Flow Injection Analysis and Liquid Chromatography for Multifunctional

Laboratory Experiment pubs.acs.org/jchemeduc

Flow Injection Analysis and Liquid Chromatography for Multifunctional Chemical Analysis (MCA) Systems Ana V. Mayo, Thomas N. Loegel, Stacey Lowery Bretz, and Neil D. Danielson* Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States S Supporting Information *

ABSTRACT: The large class sizes of first-year chemistry labs makes it challenging to provide students with hands-on access to instrumentation because the number of students typically far exceeds the number of research-grade instruments available to collect data. Multifunctional chemical analysis (MCA) systems provide a viable alternative for large-scale instruction while supporting a hands-on approach to more advanced instrumentation. This study describes how the capabilities of MCA systems are extended to introduce liquid chromatography (LC) and flow injection analysis (FIA) in undergraduate laboratories. A semi-micro plastic cuvette with a Teflon tubing insert is fashioned as the flow cell for a MCA absorbance− fluorescence detector. Two MCA systems, Vernier and MeasureNet, are used in two unique experiments demonstrating the detection of salicylate in aspirin tablets by FIA and the LC separation of a mixture of riboflavin and fluorescein. Both instruments, composed of a syringe pump, T-injection valve, and the MCA detector, operated in the kinetic mode, are rugged and inexpensive permitting student construction, if desired. KEYWORDS: First-Year Undergraduate/General, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Chromatography, Fluorescence Spectroscopy, Laboratory Equipment/Apparatus, Separation Science, UV-Vis Spectroscopy

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objective to measure an unknown concentration of salicylate in a sample of aspirin after hydrolysis (experiment 1). Two different MCA systems are then used to provide an introduction to liquid chromatography (LC) through the separation of riboflavin and fluorescein on a solid-phase extraction (SPE) cartridge using both absorbance and fluorescence detection modes (experiment 2). The principles of FIA have been previously presented by Hansen and Ruzicka,10,11 as well as a published description of how to construct an FIA detector for absorbance measurements with a light-emitting diode (LED), photodiodes (PD), and Legos.12 Additional experiments describe the construction of a three-piece injector commutator made of poly(methyl methacrylate) and a spectrometer equipped with a flow cell constructed from tubing that attaches to a glass tube that crosses a cuvette for the determination of hypochlorite13 and pyridylazo resorcinol (PAR) complexes.14,15 Simple LC instruments have previously been reported using Sep-Pak cartridges and a disposable syringe,16 where both the sample (e.g., colored dyes) and the solvent were injected into the cartridge, using the human eye as the detector. Food coloring dyes were separated using a C18 solid-phase extraction (SPE) column and different mobile phases.16 SPE cartridges have also previously been used to separate dyes while demonstrating different retention mechanisms (i.e., normal, reversed, size exclusion, and ion-exchange mechanisms).

ccording to U.S. Census Bureau projections, the number of college-age individuals (ages 20−24 years) is expected to grow from 21.8 million in 2010 to 28.2 million by 2050.1 The enrollment of students in science and engineering degrees is also rapidly increasing.2 These trends suggest that chemistry educators can expect to teach larger lecture and laboratories classes in general chemistry for the foreseeable future. Providing student access to modern instrumentation during the first-year chemistry laboratory presents a logistical challenge given the ratio of large number of students to the few (if any) research-grade instruments available for students enrolled in these laboratories. One alternative is the acquisition of a multifunctional chemical analysis system (MCA)3−5 such as MeasureNet6 or Vernier.7 These systems are robust and typically use student stations connected to a remote central computer for data collection, minimizing the need for computers at every student workspace. These systems are excellent for group instruction because students can share apparatuses and solutions, but still collect their own data. MCAs offer multiple measurement capabilities, including absorbance, fluorescence, and turbidity, with detectors having kinetic options. Recently, MCAs have been used to develop several undergraduate experiments, such as measuring the absorbance of a reaction that changes color as a function of temperature8 or finding an unknown concentration of copper in various pennies of world currency.9 The experiments described herein use an MCA system for flow injection analysis (FIA) to introduce students to the Beer−Lambert law with the © 2013 American Chemical Society and Division of Chemical Education, Inc.

