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Ion Exchange and Thin Layer Chromatographic Separation and Identification of Amino Acids in a Mixture: An Experiment for General Chemistry and Biotechnology Laboratories Linda S. Brunauer,* Katelyn E. Caslavka,† and Karinne Van Groningen Department of Chemistry and Biochemistry, Santa Clara University, Santa Clara, California 95053, United States S Supporting Information *
ABSTRACT: A multiday laboratory exercise is described that is suitable for first-year undergraduate chemistry, biochemistry, or biotechnology students. Students gain experience in performing chromatographic separations of biomolecules, in both a column and thin layer chromatography (TLC) format. Students chromatographically separate amino acids (AA) in an unknown mixture using a small column of Dowex-50 resin. The AA elution profile is determined by graphical analysis of a rapid ninhydrin microplate spectrophotometric assay that converts the colorless AA into colored ninhydrin derivatives. Column fractions corresponding to elution “peaks” are further analyzed by TLC on silica gel plates alongside AA standards. Visualization of AA migration on TLC is accomplished with a ninhydrin dipping protocol. Students use their chromatography data, structural information about the resin and the various AA, and their knowledge of intermolecular attractions and acid−base chemistry to determine the identity of the AA in their unknown mixture. KEYWORDS: First-Year Undergraduate/General, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Amino Acids, Acids/Bases, Chromatography, Thin Layer Chromatography, Ion Exchange
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INTRODUCTION Laboratory programs designed for undergraduate first-year undergraduate general chemistry courses often feature a significant introduction to analytical, physical, and inorganic chemistry. While the exercises employed are certainly important vehicles for enhancing and reinforcing concepts introduced in lecture and are essential for development of many basic laboratory skills, they typically do not completely embrace the idea of “general” chemistry. Techniques, such as chromatographic separations, commonly utilized in organic chemistry and biochemistry, are often lacking in a laboratory curriculum. A suite of experimental protocols was previously developed to introduce students to a common form of column chromatography, gel filtration.1 The protocols were designed to be appropriate for either the high school AP chemistry/biology or undergraduate general chemistry environment. The current study expands on previous work by employing chromatographic separations, including both column and thin layer formats, to analyze mixtures of amino acids (AAs), molecules of biological significance. In the 1950s, column chromatographic techniques were developed to separate all 20 standard AAs, allowing complete analysis of the AA composition of protein hydrolysates.2 However, the established protocols require instrumentation that is seldom found in college first-year/introductory laboratories and include elution at elevated temperatures for a prolonged © XXXX American Chemical Society and Division of Chemical Education, Inc.
period of time for separation. While other references describe chromatographic separations of AAs that are more appropriate for the general chemistry or biochemistry environment, they are either very limited in the specific AAs that may be included in student unknown mixtures3−8 or do not include spectrophotometric methods to monitor the elution of AAs from columns.9−13 In the current study, previous work is elaborated upon to provide chromatographic behavior information for all 20 standard AAs, greatly increasing an instructor’s choices in preparing unknown mixtures for subsequent student analysis. Students are able to separate mixtures of 3−5 AAs fully or partially using columns of sulfonated polystyrene resin (Dowex-50). The columns are developed using simple gravity-fed delivery of buffers over the course of 2 h at ambient temperature and the elution profile subsequently determined by use of a spectrophotometric microplate assay. The identity of the AAs in each column absorbance “peak” is determined by thin layer chromatography (TLC). The analytical protocols yield colored ninhydrin derivatives that may be detected visually. As a result, an exploration of the relationship between AA side chain (“R-group”) structure and chromatographic behavior may be accomplished in student laboratories equipped with standard instrumentation over the course of three 3 h laboratory periods.
