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Exploring Liquid Sequential Injection Chromatography To Teach Fundamentals of Separation Methods: A Very Fast Analytical Chemistry Experiment C. Penteado* and Jorge Ce sar Masini Jose Instituto de Química, Universidade de S~ ao Paulo, Av. Prof. Lineu Prestes, 748, 055513-970, S~ ao Paulo, Brazil *
[email protected] Liquid sequential injection chromatography (SIC) is a novel analytical technique evolved from sequential injection analysis (SIA) to perform multianalyte determinations and chromatographic separations (1). Development of SIC methodologies and instrumentation was possible because of the availability of reversed-phase monolithic columns (2). The performance of the reversed-phase monoliths is equivalent to typical spherical particulate HPLC packed columns. The main feature of these monolithic columns is the low pressure drop, allowing for high flow rates to be imposed on the system, leading to reduced time of analysis in conventional HPLC instrumentation (3). Syringe pumps such as those used in automatic burettes are able to pump mobile phase through short monolithic columns at flow rates around 1.0 mL min-1. The present experiment was designed to illustrate key factors in the development of liquid chromatographic separations exploring the features of sequential injection instrumentation. Background The interaction of ionogenic analytes in HPLC separations is strongly dependent on the solvent strength (determined by addition of organic modifiers) and pH of the mobile phase (4, 5). Addition of organic modifiers such as methanol, acetonitrile, and tetrahydrofuran decrease the polarity of the aqueous mobile phase. This addition modifies the interaction between solute and stationary phase, influencing the retention time of the solute in the column. Increase in the concentration of organic modifier in the mobile phase results in a decrease in interaction of less polar solutes with the stationary phase, decreasing their retention times. Another important variable is the pH of the mobile phase. Hydrogen ion activity has strong influence on the solubility and ionization degree of polar solutes, especially weak organic acids. A generic weak acid in aqueous solution will be distributed between its protonated (HA) and unprotonated (A-) forms, with the proportions between them depending on the hydrogen ion activity, as given by the Henderson-Hasselbalch equation: - ½A ð1Þ pH ¼ pKa þ log ½HA If the weak acid is equally distributed between its two forms, [A-]/[HA] = 1, log([A-]/[HA]) = 0, and pH = pKa. At pH < pKa, a weak acid exists primarily in its protonated form and, the uncharged form has an increased affinity for a nonpolar
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stationary phase. On the other hand, if the weak acid exists in its unprotonated form at pH > pKa, the negatively charged species will exhibit increased affinity for a polar mobile phase. In the present experiment, we show how a sequential injection system can be used for fast investigation of the effects of pH and organic modifier on separation of two food additives, sorbic acid (λmax = 254 nm, εmax = 24120, and pKa = 4.76) and vanillin (pKa = 7.38), which are substances used to preserve flavor or improve taste and appearance, as well as to promote food safety. The use of a sequential injection system to perform chromatographic separations is only possible due to the use of monolithic stationary phases, which consist of monolithic rods of silica with bimodal pore structure of macro- and mesopores. Macropores have average size of 2 μm and allow for the rapid flow of mobile phase at low pressure. Mesopores form a fine porous structure (average pore size of 13 nm) where adsorption takes place enabling high-performance separations. The column is no longer packed with small particles, but consists of a single piece of high-purity polymeric silica (1-3). Experimental Section Instrumentation A sequential injection system configured as a liquid chromatograph is shown in Figure 1, where SP is a syringe pump with capacity of 4.0 mL. A Cheminert Valco10-port multiposition valve (MPV, model C25 stream selector C25-3180 EMH) drives sample solution and mobile phases into a holding coil (HC, made of 4 m of 0.8 mm i.d. Teflon tubing) and to the chromatographic column (CC), consisting of a 10 mm long monolithic column with 4.6 mm diameter from Merck (Darmstadt, Germany). The detection system (D) consists of a flow-through spectrophotometric cell from FIAlab Instruments (Bellevue, WA) coupled to an USB 4000 spectrometer and a DH 2000 Deuterium source. Two 600 μm diameter optical fibers (20 in. long) transmit the source radiation to the flow cell and then to the spectrometer. The frontal port (FP) of SP is connected to the central port of MPV by the holding coil (HC). The rear port (RP1) of SP is connected to the carrier reservoir (deionized water) through ports 9 and 10 of MPV (Figure 1). Another port in the pump body (RP2) is connected to a secure over-pressure valve (limit of 500 psi). Port 2 of the MPV is connected first to the column and them to the detector D. Port 5 of the MPV is connected to the
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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 2 February 2011 10.1021/ed100048r Published on Web 12/09/2010
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In the Laboratory Table 1. Composition of the Mobile Phase and the Design of the Experiments Levelb Experiment
pH
A
3.0
B
5.0
C
3.0
D
5.0
E1
ACN (%)a
pH
Organic Modifier
-
-
5
þ
-
15
-
þ
15
þ
þ
4.0
10
0
0
E2
4.0
10
0
0
E3
4.0
10
0
0
5
a
Mobile phases are prepared in 5 mmol L-1 ammonium acetate. b The þ and - represent the highest and lowest levels, respectively, and 0 represents the middle point.