Published: March 15, 2013 500

dx.doi.org/10.1021/ed300340x | J. Chem. Educ. 2013, 90, 500−505

Journal of Chemical Education

Laboratory Experiment

Figure 1. (A) Flow cell (photos) requires 20 cm length (going in and out of a low-volume cuvette). (B) FIA experiment 1 setup. (C) LC experiment 2 setup: pump with mobile phase (MP), syringe containing sample to be injected, SPE (C18 Sep-Pak cartridge), S (sample cuvette), R (reference cuvette), second syringe containing MP for reference cuvette. This setup utilizes the MeasureNet detector. The Vernier detector contains only one cuvette; therefore, subtracting the background of mobile phase must be done before taking sample readings.

Table 1. Specifications for Detectors and Light Sources for the MCAs Component

MeasureNeta

Vernierb

Detector Spectrophotometer Fluorimeter

Two photodiode sensors. Allow for dual beam ratio metric measurements. Three LEDs: red (630 nm), green (525 nm), and blue (472 nm) 370 nm at 90°

Linear CCD array Tungsten halogen, wavelength range:380−950 nm Two LEDs (405 and 500 nm) at 90°

a MeasureNet’s specifications were obtained from local representatives and company Web site.6 bVernier’s specifications were obtained from the company Web site7 and Czegan and et al.5

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Students inject both the sample and appropriate mobile phase to elute different fractions; the colored dyes or precipitated anions are easily distinguished after the separation of the mixture.17 Homemade columns for separation have also been created from a 40 cm Pyrex tubing glass filled with packing material reclaimed from C18 Sep-Paks and then attached to a conventional liquid chromatograph for the separation of food coloring.18 To the best of our knowledge, inexpensive FIA and LC instrumentation has not been developed for MCA systems. The driving force for the work reported below is to describe how the current capabilities of MCAs can be extended through the addition of (i) FIA and (ii) LC, using two different MCA systems. The development of an inexpensive flow cell used with the MCA detector in the kinetic mode provided the means to create two laboratory experiments using important, but underutilized, low-pressure LC components. These experiments provide an economically viable alternative to FIA and high-performance LC instruments for large numbers of students through use of an MCA system. Detailed student and teacher guidelines for the FIA and LC experiments are provided in the Supporting Information. Given that LEDs are the major sources for MCA colorimeter−fluorometer detectors, polychromatic calibration curves19 based on wavelength emission bandwidths for different LEDs were explored by the students. Postlab questions targeted the effects of non-monochromatic light on the Beer−Lambert law, making these nonintuitive deviations of the Beer−Lambert law more student-friendly.

APPARATUS

Both experiments share a similar experimental setup (Figure 1). A homemade flow cell was constructed from Teflon tubing and a low-volume semi-micro plastic cuvette (Figure 1A). The light crosses the Teflon tube twice giving a path length of 3.2 mm. A semi-micro VWR disposable cuvette K1948 PMMA was used for all our measurements. Other cuvettes were tested and compared (see FIA teacher guidelines in the Supporting Information). The pump, which can be either syringe or peristaltic, delivers a constant low flow rate between 0.5−1.5 mL/min of the carrier solution or mobile phase. A 10 mL plastic disposable syringe is used to inject the sample through a low-pressure T injection valve that is connected with Tygon tubing to the lowvolume plastic cuvette flow cell housed in the colorimeter detector (Figure 1B). The vertical orientation permits an even separation. The specifications for the spectrophotometer− fluorimeter detectors in the MCAs used in this study can be found in Table 1. In the LC experiment 2, an SPE C18 cartridge is introduced between the injector and flow cell setup described in the FIA experiment, thereby providing LC capabilities to separate riboflavin and fluorescein (Figure 1C). Two SPE (Alltech) syringe cartridges were compared: (i) Prevail C18 modified silica, 11% C, 100% water wettable, 900 mg bed weight, 4 mL column size and (ii) Maxi-Clean C18 modified silica, 6% C, general purpose phase, 900 mg bed weight, 4 mL column size. Both cartridges are available from Grace (Deerfield, IL). 501