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Table 1. Dowex-50 Column Elution Position and TLC Rf Values for the 20 Standard Amino Acids
EXPERIMENTAL SUMMARY Students worked in groups of 2−4. A detailed description of the experiment is in the Supporting Information. Materials
Dowex-50 columns were prepared and equilibrated in 70 mM sodium citrate, pH 2.2 (buffer A). Individual AA stock solutions were prepared by dissolving samples of the AAs in buffer A to a final concentration of 1−10 mg/mL. Student unknown mixtures were prepared by combining equal molar amounts of 3−5 different AAs. Chromatographic Separation of AA “Unknown” Mixtures
AA mixtures were applied to Dowex-50 columns, and the columns were developed using a gravity-fed 5-step gradient14 with buffers of increasing pH (2.2, 3.5, 4.1, 6.5, and 10) and varying sodium ion concentration. Forty-five fractions of approximately 0.75 mL were collected over the course of 2 h. Ninhydrin Analysis of Column Fractions
The AA elution profile was determined using a microscale ninhydrin analysis. Ten microliter aliquots of each column fraction were transferred into the wells of a 96-well microplate preloaded with 165 μL of buffer A. The production of colored reaction products was initiated by addition of 50 μL of ninhydrin reagent. The plate was covered and heated at 60 °C for 15−30 min. Once cooled to room temperature, the AA elution profile was analyzed visually or by scanning in a microplate reader at 570 and 365 nm.
Amino Acid
Abbreviation
pH of Step Gradient Elution Buffera
Aspartic acid Threonine Serine Asparagine Glutamine Glutamic Acid Glycine Cysteine Proline Alanine Valine Isoleucine Leucine Methionine Phenylalanine Tyrosine Lysine Histidine Tryptophan Arginine
D T S N Q E G C P A V I L M F Y K H W R
4.1 4.1 4.1 4.1 4.1 4.1 4.1 4.1 4.1 4.1 6.5 6.5 6.5 6.5 ≥10 ≥10 ≥10 ≥10 ≥10 ≥10
Elution Peak Fractionsa
Rf on TLCa
18−22 18−21 20−23 20−24 21−25 22−25 22−24 22−25 22−26 23−25 24−28 25−31 27−34 27−33 38−42 39−44 40−43 41−43 42−45 42−45
0.50 ± 0.02 0.58 ± 0.01 0.58 ± 0.02 0.49 ± 0.01 0.50 ± 0.01 0.54 ± 0.03 0.52 ± 0.02 0.65 ± 0.01 0.42 ± 0.01 0.55 ± 0.02 0.63 ± 0.01 0.73 ± 0.02 0.75 ± 0.02 0.68 ± 0.02 0.76 ± 0.03 0.81 ± 0.02 0.25 ± 0.04 0.29 ± 0.03 0.81 ± 0.03 0.30 ± 0.05
a
Elution positions and TLC Rf values and standard deviations were determined using data from 3 class sets (approximately 48 students working in groups of 2−4).
TLC Analysis of AA Standards, “Unknown” Mixtures, and Selected Column Absorbance “Peak” Fractions
Students were given a list of 8 possible AAs that could be found in their unknown mixture. Each mixture was composed of AAs whose side chains or “R-groups” varied greatly in polarity and charge, thus ensuring significant spacing of their elution positions. A representative group of 8 AAs (aspartic acid (D), threonine (T), proline (P), valine (V), methionine (M), phenylalanine (F), histidine (H), and lysine (K)) was used for the figures presented in the current study. The Dowex-50 elution profiles for two different AA mixtures are shown in Figure 1. The relative elution order reflects the importance of electrostatic interactions associated with binding to the resin sulfonic acid groups. The AAs whose R-groups are expected to bear full or partial negative charges (D, T) at low pH elute first from the cation-exchange resin. AAs whose R-groups bear a positive charge at low pH (H, K) are expected to bind tightly to the resin and thus exit the column only at high pH/high ionic strength. The aromatic AA (F) also elutes at high pH/high ionic strength, presumably due to hydrophobic interactions with the resin’s polystyrene backbone. AAs with small hydrophobic R-groups (M, V, P) elute at positions intermediate to the other groups. With the exception of aromatic AAs, the AAs tend to elute in order of increasing isoelectric point. Although phenylalanine coelutes from the column with lysine or histidine, these resolve well by TLC since lysine and histidine have very low Rf values under these running conditions due to the greater polarity of their R-groups (Figure 2; lower right panel). In addition to the sample group of 8 AA mentioned above, many other combinations are possible, using the elution position and TLC Rf data in Table 1 to guide the selection. The eight AAs in these studies exhibit different mobilities in the TLC system (Figure 2; lower left panel), allowing students to identify the AAs in their “unknown” mixtures by comparison of relative migration of the components in the mixture to those of standards. Dowex-50 column fractions exhibiting high
Aliquots of AA standards, student “unknown” AA mixtures, and selected column fractions corresponding to absorbance peaks detected in the microplate analysis were applied to 10 cm × 10 cm silica gel TLC plates. The plates were then developed with n-propanol/water (70:30 v/v) for 90 min and air-dried. AA migration was visualized by dipping plates into a ninhydrin “dip” reagent followed by gentle heating. The Rf values for the colored ninhydrin−AA products were determined immediately.
