Laboratory Experiment pubs.acs.org/jchemeduc
Fast Analytical Separations with High-Pressure Liquid Chromatography Trandon A. Bender, Jenacee Booth, and Edward B. Walker* Chemistry Department, Weber State University, Ogden, Utah 84408-2503, United States S Supporting Information *
ABSTRACT: High-pressure liquid chromatography (HPLC) has established itself as a critically important analytical method for many research and commercial laboratories. Employers expect today’s chemistry graduates to have a working knowledge of liquid chromatography techniques including HPLC. In addition to HPLC, it is becoming more important to educate students about newer separation technologies such as ultra-highpressure liquid chromatography (UHPLC). Unfortunately, these systems cost significantly more than traditional HPLC instruments, preventing their broad utilization in instrumental teaching laboratories. We have developed an ultra-fast isocratic separation method using traditional HPLC instrumentation that separates five compounds in 1 min. When utilized as a teaching activity, this ultra-fast separation allows students to develop an analytical method, generate standard calibration curves, and analyze unknown samples in a single teaching laboratory period. Furthermore, chromatograms obtained with this new method are similar to UHPLC, allowing students to experience separations similar to those obtained on newer UHPLC systems using a traditional HPLC. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, HPLC, Laboratory Equipment/Apparatus
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water) on a short reverse-phase (C18) column, generate multilevel standard calibration plots, and analyze unknown solutions in one 3-h laboratory session. The short column is actually a C18 guard column that costs only 1/10 as much as a standard analytical column. Because the optimum mobile phase requires only 1 min to separate five compounds, students have time to make numerous injections while consuming relatively small quantities of mobile-phase solvents. Furthermore, the resulting chromatograms are similar to the results that would be obtained from a more advanced ultra-high-pressure liquid chromatography (UHPLC) system. UHPLC has become a dominant method for analytical separations because it offers enhanced selectivity and reduced run times.10 The method proposed here simulates separations seen on modern UHPLC systems using a traditional HPLC because of the short run times and small solvent volumes used. However, the column used for this procedure has much larger solid-phase particles. Therefore, ultra-high pressures are not required and the analytical column used is affordable compared to UHPLC columns. The chemical compounds in this activity are common analytes in a commercial laboratory, caffeine, β-naphthol, and methyl-, ethyl-, and propyl parabens, and exhibit excellent stability in aqueous solutions.11
ver the past two decades, high-pressure liquid chromatography (HPLC) has become a common separation technique in most analytical laboratories. Industries ranging from food to pharmaceuticals use this technology for testing their products.1,2 As a result of the increased use of these systems, the pressure for educators to introduce and teach HPLC in undergraduate teaching laboratories has been amplified. Many papers in this Journal have answered the call by developing undergraduate lab exercises that can be performed at most institutions.3−8 In 1997, Van Arman and Thomsen designed an experiment for students that involved the separation of five nucleosides in under four minutes.4 A year later, Ferguson devised an experiment that separated caffeine and an analgesic powder in three minutes.5 A 2-min separation of soft-drink ingredients was described for capillary electrophoresis.9 Each of these papers offers a novel approach for teaching rapid separations. A shorter chromatographic run time allows for more experimental parameters to be investigated during a single teaching laboratory session and reduces mobile-phase consumption. For example, one important aspect of liquid chromatography, the effect of mobile-phase composition on analyte retention and separation, can be studied in more detail by utilizing a procedure with shorter run times. We have developed a rapid HPLC method that allows students in an undergraduate instrumental chemistry course to determine an optimal mobile-phase solvent mixture (methanol/ © XXXX American Chemical Society and Division of Chemical Education, Inc.
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Laboratory Experiment
elution profiles of the primary standard solution at a flow rate of 2 mL/min. Resulting chromatograms were processed to obtain peak areas, heights, widths, and retention times. Students were instructed to select an optimal mobile phase based upon the criteria of both speed and selectivity for all four analytes and the internal standard. After selecting their optimal mobile phase, calibration standards and unknowns were injected. Peak areas were used to calibrate the instrument and determine concentrations of unknown samples.