Mobile Phase and Sample Preparation Figure 1. SIC manifold to perform reversed-phase liquid chromatography separation and UV-vis detection: SP = syringe pump, P = piston; RP1 = rear port 1, RP2 = rear port 2, FP = frontal port, HC = holding coil (4 m of 0.8 mm i.d. PTFE tubing), W = waste, MPV = 10 port multiposition selection valve, CC = C18 monolithic guard column, D = double wavelength detector (λ1 = 280 nm, λ2 = 254 nm), S = sample or standard solution, MP1 = mobile-phase composed of 95% (v/v) 5 mmol L-1 ammonium acetate buffer pH 3.0 or 5.0 (5% (v/v) ACN) or pH 4.0 (10% (v/v) ACN), and MP2 = 100% ACN. MPV is shown in the fill-position, in which an internal groove (semicircle) connects ports 9 and 10, allowing one to fill the solvent compartment. In any other rotor position, this groove does not connect adjacent ports, but the straight groove connects the selected port to the central one and to the holding coil.
sample reservoir (S). Ports 6 and 8 are connected to solvent reservoirs of MP1 and MP2, respectively. MP1 is a mobile phase that may be composed of 5% or 10% (v/v) in acetonitrile (ACN) at pH 3.0, 4.0, or 5.0 (see the supporting information for details); MP2 is pure ACN, which is used to create a gradient inside HC. The system used in these experiments is commercialized with the name SIChrom Accelerated Liquid Chromatography System (FIAlab Instruments) and is controlled by external software (FIAlab for Windows version 5.9.269). The elution is made using five different mobile phases, prepared with the following composition: (i) 5 mmol L-1 ammonium acetate buffer at pH 3.0, or (ii) 5.0 containing 5% (v/v) of acetonitrile and (iii) pH 4.0 containing 10% (v/v) of acetonitrile (see Table 1); two other mobile phases composed of 5 mmol L-1 ammonium acetate buffer at pH (iv) 3.0 or (v) 5.0 containing 15% (v/v) of acetonitrile are automatically prepared in line by software (see the supporting information). All solvents are HPLC-grade, purchased from J. T. Baker (Philipsburg, NJ). Buffer solutions are prepared using analyticalgrade ammonium acetate, purchased from Merck (Rio de Janeiro, RJ, Brazil). Water used in these solutions is distilled in glass distillation equipment from Marconi (Piracicaba, SP, Brazil) and is deionized to 18.2 MΩ cm resistivity using a Simplicity 185 system from Millipore (S~ao Paulo, SP, Brazil) coupled to an UV lamp. Mobile phases are filtered through 0.45 μm PTFE membrane and are degassed by ultrasound for about 10 min prior to use. 236
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A two-level (22), two-factor, full-factorial experimental design and triplicate central point (middle average point) (Table 1) is used to specify five elution programs, corresponding to combinations of pH (3.0, 4.0, 5.0) and (v/v) acetonitrile percentage (5, 10, and 15%). The order of mobile-phase programs investigated is randomized to avoid the confusion of time trends with factor effects (6). Three replicate injections are performed at the central point to evaluate the precision of the system. Hazards Appropriate safety goggles should be worn throughout the course of this experiment. Handling of organic solvents and preparation of solutions should be made inside fume-hoods. Special care is recommended for handling acetonitrile, acids, and bases during the preparation of the mobile phases. If any chemical is spilled on the body, the affected area should be washed with copious quantities of water for at least 15 min. Consulting the MSDS for complete information regarding toxicology of all chemicals is recommended. After the experiments, extensive washing with deionized water is necessary to avoid crystallization of buffer salts in instrument components such as pump, valve, or column. When not in use, the column may be kept in medium of 50% (v/v) ACN/H2O. Results and Discussion The separations obtained for the studied solutes in less than 50 s are shown in Figure 2. Optimization of separation is more efficient and precise if multivariate statistical techniques are employed (7, 8). Two-level factorial designs, such as multivariable tools, test the response values for better separation at all combinations of these levels for the investigated factors (pH and solvent strength). The range of investigated pH is the major factor governing the retention time, tR, of sorbic acid (Figure 2A,B). However, retention time of vanillin remains unchanged at around 25 s (Figure 2A,B), independent of the pH. The range of mobile-phase pH explored in this work is below the pKa of vanillin, so that its protonated form will predominate in any of the studied mobile phases. As a consequence, interactions of vanillin with either the mobile or stationary phases are not significantly affected in the experiments. On the other hand, the
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In the Laboratory
Figure 2. Sequential injection chromatograms using (A) pH 3.0 and 5% acetonitrile/95% buffer (- -); (B) pH 5.0 and 5% acetonitrile/95% buffer (þ -); (C) pH 3.0 and 15% acetonitrile/85% buffer (- þ); (D) pH 5.0 and 15% acetonitrile/85% buffer (þ þ); (E) pH 4.0 and 10% acetonitrile/90% buffer (0 0) mobile phase. The -, þ, 0 refer to the levels (see Table 1). Solid lines at 254 nm and dotted lines at 280 nm.