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HAZARDS Information on safety and hazards are included in the detailed protocol in the Supporting Information. All work should be done wearing appropriate personal protective equipment (e.g., chemical splash goggles, appropriate gloves, and lab coat). Instructors should familiarize themselves with the MSDS information readily available for each compound prior to the start of the experiment. Ninhydrin microplate analysis, TLC development and ninhydrin dip analysis should be carried out in a fume hood to avoid exposure to volatile solvents. 1-Butanol and 1-propanol are flammable liquids; take extreme care in using a heat gun or blow dryer on TLC plates that have traces of the flammable ninhydrin dip reagent. Glacial acetic acid is a flammable and corrosive liquid. Dimethyl sulfoxide is a combustible liquid that is an irritant. Dowex-50, aspartic acid, and ninhydrin are irritants; contact with ninhydrin will discolor the skin.
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RESULTS AND DISCUSSION AA “unknown” mixtures were carefully selected to feature AAs expected to elute from Dowex-50 columns at different positions in the elution schedule and/or ones that exhibit significant differences in behavior on TLC. Table 1 shows the relative Dowex-50 column elution positions and TLC analysis Rf values for all 20 standard AAs. B
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Figure 1. Ninhydrin microplate analysis of column fractions for 2 representative mixtures of AAs. Mixtures were separated by Dowex-50 chromatography, and relative elution positions were determined by ninhydrin analysis. Absorbance was measured at 570 nm (filled circles); samples containing proline, whose ninhydrin derivative has a low extinction coefficient at 570 nm but a high value at 365 nm, were also measured at 365 nm (open circles). The AA mixture used for each column and the elution position for the component AAs are noted using single letter abbreviations.
Table 2. Student Achievement of Learning Goals and Objectives Measured by Formal Written Examination, Laboratory Reports and Classroom Observation Learning Goals
Learning Objective
Goal 1 Goal 2 Goal 3 Goal 4 Goal 5 Learning Goal Tested
Acquire an understanding of the general features of an amino acid and understand how intermolecular forces influence the behavior of molecules Appreciate the importance of solvent pH in influencing the charge state of ionizable groups Become familiar with collection and analysis of spectrophotometric data Appreciate the importance of deductive reasoning in using a variety of pieces of analytical data to analyze the composition of an “unknown” Develop the ability to carry out microscale analytical procedures Assessment Student Success Rate Instrument Learning Objective Assessed (SD)a
Goal 1
Written exam
Be able to correctly predict the relative elution order for a set of 5 AAs and explain the behavior in terms of intermolecular forces when given information on the structure and isoelectic point of each amino acid as well as the structure of Dowex-50 resin Be able to correctly define the term “Rf ” as applied to TLC and consider R group structure to explain the migration order of 5 amino acids on TLC Be able to make logical suggestions on how to alter the protocol to try to separate coeluting aromatic and basic amino acids Be able to explain why we use buffers of decreasing pH with anion exchange resins (compared to cation exchange resins using buffers of increasing pH) Be able to explain the reason for measuring ninhydrin-AA absorbance at two different wavelengths Be able to use graphing software to generate high-quality graphs of absorbance vs elution position data
85% (1.6)
Goal 1
Written exam
Goal 2
Written exam
Goals 1 and 2
Written exam
Goal 3 Goal 3 Goal 4
Written exam Laboratory report and classroom observation Laboratory report
Be able to correctly determine the composition of an unknown mixture of 3 amino acids based on Dowex-50 elution and TLC data
77% (no errors), 16% (1 error), 7% (multiple errors)c 59% (no errors), 28% (1 error), 13% (multiple errors)c b 100%
Goal 4
Laboratory report
Be able to correctly determine the composition of an unknown mixture of 4−5 amino acids based on Dowex-50 elution and TLC data
Goal 5
Laboratory report and classroom observation
Be able to carry out microplate spectroscopic analyses with good precision and exhibit an understanding of the use of micropipettes