EQUIPMENT AND MATERIAL HPLC experiments were performed using a Waters Alliance HPLC System equipped with a Waters 996 diode array detector and automated injection. UV spectra were collected across the range of 200−400 nm, extracting 254 nm for chromatograms. Waters Empower software was utilized for instrument control, data collection, and data processing. The column was a Waters 4 μm C18 Nova Pak 3.9 mm × 20 mm guard column (Figure 1). Mobile-phase flow rate was 2 mL/min and injection volumes were all 10 μL. Any standard isocratic HPLC system with 254 nm UV detection will work well for this experiment.
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HAZARDS Standard safety precautions and appropriate chemical disposal methods should be used throughout this activity. Methanol is flammable and toxic by inhalation. The parabens are slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, and of inhalation. All analytes were selected because of their safety, stability, and common occurrence in retail products.
Figure 1. Comparison of a standard, full-sized analytical C18 HPLC column to a much shorter guard column. Pictured is a (top) Nova-Pak C18 column, 4 μm, 3.9 mm × 150 mm and (bottom) the shorter Waters Nova-Pak C18 guard column, 4 μm, 3.9 mm × 20 mm.
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RESULTS AND DISCUSSIONS Students obtained excellent separations and linear calibrations over an order of magnitude of concentration for each of the four analytes and internal standard (Figure 2). Utilization of an
Caffeine, methyl 4-hydroxybenzoate (methyl paraben), ethyl 4-hydroxybenzoate (ethyl paraben), propyl 4-hydroxybenzoate (propyl paraben), and naphthalen-2-ol (β-naphthol) were reagent grade, obtained from Sigma Aldrich (St. Louis, MO), and used without further purification. HPLC-grade formic acid and methanol were supplied by Fisher Scientific (Pittsburgh, PA), and water was prepared using a Milli-Q filtration system (Millipore, Bedford, MA). Mobile phases, standards, and samples were all filtered through 0.45 μm polycarbonate filters.
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PRELAB PREPARATIONS All mobile phases and primary standards were prepared by the instructor prior to the start of the laboratory session. Four different isocratic mobile phases were prepared by mixing appropriate volumes of methanol (35%, 40%, 45%, and 50% methanol) and 0.1% formic acid. A separate portion of the 45% methanol mobile phase was also used as the solvent to prepare primary standard solutions and student unknowns. An internal standard solution of β-naphthol at 0.5 mg/mL was prepared using the 45% methanol mobile phase. Primary solutions of caffeine at 0.25 mg/mL, methyl 4-hydroxybenzoate (methyl paraben), ethyl 4-hydroxybenzoate (ethyl paraben), and propyl 4-hydroxybenzoate (propyl paraben) all at 0.13 mg/mL were then prepared using the internal standard solution as the solvent. A primary standard mixture containing internal standard, caffeine, and the three parabens was also prepared at the same concentrations as the individual standards, also using internal standard solution as the solvent. Student unknowns of various analyte combinations and concentrations were also prepared in advance by the instructor.
Figure 2. (Left) Elution profile of caffeine, methyl paraben, ethyl paraben, β-naphthol, and propyl paraben obtained by students using optimal mobile phase (45% methanol/0.1% formic acid in water). (Right) An example of a typical student calibration plot for HPLC analysis of caffeine, methyl paraben, ethyl paraben, and propyl paraben. The response factor is the peak area ratio of analyte to internal standard.
