In the Laboratory
Investigating the Retention Mechanisms of Liquid Chromatography Using Solid-Phase Extraction Cartridges Mary E. O’Donnell, Beata A. Musial, Stacey Lowery Bretz, and Neil D. Danielson* Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056; *
[email protected] Diep Ca Department of Chemistry, Shenandoah University, Winchester, VA 22601
Liquid chromatography (LC), gas chromatography, and capillary electrophoresis are generally considered the most important instrumental separation techniques. While LC separations are based on four retention mechanisms (normal-phase, reverse-phase, ion-exchange, and size-exclusion), experiments for undergraduate laboratory courses often focus on a single retention mechanism, usually reverse-phase. Furthermore, the rationale for the observed retention order is not always apparent. The experiments described below have been used in both firstyear BS chemistry major laboratory courses and BA chemistry– biological science major laboratory courses over the past seven years. Collectively, the current experiments have demonstrated an effective, yet inexpensive way for students to investigate LC retention mechanisms. The experiments demonstrating the four retention mechanisms can be completed in a standard 3–4 hour laboratory period by students working in pairs. These experiments do not focus solely on instrumentation or techniques, nor on just the application of chromatography. Rather, students answer questions during the experiment for each type of retention mechanism to demonstrate that they understand why certain mobile-phase solvents are effective and to explain analyte retention order based on structure. Through this experiment, students are also introduced to the concept of polarity as it applies to both solvent and solute molecules in both normaland reverse-phase separations. The structures of all analytes, stationary phases, and mobile phases are provided; students are expected to use this information to rationalize the retention orders observed. Experimental results and answered questions are handed in as the lab report at the end of the period. There is a post-lab assignment, due the following week, for students to practice predicting retention order based on analyte structure and to diagram the components of a high-performance liquid chromatography (HPLC) instrument. Background Laboratory experiments that demonstrate reverse-phase HPLC usually focus on the separation of real samples, such as analgesics (1–4), additives in beverages (5, 6), chlorophyll pigments (7), and other complex mixtures. For example, glass columns used with a HPLC instrument permitted the visual separation of dyes by reverse phase (8). In the aforementioned experiments, however, the rationale for the separation order is often difficult for students to ascertain. Ion chromatography teaching experiments focusing on the separation of phosphate (9), chloride (10, 11), sulfate (12), and sugars (13) are also well-represented.
60
Several reports of self-prepared columns have been described in this Journal (14–16). These experiments describe the normal-phase separation of dyes, such as fluorescein and methylene blue (16), using silica or alumina packed in Pasteur pipet columns. These experiments demonstrate only normaland reverse-phase separation mechanisms. McKay (17) has described the use of a commercially available Sephadex-packed column for the size-exclusion separation of blue dextran and phenol red. Solid-phase extraction (SPE) cartridges provide an alternative method for investigating HPLC separations. Bidlingmeyer (18) introduced SPE cartridges in 1984 as an inexpensive and safe means of demonstrating the fundamentals of HPLC. The HPLC components are mimicked by the SPE cartridge (column), plastic syringes (injector and pump), 10 mL test tubes to collect fractions, and the student’s eye (detector). Bidlingmeyer described five separations of the red and blue dyes in grape KoolAid involving both normal-phase and reverse-phase mechanisms, along with the use of both isocratic and step-gradient mobile phases. However, the dyes are quite similar in structure with respect to polar and non-polar groups, making prediction of retention order difficult. Ion-exchange and size-exclusion separation mechanisms were not described. Brenneman and Ebeler (19) demonstrated the separation of classes of wine phenols using a reverse-phase SPE column and mobile phases of different polarity. Here, we have shown that SPE cartridges can be used to investigate all four major retention