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°C (simulated stomach temperature of a healthy or feverish child), for at least 1 h. The control aspirin sample showed 1.14% and the experimental aspirin sample 1.23%. The limit of detection for MeasureNet is about 2 ppm. The students can obtain larger percent salicylate conversions of about 15−25% at elevated temperatures ∼70 °C at 20−30 min. This experiment can be completed in one, 3-h laboratory (see teacher guidelines in the Supporting Information). The linear regression analysis equation (absorbance, y, versus concentration in ppm, x, for n = 6) was y = 6.4 × 10−4x + 1.8 × 10−3 with R2 = 0.99202. The actual plot is shown in the FIA teacher guidelines in the Supporting Information. Given that LEDs are the major components of the colorimeter detector used in MeasureNet MCA system, the effects of non-monochromatic light are discussed in both the prelab and postlab assignments. Student-generated Excel spreadsheets in the prelab assignment provide the opportunity to explore the effects of changing both the molar absorptivity and the concentration of a substance on the transmittance. The prelab also incorporates a student-friendly model that uses the corpuscular theory of light22 to consider the probabilities of photons being transmitted through one layer of a finite path length and their capture in a second layer. The FIA experiment can conveniently introduce this concept. In the case of the Vernier MCA system (a CCD array), the wavelength interval specifies 1 nm between reported values (collects 570 values).7 This wavelength interval is the source of nonmonochromatic effect at the detector and ultimately what limits linearity. In the postlab assignments, the effects of the bandwidths of green LED versus red LED sources on the molar absorptivities (slopes) for Fe(III)−salicylate are analyzed at different wavelengths and concentrations, similar to Figure 3. After modeling the changes in molar absorptivities, it is clear that although either LED could be selected in the MCA colorimeter, less variance in the slope is more desirable in order to reduce non-monochromatic effects on the Beer−Lambert law. In addition, bromothymol blue, a dye more difficult to measure spectrophotometrically using a LED source, is evaluated as a written exercise (see FIA student and teacher guidelines in the Supporting Information).

EXPERIMENTAL PROCEDURE AND RESULTS

Flow Injection Analysis Experiment

Although aspirin is commonly used as a preventive measure against heart attacks and stroke, particularly for men, accidental salicylate poisoning can occur in children from an overdose of aspirin. The Food and Drug Administration (FDA) has an established tolerance of 0.10% salicylic acid in unbuffered aspirin.20 Salicylic acid is formed from the hydrolysis of an aspirin tablet in stomach acid. A purple complex is formed when salicylic acid reacts with ferric ion (Scheme 1); the resulting absorbance of visible light by the complex can be measured. Scheme 1. Reaction of Salicylic Acid with Iron(III) To Form a Purple Complex

A series of known salicylate concentrations are injected into the carrier stream composed of ferric nitrate dissolved in 0.01 M nitric acid. In the flow cell, the reactants mix virtually instantaneously21 to form the iron(III) complex (Scheme 1). The spectrum of the iron complex formed in Figure 2 permits

Liquid Chromatography Experiment

The FIA instrument in experiment 1 can be easily modified by the addition of an SPE cartridge C18 after the injection site, converting it into the LC instrument (Figure 1). This instrument is used to separate two similar light yellow-colored dyes, riboflavin and fluorescein, through a reverse-phase separation mechanism using an optimum 60:40 water:methanol degassed mobile phase propelled through the SPE cartridge using a syringe pump. If a 50:50 water/methanol mobile phase is used, then both dyes have shorter retention times, and a lower peak resolution with peak overlap is observed, particularly using the lower percent carbon (C) stationary phase cartridge. The class should be divided into groups, with each group trying a different mobile-phase composition. The chromatograms from each group can then be shared among the class. Experiment 2 requires one, 3-h lab, working in groups of 2 to 3 students, requiring duplicate runs for mixture separation. In the prelab assignment, students are provided background chromatography information with references and asked to predict retention orders based on the chemical structures provided (see LC student and teacher guidelines in the Supporting Information). Both absorbance and fluorescence

Figure 2. UV−vis of iron(III) salicylic acid complex (purple solution) in FIA experiment 1.

identification of the wavelength of maximum absorbance and can be matched to the best wavelength available in the MeasureNet MCA, the green LED. A calibration curve of absorbance versus concentration of salicylate can be quickly generated and used to calculate the unknown concentration of salicylic acid in one or more tablets of aspirin. The aspirin should be hydrolyzed in 0.1 M HCl (simulated stomach acid) at an elevated temperature, between 37 and 43 502




Figure 3. Author’s generated graph showing polychromatic effects on iron complex absorbance using red and green LEDs. MeasureNet specifications, maximum emission wavelength with full width at half-maximum (fwhm) of light intensity emission peak: red LED = 635 (fwhm 15 nm) and green LED = 515 (fwhm 30 nm).

intensity were measured using two different MCA systems, namely, MeasureNet and Vernier. All samples are suggested to be run at least in duplicate (Figure 4); modest reproducibility

Figure 5. Student’s separation of 2:1 mixture of riboflavin and fluorescein using the MeasureNet detector in the absorbance mode and the Prevail SPE cartridge. 60:40 water/methanol; 1 mL/min.

