Ion Exchange and Thin Layer Chromatographic Separation andIdentification of Amino Acids in a Mixture: An Experiment forGeneral Chemistry and Biotechnology LaboratoriesLinda 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 forfirst-year undergraduate chemistry, biochemistry, or biotechnology students.Students gain experience in performing chromatographic separations of bio-molecules, in both a column and thin layer chromatography (TLC) format.Students chromatographically separate amino acids (AA) in an unknown mix-ture using a small column of Dowex-50 resin. The AA elution profile is deter-mined by graphical analysis of a rapid ninhydrin microplate spectrophoto-metric assay that converts the colorless AA into colored ninhydrin derivatives.Column fractions corresponding to elution “peaks” are further analyzed byTLC on silica gel plates alongside AA standards. Visualization of AA migrationon TLC is accomplished with a ninhydrin dipping protocol. Students use theirchromatography data, structural information about the resin and the variousAA, and their knowledge of intermolecular attractions and acid−base chem-istry 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

■ INTRODUCTION

Laboratory programs designed for undergraduate first-yearundergraduate general chemistry courses often feature a sig-nificant introduction to analytical, physical, and inorganicchemistry. While the exercises employed are certainlyimportant vehicles for enhancing and reinforcing conceptsintroduced in lecture and are essential for development ofmany basic laboratory skills, they typically do not completelyembrace the idea of “general” chemistry. Techniques, such aschromatographic separations, commonly utilized in organicchemistry and biochemistry, are often lacking in a laboratorycurriculum.A suite of experimental protocols was previously developed to

introduce students to a common form of column chromatog-raphy, gel filtration.1 The protocols were designed to beappropriate for either the high school AP chemistry/biology orundergraduate general chemistry environment. The currentstudy expands on previous work by employing chromatographicseparations, including both column and thin layer formats, toanalyze mixtures of amino acids (AAs), molecules of biologicalsignificance.In the 1950s, column chromatographic techniques were

developed to separate all 20 standard AAs, allowing completeanalysis of the AA composition of protein hydrolysates.2 How-ever, the established protocols require instrumentation thatis seldom found in college first-year/introductory laboratoriesand include elution at elevated temperatures for a prolonged

period of time for separation. While other references describechromatographic separations of AAs that are more appro-priate for the general chemistry or biochemistry environment,they are either very limited in the specific AAs that may beincluded in student unknown mixtures3−8 or do not includespectrophotometric methods to monitor the elution of AAsfrom columns.9−13

In the current study, previous work is elaborated upon toprovide chromatographic behavior information for all 20standard AAs, greatly increasing an instructor’s choices inpreparing unknown mixtures for subsequent student analysis.Students are able to separate mixtures of 3−5 AAs fully orpartially using columns of sulfonated polystyrene resin(Dowex-50). The columns are developed using simplegravity-fed delivery of buffers over the course of 2 h atambient temperature and the elution profile subsequentlydetermined by use of a spectrophotometric microplate assay.The identity of the AAs in each column absorbance “peak” isdetermined by thin layer chromatography (TLC). Theanalytical protocols yield colored ninhydrin derivatives thatmay be detected visually. As a result, an exploration of therelationship between AA side chain (“R-group”) structure andchromatographic behavior may be accomplished in studentlaboratories equipped with standard instrumentation over thecourse of three 3 h laboratory periods.

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■ EXPERIMENTAL SUMMARYStudents worked in groups of 2−4. A detailed description of theexperiment is in the Supporting Information.Materials

Dowex-50 columns were prepared and equilibrated in 70 mMsodium citrate, pH 2.2 (buffer A). Individual AA stock solutionswere prepared by dissolving samples of the AAs in buffer A to afinal concentration of 1−10 mg/mL. Student unknown mixtureswere prepared by combining equal molar amounts of 3−5different AAs.Chromatographic Separation of AA “Unknown” Mixtures

AA mixtures were applied to Dowex-50 columns, and thecolumns were developed using a gravity-fed 5-step gradient14

with buffers of increasing pH (2.2, 3.5, 4.1, 6.5, and 10) andvarying sodium ion concentration. Forty-five fractions ofapproximately 0.75 mL were collected over the course of 2 h.Ninhydrin Analysis of Column Fractions

The AA elution profile was determined using a microscaleninhydrin analysis. Ten microliter aliquots of each column fractionwere transferred into the wells of a 96-well microplate preloadedwith 165 μL of buffer A. The production of colored reactionproducts was initiated by addition of 50 μL of ninhydrin reagent.The plate was covered and heated at 60 °C for 15−30 min. Oncecooled to room temperature, the AA elution profile was analyzedvisually or by scanning in a microplate reader at 570 and 365 nm.TLC Analysis of AA Standards, “Unknown” Mixtures, andSelected Column Absorbance “Peak” Fractions

Aliquots of AA standards, student “unknown” AA mixtures, andselected column fractions corresponding to absorbance peaksdetected in the microplate analysis were applied to 10 cm ×10 cm silica gel TLC plates. The plates were then developedwith n-propanol/water (70:30 v/v) for 90 min and air-dried. AAmigration was visualized by dipping plates into a ninhydrin “dip”reagent followed by gentle heating. The Rf values for the coloredninhydrin−AA products were determined immediately.