mechanisms: normal-phase, reverse-phase, size-exclusion, and ion-exchange. Experimental Procedure and Typical Student Results Part 1: Polarity Students are introduced to the concept of polarity for the analytes and for both the stationary and mobile phases of interest. The structures of the organic analytes (Figure 1) and stationary and mobile phases (Figure 2) are available to the students in their lab manual. Students are also provided the degree of polarity (P′) of each mobile phase. The students work with mobile phases that range from highly non-polar, hexane (P′ = 0.1), to highly polar, water (P′ = 10.1), with methanol (P′ = 5.1) and isopropanol (P′ = 3.9) in between. For mobilephase mixtures, students calculate the degree of polarity based on the equation
P AB b f A P Ab f B P bB
(1)
where f is the volume fraction of either solvent A or B. Students are asked to predict retention orders based on polarity and structure.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Part 2: Reverse-Phase, Normal-Phase, and SizeExclusion Liquid Chromatography Separations Silica and C18 SPE cartridges, compatible for attachment of luer lock plastic syringes, can be purchased from Alltech Associates. To increase porosity and mobile-phase permeability for size-exlusion separation, three silica SPE cartridges were treated with 1 M NaOH for 2 days. β-Carotene, thymol blue, alizarin red, and blue dextran were purchased from Sigma-Aldrich. An alternative source for β-carotene can be pharmaceutical capsules. Other analytes and solvents are commonly available. Organic dye analyte solutions were prepared as 2% (w/v) in methanol or isopropanol. A 5% blue dextran solution was prepared in water. Cartridges are equilibrated (pushing mobile phase through cartridge) with 10 mL of the mobile phase prior to loading the analyte mixture. For the reverse-phase separation, the C18 cartridge is equilibrated with isopropanol before it is equilibrated with the water–methanol mobile phase to reduce the collapse of the hydrophobic C18 chains. Students pump the mobile phase through the cartridges at a maximum flow rate of one drop per second using a 10 mL syringe. Students then load 5–6 drops (about 0.3 mL) of a 1:1 mixture of the analytes onto the cartridges with 1 mL syringes. (Students should take care not to overload the column because excess analyte will not be retained on the column and it will elute with the front of the mobile phase.) All fractions are collected in 10 mL test tubes. Students determine how much eluent to collect in each fraction and are told to start collecting a new fraction if the color of the eluent changes. They should be encouraged to monitor color changes along the cartridge. Students will see the colored analytes start to separate from a single band to a gradient of color along the column as the separation occurs. All fractions collected in the reverse-phase and normal-phase separations are developed with NaOH. Thymol blue turns blue when developed post-column with NaOH. Alizarin red also changes color to purple in the presence of NaOH because of the impurity of alizarin in the dye. In the size-exclusion separations, three SPE columns, previously treated with 1 M NaOH were connected together. The salicylate is developed post-column with Fe(NO3)3. Iron(III) will complex with salicylate, changing the color of the solution to purple. Most students are able to see at least partial resolution of the two analytes. A slow flow rate is critical for a successful separation. A summary of the experimental conditions for each retention mechanism is given in Table 1. Part 3: Ion-Exchange Liquid Chromatography The ion-exchange experiments demonstrate the ability of a hydrophobically coated charged polymer to retain and exchange ions. The two ion-exchange experiments are designed to illustrate proof-of-concept and not a separation. Students observe no interactions between ions and the C18 stationary phase; however, they will observe an interaction with the C18 stationary phase after it is modified with an ion-exchange polymer. Due to time constraints, each student pair completes either the anion- or cation-exchange procedure. Students who complete the anion-exchange experiment share data with a group that completes the cation-exchange experiments.
C3H7
C3H7
HO
O
O
OH OH
CH3 SO3ź
CH3
SO3ź O
thymol blue
alizarin red
ß-carotene O
NH2 SO3ź
O OH
O
OH
HN
salicylic acid O2S H HO
CH2
OH O
OH H H H OH CH2
H
OH
O
O
OH H H H OH CH2
H
OH
HO
O OH H H H OH CH2
n
blue dextran Figure 1. Structures of organic analytes.