Figure 4. Three trial runs of the author’s separation of a 2:1 mixture of riboflavin and fluorescein using the absorbance mode in Vernier system and Maxi-Clean SPE cartridge: 60:40 water/methanol mobile phase, 1 mL/min flow rate, and 0.2 mL sample injection.

1100 times greater than that for Vernier in the absorbance spectra. Figure 6 shows a student’s chromatographic separation of 10−4 M riboflavin and 10−3 M fluorescein using fluorescence detection. A 370 nm excitation wavelength was used for MeasureNet, specified by the LED. Similarly, a 405 nm excitation wavelength was specified by the Vernier detector although the 530.5 nm emission wavelength was chosen. The chromatogram using the Vernier detector in the fluorescence mode is shown in the LC teacher guidelines. MeasureNet provided a 7.7 times greater signal-to-noise ratio in the fluorescence spectra.

was likely due to manual loadings and possible slight initial positioning differences of the sample band in the small SPE column after each injection (Table 2). Figures 4 and 5 show chromatographic separations of 10−4 M riboflavin and 10−3 M fluorescein using both MCA systems, with the same mobile phases, but with different SPE cartridges. Note that the higher C18 coverage for the Prevail SPE cartridge as compared to the Maxi-Clean cartridge proportionally increases the retention time for fluorescein (Figures 4, 5). Differences in the signal-to-noise ratio were compared for both MCA systems with the MeasureNet signal-to-noise ratio being

Table 2. Comparison of the MCA Systems for the Separation of Riboflavin and Fluorescein Absorbance Detector k′R

MCA MeasureNet Verniere

d

a,b

2.47 ± 0.53 0.78 ± 0.17

Fluorescence Detector

k′Fa,b

α

7.86 ± 1.10 3.35 ± 0.48

3.22 ± 0.33 3.29 ± 0.34

c

k′Ra,b

k′Fa,b

α′c

1.73 ± 0.22 0.94 ± 0.27

5.72 ± 1.1 3.16 ± 0.53

4.3 ± 0.60 3.56 ± 1.3

a k′R and k′F are riboflavin and fluorescein retention factors, respectively, averaging triplicate runs. Author’s results were used for all calculations. bThe retention factor was calculated using k′ = (t − tM)/tM, where t is the retention time of the dye and tM is the unretained peak, the refractive index mismatch of sample solvent and mobile phase. k′R and k′F calculations should not vary if same cartridge is used for all runs. cα is the column selectivity α = k′R/k′F. dMeasureNet absorbance and fluorescence data: Prevail SPE cartridge. eVernier absorbance and fluorescence data: MaxiClean SPE cartridge.

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may have to troubleshoot multiple variables to increase resolution or solve unforeseen problems. Some students also tried to separate other dyes, such as thymol blue and methyl orange, taking advantage of the full spectrum that the Vernier provides using the CCD and selecting the most viable LED available using MeasureNet. This study not only shows experiment versatility and compatibility with multiple MCA systems, but also extends the capabilities of an MCA system using rugged and inexpensive materials that can easily be built by the student, if desired, to create interesting experiments.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed student and teacher guidelines for the FIA and LC experiments. This material is available via the Internet at http://pubs.acs.org.

Figure 6. Students’s separation of 2:1 mixture of riboflavin and fluorescein using the MeasureNet detector in the fluorescence mode and the Prevail SPE cartridge. 60:40 water/methanol; 1 mL/min.