■ HAZARDSInformation on safety and hazards are included in the detailedprotocol in the Supporting Information. All work should be donewearing appropriate personal protective equipment (e.g.,chemical splash goggles, appropriate gloves, and lab coat).Instructors should familiarize themselves with the MSDSinformation readily available for each compound prior to thestart of the experiment. Ninhydrin microplate analysis, TLCdevelopment and ninhydrin dip analysis should be carried out ina fume hood to avoid exposure to volatile solvents. 1-Butanol and1-propanol are flammable liquids; take extreme care in using aheat gun or blow dryer on TLC plates that have traces of theflammable ninhydrin dip reagent. Glacial acetic acid is a flammableand corrosive liquid. Dimethyl sulfoxide is a combustible liquidthat is an irritant. Dowex-50, aspartic acid, and ninhydrin areirritants; contact with ninhydrin will discolor the skin.

■ RESULTS AND DISCUSSIONAA “unknown” mixtures were carefully selected to feature AAsexpected to elute from Dowex-50 columns at different positionsin the elution schedule and/or ones that exhibit significantdifferences in behavior on TLC. Table 1 shows the relativeDowex-50 column elution positions and TLC analysis Rf valuesfor all 20 standard AAs.

Students were given a list of 8 possible AAs that could be foundin their unknown mixture. Each mixture was composed ofAAs whose side chains or “R-groups” varied greatly in polarityand charge, thus ensuring significant spacing of their elutionpositions. 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 forthe figures presented in the current study. The Dowex-50 elutionprofiles for two different AA mixtures are shown in Figure 1.The relative elution order reflects the importance of elec-

trostatic interactions associated with binding to the resin sulfonicacid groups. The AAs whose R-groups are expected to bear fullor partial negative charges (D, T) at low pH elute first fromthe cation-exchange resin. AAs whose R-groups bear a positivecharge at low pH (H, K) are expected to bind tightly to the resinand 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’spolystyrene 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 inorder of increasing isoelectric point. Although phenylalaninecoelutes from the column with lysine or histidine, these resolvewell by TLC since lysine and histidine have very low Rf valuesunder these running conditions due to the greater polarity oftheir R-groups (Figure 2; lower right panel). In addition to thesample group of 8 AA mentioned above, many othercombinations are possible, using the elution position and TLCRf 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 studentsto identify the AAs in their “unknown” mixtures by comparisonof relative migration of the components in the mixture to thoseof standards. Dowex-50 column fractions exhibiting high

Table 1. Dowex-50 Column Elution Position and TLC RfValues for the 20 Standard Amino Acids

Amino Acid Abbreviation

pH of StepGradient

Elution Buffera

ElutionPeak

Fractionsa Rf on TLCa

Aspartic acid D 4.1 18−22 0.50 ± 0.02Threonine T 4.1 18−21 0.58 ± 0.01Serine S 4.1 20−23 0.58 ± 0.02Asparagine N 4.1 20−24 0.49 ± 0.01Glutamine Q 4.1 21−25 0.50 ± 0.01Glutamic Acid E 4.1 22−25 0.54 ± 0.03Glycine G 4.1 22−24 0.52 ± 0.02Cysteine C 4.1 22−25 0.65 ± 0.01Proline P 4.1 22−26 0.42 ± 0.01Alanine A 4.1 23−25 0.55 ± 0.02Valine V 6.5 24−28 0.63 ± 0.01Isoleucine I 6.5 25−31 0.73 ± 0.02Leucine L 6.5 27−34 0.75 ± 0.02Methionine M 6.5 27−33 0.68 ± 0.02Phenylalanine F ≥10 38−42 0.76 ± 0.03Tyrosine Y ≥10 39−44 0.81 ± 0.02Lysine K ≥10 40−43 0.25 ± 0.04Histidine H ≥10 41−43 0.29 ± 0.03Tryptophan W ≥10 42−45 0.81 ± 0.03Arginine R ≥10 42−45 0.30 ± 0.05aElution positions and TLC Rf values and standard deviations weredetermined using data from 3 class sets (approximately 48 studentsworking in groups of 2−4).

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Figure 1. Ninhydrin microplate analysis of column fractions for 2 representative mixtures of AAs. Mixtures were separated by Dowex-50chromatography, and relative elution positions were determined by ninhydrin analysis. Absorbance was measured at 570 nm (filled circles); samplescontaining 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 Reportsand Classroom Observation

Learning Goals Learning Objective

Goal 1 Acquire an understanding of the general features of an amino acid and understand how intermolecular forces influence the behavior of moleculesGoal 2 Appreciate the importance of solvent pH in influencing the charge state of ionizable groupsGoal 3 Become familiar with collection and analysis of spectrophotometric dataGoal 4 Appreciate the importance of deductive reasoning in using a variety of pieces of analytical data to analyze the composition of an “unknown”Goal 5 Develop the ability to carry out microscale analytical procedures