H2 H C C
Ná H3C
CH3
SO3ź
n
silica particle
Si
CH3
silica particle
OH
n
poly(styrenesulfonate) (PSS)
poly(diallyldimethyl) ammonium chloride (PDDAC)
Si
O
Si
(CH2)17 CH3
CH3
reverse-phase octadecyl (C18) modified silica
normal-phase silica
H3C OH
H3C
hexane
CH3
H3C
OH
methanol
H3C
isopropanol
Figure 2. Structures of stationary and mobile phases.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
61
In the Laboratory Table 1. Experimental Conditions for Liquid Chromatography Retention Mechanisms Retention Mechanism
Stationary Phase
Mobile Phase
Analytes
Reverse-Phase
Non-polar octadecyl hydrocarbon (C18) modified silica
Polar (step gradient) 1. Water–alcohol 2. Isopropanol
Thymol blue and alizarin red
Normal-Phase
Polar silica
Non-polar (step gradient) 1. Hexane–isopropanol 2. Isopropanol–methanol 3. Methanol
Thymol blue and ß-carotene
Cation-Exchange
C18 column with adsorbed polymer, e.g., PSS (–SO3–)
Water
Ag+ or Cu2+
Anion-Exchange
C18 column with adsorbed polymer, e.g., PDDAC [–N(CH3)3+]
Water
Cl– or I–
Size-Exclusion
Polar silica
Water
Blue dextran and salicylate
Table 2. Typical Student Results Retention Mode
Chromatographic Result
Separation Mechanism
Reverse-Phase
Alizarin red elutes before thymol blue
Stationary phase retains more non-polar hydrophobicor hydrocarbon-like analytes (thymol blue).
Normal-Phase
ß-carotene elutes before thymol blue
Stationary phase retains organic compounds with polar functional groups (–OH, –NH2, –COOH).
Ion-Exchange
Ag+, Cu2+, Cl–, or I– retained by polymer-coated C18 phase
Polymer-modified stationary phase retains ions of opposite charge based on ion size and charge magnitude.
Size-Exclusion
Blue dextran elutes before salicylate
Pores in stationary phase retain smaller molecules (salicylate).
Students first equilibrate a C18 column with water, load the ion of interest (Table 1), and observe the lack of retention of the ion using water as a mobile phase. The ions are detected post-column through a precipitation reaction (Ag+, I–, and Cl–) or a complexation reaction (Cu2+). The silver ion is precipitated as silver iodide. The chloride and iodide ions are precipitated as silver chloride and silver iodide, respectively. The copper(II) ion is complexed post-column with ammonia to form the light blue Cu(NH3)42+ species. A C18 column does not retain any ions, and the analyte will be present in the first few fractions. Students then modify the C18 column with a 20% solution of either poly(diallyldimethyl)ammonium chloride (PDDAC) or poly(styrenesulfonate) (PSS) polymers for anion or cation exchange, respectively. A column pre-wash with either cetyltrimethyl bromide for anion exchange or sodium dodecyl sulfate for cation exchange is recommended to increase sample retention. Students again load the ions onto the column and observe the retention using water as the mobile phase. (Students should take care not to overload the column for the same reasons given previously.) The ions are retained on the column coated with the ion-exchange polymer, although excess ions may be observed in the first collected fraction. Lastly, students will use an aqueous solution of MgSO4 as the mobile phase to illustrate the influence of ionic strength and speed elution of the analyte ion. For the cation exchange, students will observe that both the silver ion and the copper(II) ion are exchanged on the column by the magnesium ion. In contrast, students will observe that the chloride ion is exchanged by the sulfate, but the iodide ion 62