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STUDENT LABORATORY ACTIVITY Students prepared their own multilevel calibration standards by diluting the previously prepared primary standards. They were also issued individual unknowns containing internal standard and various concentrations of analytes. All standards and samples were filtered through a 0.45 μm polycarbonate syringe filters into HPLC vials, before injecting 10 μL of each solution for analysis. Students examined the effect of increasing methanol concentrations in the mobile phase (35%, 40%, 45%, and 50% methanol) on a reverse phase C18 column by observing
internal standard is a common technique in many chromatographic methods. Normalized response factors for each analyte were calculated by dividing their respective peak areas by the peak area of the internal standard for each chromatogram. The resulting response factors were plotted as a function of concentration to generate calibration curves for each analyte. Typically, a pair of students working together requires about one hour to select their optimal mobile phase and about 45−60 min to calibrate and analyze their unknown. The extremely B
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(5) Ferguson, G. K. Quantitative HPLC analysis of an analgesic/ caffeine formulation: determination of caffeine. J. Chem. Educ. 1998, 75 (4), 467−469. (6) Batchelor, J. D.; Jones, B. T. Determination of the Scoville heat value for hot sauces and chilies: An HPLC experiment. J. Chem. Educ. 2000, 77 (2), 266−267. (7) Leacock, R. E.; Stankus, J. J.; Davis, J. M. Simultaneous determination of caffeine and vitamin B6 in energy drinks by highperformance liquid chromatography (HPLC). J. Chem. Educ. 2010, 88 (2), 232−234. (8) Gandía-Herrero, F.; Simón-Carrilo, A.; Escribano, J.; GarcíaCarmona, F. Determination of beet root betanin in dairy products by high-performance liquid chromatography (HPLC). J. Chem. Educ. 2012, 89 (5), 660−664. (9) Walker, J. C.; Zaugg, S. E.; Walker, E. B. Analysis of beverages by capillary electrophoresis. J. Chromatogr., A 1997, 781 (1−2), 481−485. (10) Wu, N.; Collins, D. C.; Lippert, J. A.; Xiang, Y.; Lee, M. L. Ultrahigh pressure liquid chromatography/time-of-flight mass spectrometry for fast separations. J. Microcolumn Sep. 2000, 12 (8), 462− 469. (11) The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 14th ed.; O’Neil, M. J., Ed.; Merck & Co.: Whitehouse Station, NJ, 2006; entry 6107.
short retention times on the small guard column give separation times of 0.5 min at 50% methanol and a maximum of only 1.7 min with 35% methanol. Column equilibration between mobile phase changes requires less than 5 min at a flow rate of 2 mL/min. As a result, the total time for students to select a mobile phase, calibrate, and analyze an unknown mixture is generally less than 2 h. This leaves ample time for HPLC power-up, priming, and other challenges sometimes encountered with instruments during teaching laboratories. The novel application of a short, guard column offers significant cost benefits and saves time compared to traditional HPLC columns. A variety of different HPLC guard columns were tested during development of this activity. Some guard columns offer little or no separation of the analytes, acting more like guard filters than columns. The best column for separating the analytes in this activity was the Nova-Pak C18 guard column, 4 μm, 3.9 mm × 20 mm.
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CONCLUSION This novel method for teaching HPLC makes it possible for students to experience important HPLC techniques of selecting an optimal isocratic mobile phase, multilevel calibration, and sample analysis during a single laboratory session. Separations achieved with this method are also dramatically shorter than traditional HPLC and mimic separation times achieved using UHPLC. This lets students experience fast separations without purchasing new instrumentation for undergraduate teaching labs. The novel technique of using a short guard column for analytical separations dramatically shortens run times, moderates column costs, and significantly reduces solvent consumption for teaching laboratories. Students can make up to 50 HPLC runs while consuming less than 100 mL of mobile-phase methanol during a single laboratory session.
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ASSOCIATED CONTENT
S Supporting Information *
Instructor notes including eluent preparation and student unknown preparation. Student instruction for basic HPLC operation, sample preparation, and data analysis. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank the Weber State University Center for Chemical Technology for support of this project.
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REFERENCES
(1) Gratzfeld-Husgen, A.; Schuster, R. HPLC for Food Analysis; Agilent Technologies Company: Germany, 1996. (2) Chesnut, S.; Salisbury, J. The role of UHPLC in pharmaceutical development. J. Sep. Sci. 2007, 30 (8), 1183−1190. (3) Bidlingmeyer, B. A.; Schmitz, S. The analysis of artificial sweeteners and additives in beverages by HPLC: An undergraduate experiment. J. Chem. Educ. 1991, 68 (8), A195−200. (4) Van Arman, S. A.; Thomsen, M. W. HPLC for undergraduate introductory laboratories. J. Chem. Educ. 1997, 74 (1), 49−50. C
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