Learning GoalTested

AssessmentInstrument Learning Objective Assessed

Student Success Rate(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 termsof intermolecular forces when given information on the structure and isoelectic point of each aminoacid as well as the structure of Dowex-50 resin

85% (1.6)

Goal 1 Written exam Be able to correctly define the term “Rf ” as applied to TLC and consider R group structure to explainthe migration order of 5 amino acids on TLC

97% (0.86)

Goal 2 Written exam Be able to make logical suggestions on how to alter the protocol to try to separate coeluting aromaticand basic amino acids

96% (1.1)

Goals 1 and 2 Written exam Be able to explain why we use buffers of decreasing pH with anion exchange resins (compared to cationexchange resins using buffers of increasing pH)

92% (1.7)

Goal 3 Written exam Be able to explain the reason for measuring ninhydrin-AA absorbance at two different wavelengths 89% (1.7)Goal 3 Laboratory report

and classroomobservation

Be able to use graphing software to generate high-quality graphs of absorbance vs elution position data 100%b

Goal 4 Laboratory report Be able to correctly determine the composition of an unknown mixture of 3 amino acids based onDowex-50 elution and TLC data

77% (no errors), 16%(1 error), 7%(multiple errors)c

Goal 4 Laboratory report Be able to correctly determine the composition of an unknown mixture of 4−5 amino acids based onDowex-50 elution and TLC data

59% (no errors), 28%(1 error), 13%(multiple errors)c

Goal 5 Laboratory reportand classroomobservation

Be able to carry out microplate spectroscopic analyses with good precision and exhibit an understandingof the use of micropipettes

b100%

aExcept where noted, data reflect aggregate scores for a total of 48 lower and upper-division students. bStudents were allowed to consult with theinstructor on construction of graphs and use of micropipettes and all showed competence in these areas. In the few instances where students madesignificant errors in microplate assays and requested to repeat the assays, the new analyses were carried out correctly. cData reflect a representativegeneral chemistry class set.

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absorbance (“peaks”) from each of the separations were alsosubjected to TLC analysis (Figure 2; lower right panel) andprovided students with further information to aid in theirdetermination of the composition of their “unknown”mixtures. Several of the ninhydrin derivatives exhibit uniquecolors that provide an additional method of confirmation ofthe identity of the components in the mixture. For example,while most of the derivatives are purple in color, the prolinederivative is yellow and histidine yields a brownish derivativeon TLC. TLC analysis of all 20 standard AAs is shown forreference (Figure 2; upper panel).The experimental protocols have been incorporated into

honors sections of general chemistry (16−20 students/term) forthe past five academic years. In addition, for the past twoacademic years, we have featured the experiment in an upper-division biochemical techniques course (14−15 students/term).The entire suite of protocols required 8−9 h of laboratory time tocomplete. Working in small groups, students identified the AAsin their unknown mixtures with a high success rate (Table 2). Inmost cases, errors involved incorrect assessment of AAs with verysimilar chromatographic behavior (e.g., confusing M with V).Students came to their conclusions based on a combinationof visualization of the color of ninhydrin−AA derivatives, relativeelution position from Dowex-50 columns, TLC migration pat-terns, and comparison of the structural features of the R-groups

of the eight AAs employed in the experiment. Discussionsabout the structures of these R-groups enhanced and rein-forced lecture discussions on acid/base chemistry, buffers,titration of ionizable groups, intermolecular attractions, andthe concept of an organic functional group. Analysis of studentanswers to experiment-related questions on a formal writtenexamination (Table 2) indicates that students exhibited a highlevel of success in achieving learning goals. Student narrativesurveys indicated that students enjoyed the deductivereasoning aspects of the experiment and felt that the experi-ment helped them to achieve a variety of learning goals, suchas gaining experience in column and thin layer chromatog-raphy, microscale manipulations, spectrophotometric assays,and graphical analysis of data.

■ ASSOCIATED CONTENT

*S Supporting Information

Student handouts and instructor notes including detailedinformation on reagent and equipment sources and preparations,CAS numbers, color photographs of ninhydrin-stained TLCplates and ninhydrin microplate analysis of column fractions,extinction coefficients for ninhydrin−AA derivatives, andinformation regarding hazards. This material is available via theInternet at http://pubs.acs.org.

Figure 2. TLC analysis of AA standards, representative mixtures, and selected column fraction elution “peaks”. Samples were separated by TLC andstained by reaction with ninhydrin. (Upper panel) TLC analysis of all 20 standard AAs. (Lower left panel) TLC analysis of 8 AA standards and 5representative “unknown”mixtures: 1 (TMH), 2 (TVH), 3 (TPK), 4 (TPMFK), and 5 (DPFK). (Lower right panel) TLC analysis of 2 AAmixtures andDowex-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.

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■ AUTHOR INFORMATIONCorresponding Author

*E-mail: lbrunauer@scu.edu.Present Address†Department of Chemistry and Biochemistry, University ofCalifornia, Los Angeles, California 90095 United States.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank Mary Helen Mack, Linh Lam and NaushabaKhan for testing the protocol and our colleagues for helpfulcomments on this manuscript.

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