retention time of sorbic acid is strongly affected by the pH of mobile phases: at pH 3.0 or 4.0, sorbic acid is predominantly protonated and elutes at a retention time greater than that of vanillin (Figure 2A,E). As the pH increases to 5.0, a value that is above the pKa of sorbic acid, the predominant form of this compound changes from protonated to unprotonated, decreasing its affinity for the nonpolar stationary phase and decreasing its retention time, inverting the elution order in comparison with that observed at pH 3.0 and 4.0 (Figure 2B). The second variable, solvent strength (determined by addition of organic modifiers), showed the negative effect caused by high levels of ACN concentrations on selectivity (Figure 2C,D). At the central point, the concentration of ACN is 10% (v/v) and pH is 4.0. Under these conditions, the elution order was unaffected in relation to the experiment using 5% (v/v) ACN and pH 3.0, but changes in retention time were observed. The strength of mobile phase affects hydrophobic interactions between the solute and the stationary phase. At 15% (v/v) ACN, however, interaction of the analytes with the stationary phase is negligible, and they eluted at a retention time corresponding to the dead volume of the chromatographic column. The replicates at the central point showed negligible variations in retention time (Table 1; E1, E2, and E3), proving the robustness of this separation even using a short 10 mm long separation column, which is a guard column usually employed in conventional HPLC instrumentation (9, 10). The fast column equilibration achieved upon changing the mobile phase, the low back pressure provided by monolithic columns, and the capabilities of a sequential injection system for precise manipulation of liquids allow one to quickly explore the fundamentals of liquid chromatography separations by using the factorial experimental design to teach the effects of pH and solvent strength on the separation.
vanillin peaks is inverted as one changes the pH from the low to the high levels, but resolution is never achieved when high levels of organic modifier are used in the mobile phase. In general, as the percentage of organic modifier increases, the retention times of both analytes decrease (independent of pH). Factorial experimental design and sequential injection chromatography allow one to quickly observe the effects of pH and content of organic modifier on this separation (about 50 s per chromatographic run). The instrumental cost is significant lower than that of an HPLC instrument, especially if one is able to assemble the apparatus from its components (pump, valve, detector, and accessories). Consumption of mobile phases is significantly reduced in comparison with HPLC, attending the green chemistry demand for minimizing wastes in laboratory experiments. These features led us to suggest sequential injection chromatography as an interesting technique to discuss not only acid-base and separation fundamentals, but also some aspects of modern analytical chemistry such as experimental design, instrumentation, and green chromatography. Acknowledgment The authors express their gratitude to Fundac-~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq) for financial assistance and fellowships. Literature Cited
Summary Separation of sorbic acid and vanillin is achieved with three of the five mobile phases studied. Elution order of sorbic acid and
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1. Chocholous, P.; Solich, P.; Satinski, D. Anal. Chim. Acta 2007, 600, 129–135. 2. Satinsky, D.; Neto, I.; Solich, P.; Skalenarova, H.; Conceic-~ao, M.; Montenegro, B. S.; Araujo, A. N. J. Sep. Sci. 2004, 27, 529–536. 3. Huclova, J.; Satinski, D.; Karlicek, R. Anal. Chim. Acta 2003, 494, 133–140. 4. Snyder, R. L.; Kirkland, J. J.; Glaich, J. L., Pratical HPLC Method Development, 2nd ed; Wiley-Interscience: New York, 1997; pp 292-349. 5. Joseph, S. M.; Palosota, J. A. J. Chem. Educ. 2001, 78, 1381–1383.
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Supporting Information Available An equipment list; a summary of the lab procedure; the suggested format for the lab report. This material is available via the Internet at http://pubs.acs.org.
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