is not exchanged. Students should explain these observations based on the relative sizes of the ions and the magnitudes of their charges. The polymer resin is removed from the C18 cartridge using methanol. Students may observe a precipitate in fractions collected from the methanol wash if there are still ions retained by the polymer. Hazards Standard laboratory safety precautions should be followed, including the use of goggles and gloves. Methanol, isopropanol, and hexane are flammable solvents and harmful if inhaled, ingested, or absorbed through the skin. Aqueous NaOH is corrosive. Thymol blue, alizarin red, β-carotene, blue dextran, salicylic acid, potassium iodide, copper(II) nitrate, iron(III) nitrate, silver nitrate, and ammonia are eye and skin irritants; avoid contact and inhalation. Silver nitrate will discolor skin. Concentrated ammonia solution is poisonous and corrosive. Magnesium sulfate may be harmful if swallowed. Avoid contact and inhalation for poly(diallyldimethylammonium)chloride and sodium poly(styrenesulfonate). Typical Student Results A summary of typical student results is found in Table 2. Students are able to directly observe the elution of the colored analytes in the reverse-phase, normal-phase, and size-exclusion separations. In post-lab questions, students do particularly well
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
in predicting retention order for a new set of analytes. About 75% of the students are able to correctly predict the elution order of three analytes such as benzoate, toluene, and phthalate based on structure and polarity for normal and reverse phase. In the ion-exchange separations, virtually all the students can accurately predict the effect of charge on the elution order of inorganic ions in post-lab questions but size is more difficult. For size exclusion, accurate prediction of the elution order of a set of proteins based on molecular weight was achieved by virtually all the students. Discussion This experiment allows students to investigate the retention mechanisms involved in LC using two types of inexpensive SPE cartridges. Through the normal-phase and reverse-phase separations, students are introduced to the concept of analyte, stationary-phase, and mobile-phase polarities as important variables in LC. Students are also able to explore size-exclusion separations using the silica SPE cartridges. Coating the C18 SPE cartridges with an ion-exchange polymer allows the students to investigate the mechanism of ion-exchange separations. If time allows, many of these experiments could be modified or extended to give the students more freedom in the investigation of the retention mechanisms. For example, isocratic mobile-phase conditions are sometimes not optimal for reverse- or normalphase chromatography, and students could be asked to develop a step-gradient set of mobile phases for either or both types of separations. Students could also further investigate the role of ionic strength in ion-exchange separations. Literature Cited 1. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447. 2. Ferguson, G. K. J. Chem. Educ. 1998, 75, 467–469. 3. Ferguson, G. K. J. Chem. Educ. 1998, 75, 1615–1618.
4. Mueller, B. L.; Potts, L. W. J. Chem. Educ. 1988, 65, 905–906. 5. Bidlingmeyer, B. A.; Schmitz, S. J. Chem. Educ. 1991, 68, A195–A200. 6. Haddad, P.; Hutchins, S.; Tuffy, M. J. Chem. Educ. 1983, 60, 166–168. 7. Schaber, P. M. J. Chem. Educ. 1985, 62, 1110–1113. 8. McBride, P. Revitalizing Chemistry Laboratory Instruction. Ph.D. Thesis, Miami University, Oxford, OH, 2003. 9. Bello, M. A.; Gonzalez, A. G. J. Chem. Educ. 1996, 73, 1174–1176. 10. Kieber, R. J.; Jones, S. B. J. Chem. Educ. 1994, 71, A218–A222. 11. Drossman, H. J. Chem. Educ. 2007, 84, 124–127. 12. Koubek, E.; Stewart, A. E. J. Chem. Educ. 1992, 69, A146–A148. 13. Luo, P. F.; Luo, M. Z.; Baldwin, R. P. J. Chem. Educ. 1993, 70, 679–681. 14. Sander, L. C. J. Chem. Educ. 1988, 65, 373–374. 15. Svoronos, P.; Sarlo, E. J. Chem. Educ. 1993, 70, A158–A159. 16. Ruekberg, B. J. Chem. Educ. 2006, 83, 1200. 17. McKay, P. An Introduction to Chromatography. http://www.accessexcellence.org/LC/SS/chromatography_background.html (accessed Oct 2008). 18. Bidlingmeyer, B. A.; Warren, F. V. J. Chem. Educ. 1984, 61, 716–720. 19. Brenneman, C. A.; Ebeler, S. E. J. Chem. Educ. 1999, 76, 1710–1711.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Jan/abs60.html Abstract and keywords Full text (PDF) with links to cited URL and JCE articles Supplement Student lab manual Solutions to lab questions and after-lab assignment Notes for the instructor
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
63