97% (0.86) 96% (1.1) 92% (1.7) 89% (1.7) 100%b
a
Except where noted, data reflect aggregate scores for a total of 48 lower and upper-division students. bStudents were allowed to consult with the instructor on construction of graphs and use of micropipettes and all showed competence in these areas. In the few instances where students made significant errors in microplate assays and requested to repeat the assays, the new analyses were carried out correctly. cData reflect a representative general chemistry class set. C
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Figure 2. TLC analysis of AA standards, representative mixtures, and selected column fraction elution “peaks”. Samples were separated by TLC and stained by reaction with ninhydrin. (Upper panel) TLC analysis of all 20 standard AAs. (Lower left panel) TLC analysis of 8 AA standards and 5 representative “unknown” mixtures: 1 (TMH), 2 (TVH), 3 (TPK), 4 (TPMFK), and 5 (DPFK). (Lower right panel) TLC analysis of 2 AA mixtures and Dowex-50 column elution “peaks”. Fractions eluting in pH 6.5 or higher solvent were acidified prior to TLC. F and K coelute in the last elution step, resulting in two “spots” in the TLC for these fractions.
of the eight AAs employed in the experiment. Discussions about the structures of these R-groups enhanced and reinforced lecture discussions on acid/base chemistry, buffers, titration of ionizable groups, intermolecular attractions, and the concept of an organic functional group. Analysis of student answers to experiment-related questions on a formal written examination (Table 2) indicates that students exhibited a high level of success in achieving learning goals. Student narrative surveys indicated that students enjoyed the deductive reasoning aspects of the experiment and felt that the experiment helped them to achieve a variety of learning goals, such as gaining experience in column and thin layer chromatography, microscale manipulations, spectrophotometric assays, and graphical analysis of data.
absorbance (“peaks”) from each of the separations were also subjected to TLC analysis (Figure 2; lower right panel) and provided students with further information to aid in their determination of the composition of their “unknown” mixtures. Several of the ninhydrin derivatives exhibit unique colors that provide an additional method of confirmation of the identity of the components in the mixture. For example, while most of the derivatives are purple in color, the proline derivative is yellow and histidine yields a brownish derivative on TLC. TLC analysis of all 20 standard AAs is shown for reference (Figure 2; upper panel). The experimental protocols have been incorporated into honors sections of general chemistry (16−20 students/term) for the past five academic years. In addition, for the past two academic years, we have featured the experiment in an upperdivision biochemical techniques course (14−15 students/term). The entire suite of protocols required 8−9 h of laboratory time to complete. Working in small groups, students identified the AAs in their unknown mixtures with a high success rate (Table 2). In most cases, errors involved incorrect assessment of AAs with very similar chromatographic behavior (e.g., confusing M with V). Students came to their conclusions based on a combination of visualization of the color of ninhydrin−AA derivatives, relative elution position from Dowex-50 columns, TLC migration patterns, and comparison of the structural features of the R-groups
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ASSOCIATED CONTENT
S Supporting Information *
Student handouts and instructor notes including detailed information on reagent and equipment sources and preparations, CAS numbers, color photographs of ninhydrin-stained TLC plates and ninhydrin microplate analysis of column fractions, extinction coefficients for ninhydrin−AA derivatives, and information regarding hazards. This material is available via the Internet at http://pubs.acs.org. D
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address †
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095 United States. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Mary Helen Mack, Linh Lam and Naushaba Khan for testing the protocol and our colleagues for helpful comments on this manuscript.
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REFERENCES
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