (e.g., 605% water/40% acetonitrile). Similarly, higher percentages of water (e.g., 90% water/lO% acetonitrile) increase separation times. Summary Basic principles of reversed-phase liquid chromatography can be easilv demonstrated bv use of transuarent chromatographic col&nsand colored solutes. This ;isualapproach to chromatomaohv should urnvide a clear and direct introduction that ~ k b i d i s c u s s ~adt elementary or advanced levels.