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HAZARDS Iron nitrate is an oxidizer and should be handled with goggles, gloves, and under a vent hood. Nitric acid is very corrosive and should also be handled with goggles, gloves, and under a vent hood. Sodium salicylate might cause irritation to the respiratory tract. Riboflavin and fluorescein may be hazardous in case of ingestion. Methanol, a flammable and volatile solvent, may be harmful if swallowed, inhaled, or absorbed through the skin. It can cause eye, skin, and respiratory tract irritation.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Science and Engineering Indicators: 2010.Chapter 2. Higher Education in Science and Engineering. Undergraduate Education, Enrollment, and Degrees in the United States. http://www.nsf.gov/ statistics/seind10/c2/c2s2.htm (accessed Feb 2013). (2) Science and Engineering Indicators: 2010.Chapter 2. Higher Education in Science and Engineering. Highlights. http://www.nsf. gov/statistics/seind10/c2/c2h.htm (accessed Feb 2013). (3) Vannatta, M. W.; Richards-Babb, M.; Solomon, S. D. J. Chem. Educ. 2010, 87 (8), 770−772. (4) Sprague, E. D.; Voorhees, R.; McKenzie, P.; Alexander, J. J.; Padolik, P. J. Chem. Educ. 1998, 75 (7), 859−859. (5) Czegan, D. A. C.; Hoover, D. K. J. Chem. Educ. 2012, 89 (3), 304−309. (6) MeasureNet: The new element for your science lab. http://www. measurenet-tech.com/ (accessed Feb 2013). (7) SpectroVis Plus Spectrophotometer. http://www.vernier.com/ products/sensors/spectrometers/svis-pl/ (accessed Feb 2013). (8) Nyasulu, F.; Nething, D.; Barlag, R.; Wise, L.; Arthasery, P. J. Chem. Educ. 2012, 89 (4), 536−539. (9) Novak, M. J., Yasmin; Bear, D. D. General Chemistry Laboratory Manual CHM 144; Hayden McNeil: Oxford, OH, 2011−2012; pp 117−126. (10) Hansen, E. H.; Ruzicka, J. J. Chem. Educ. 1979, 56 (10), 677. (11) Růzǐ čka, J.; Hansen, E. H.; Ramsing, A. U. Anal. Chim. Acta 1982, 134 (0), 55−71. (12) Carroll, M. K.; Tyson, J. F. J. Chem. Educ. 1993, 70 (8), A210. (13) Ramos, L. A.; Prieto, K. R.; Cavalheiro, É. T. G.; Cavalheiro, C. C. S. J. Chem. Educ. 2005, 82 (12), 1815. (14) Betteridge, D. Fresenius’ J. Anal. Chem. 1982, 312 (5), 441−443. (15) Rocha, F. R. P.; Nóbrega, J. A. Chem. Educator 1999, 4 (5), 179−182. (16) Bidlingmeyer, B. A.; Warren, F. V. J. Chem. Educ. 1984, 61 (8), 716. (17) O’Donnell, M. E.; Ca, D.; Musial, B. A.; Bretz, S. L.; Danielson, N. D. J. Chem. Educ. 2009, 86 (1), 60. (18) Sander, L. C. J. Chem. Educ. 1988, 65 (4), 373. (19) Smith, R.; Cantrell, K. J. Chem. Educ. 2007, 84 (6), 1021. (20) Chalasani, N.; Roman, J.; Jurado, R. L. South. Med. J. 1996, 89 (5), 479−482. (21) Mansour, F.; Shafi, M.; Danielson, N. D. Talanta 2012, 95 (0), 12−17.

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DISCUSSION The 1997 National Science Foundation (NSF) report from the Analytical Science Digital Library23 called for a thorough understanding of analytical processes in an effort to improve the analytical chemistry curriculum. NSF also called for the exposure of analytical techniques early in the undergraduate laboratory in both two- and four-year schools. Exposing firstyear classes to analytical instrumentation concepts provides a rich base of knowledge early in the analytical curriculum. This study exposes students to the underutilized concepts of liquid chromatography and flow injection analysis. Both experiments can be used sequentially or independently and have been piloted both ways with first-year nonmajors and first-year honors chemistry students. They have been well received by students who appreciated the straightforward FIA experiment 1 and a more open-ended lab LC experiment 2. Although these experiments were created for first-year students, upper-level undergraduates could also carry them out, particularly at institutions that do not provide access to research-grade instruments. The primary modification of the colorimetric salicylate experiment, from a manual standard cuvette method that has yielded good student results for many years, to one with a flow cell, is that it allows the introduction of the underutilized FIA technique during a first-year laboratory course. To perform these experiments, students must literally assemble the key parts themselves, thereby removing the “black box” view of instrumentation. In addition, students are able to compare the advantages and disadvantages of both manual standard cuvette and FIA techniques if time allows. One advantage of FIA is acquisition of data in a shorter quantity of time; therefore, students can collect multiple sample data sets. One disadvantage is the loss of sensitivity due to the shorter path length of our flow cell design. For the LC experiment, students 504




(22) Bare, W. D. J. Chem. Educ. 2000, 77 (7), 929. (23) ASDL Analytical Sciences Digital Library. http://www.asdlib. org/aboutASDL.php (accessed Feb 2013).

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Flow Injection Analysis and Liquid Chromatography for Multifunctional

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