Two-dimensional nuclear magnetic resonance structure determination module for introductory biochemistry: synthesis and structural characterization of lyso-glycerophospholipids


Address for correspondence to: Teresa A. Garrett, Department of Chemistry, Vassar College, 124 Raymond Avenue, Poughkeepsie, New York 12604. Tel.: 845-437-5738; Fax: 845-437-5732; E-mail:


In this laboratory module, introductory biochemistry students are exposed to two-dimensional 1H-nuclear magnetic resonance of glycerophospholipids (GPLs). Working in groups of three, students enzymatically synthesized and purified a variety of 2-acyl lyso GPLs. The structure of the 2-acyl lyso GPL was verified using 1H-correlation spectroscopy. Students scored significantly higher on an assessment of NMR knowledge after having participated in this lab module and in comparison to a similar cohort who did not participate. Inaddition, student confidence in their NMR knowledge and abilities increased 62% following the module and correlated with their ability to apply their NMR knowledge. Based on these results, the laboratory module was very effective at providing students with a more extensive understanding of the underlying concepts of NMR as a tool for structural determination. © 2013 by The International Union of Biochemistry and Molecular Biology 41(2):87-94, 2013

A fundamental part of modern biochemical analysis is determination of the structure of biomolecules, both small molecules, such as lipids and small peptides, and complex macromolecular structures such as DNA, proteins, and protein complexes. Currently, researchers determine the structures of biological molecules routinely using techniques such as mass spectrometry, nuclear magnetic resonance (NMR), and X-ray crystallography. Of these techniques, undergraduate students are most often exposed to NMR. In organic chemistry classes, the theoretical underpinnings of NMR are covered and students often interpret NMR spectra as part of laboratory, classroom activities, or problem sets. In addition, even small undergraduate institutions often have NMR spectrometers that are used for research and upper-level chemistry classes.

Introductory biochemistry students are exposed to the concepts fundamental to structural determination using NMR to analyze biological molecules through textbooks or in class lectures, but do not gain hands on experience to these techniques in the laboratory. Given that biochemistry students have had previous exposure to the basics of one-dimensional NMR in organic chemistry, they are ready to expend upon that knowledge to use multi-dimensional NMR to determine the structure of a small biological macromolecule.

While one-dimensional (1D) 1H-NMR analysis is a powerful technique that can be used to determine molecular structure unambiguously, for molecules with a large number of protons these 1D spectra can become very difficult to interpret. Two-dimensional 1H-NMR (2D-1H-NMR) is actually a straightforward method to determine the structure of modestly sized biological macromolecules such as glycerophospholipids (GPLs). Students can be introduced to the power of multi-dimensional NMR, and increase their understanding of how it can be applied to larger macromolecules such as proteins. While the analysis of protein NMR data sets is complex and beyond the reach of an undergraduate classroom lab, the analysis and interpretation of 2D-1H-NMR of GPLs is accessible to undergraduate biochemistry students.

Here, we present a laboratory module for use in an introductory biochemistry laboratory course. This module class engages students in the synthesis and structural characterization of 2-acyl GPLs that they then analyze structurally using two-dimensional proton nuclear magnetic resonance (2D-1H-NMR), specifically 1H-correlation spectroscopy (1H-COSY). This laboratory module, which integrates concepts used in traditional chemistry courses into biochemistry courses, promotes skills necessary for success in biochemistry and molecular biology fields [[1-3]]. Specifically, students expand upon their fundamental knowledge of chemistry as it applies to biochemical questions, learn to interpret the quantitative and qualitative data obtained from 2D-NMR experiments and most importantly think in a more integrative manner [[3]].

Other lab modules that introduce 2D-NMR to students are available in the literature [[4-8]]. Some are more applicable to use in organic or advanced chemistry laboratories [[5-7][9]]. Others are useful for biochemistry courses in that they purify and characterize biochemically important compounds [[4][5]]. This lab module uses enzymes, central to undergraduate biochemistry, to synthesize phospholipid metabolites, integrating techniques used in chemistry courses, such as thin layer chromatography (TLC), organic extraction, and NMR analysis, with biochemistry.



GPLs, 16:0–18:1 phosphatidylethanolamine (PE), 16:0–18:1 phosphatidylserine (PS), 16:0–18:1 phosphatidylcholine (PC), 16:0–18:1 phosphatidylglycerol (PG), 16:0–18:1 phosphatidic acid (PA), and egg PC were obtained from Avanti Polar Lipids (Alabaster, Al). Deuterated chloroform and methanol were obtained from Cambridge Isotopes (Andover, MA). Glass-backed Silica Gel 60 high performance thin layer chromatography (HPTLC) plates were from E. Merck (Bridgeport, NJ); solvents were reagent grade from Malinckrodt (St. Louis, MO). Other reagents and supplies, including 15 mL disposable, borosilicate glass tubes with Teflon lined lids, were obtained from VWR (Bridgeport, NJ). Rhizopus arrhizus phospholipase A1 was from Sigma (St. Louis, MO).

Synthesis of 2-Acyl Lyso-GPLs

From the lipid stocks, 5 mg of each of the GPLs were dried into separate 15 mL glass tubes equipped with Teflon lined lids. The GPLs were digested in a reaction containing 2 mM GPL, 100 mM Tris-maleate pH 5.6, 20 mM CaCl2, 1 mg/mL R. arrhizus lipase. The GPL was sonicated in a water bath sonicator into the reaction mixture lacking the lipase. After the lipase was added an overlay of diethylether, which is required for activity [[10]], was applied and the mixture incubated at 37 °C for 1–2 hours.

Thin layer Chromatography Analysis of Digest Products

Digest products were analyzed by TLC. About 5 μL of the reaction before and after digestion were analyzed on a HPTLC developed in 65:25:4, CHCl3:CH3OH:H2O. Lipids were visualized by exposure to iodine vapors or by charring with sulfuric acid [[11]].

Lipid Extraction

Digestion products were purified using an acidic Bligh and Dyer extraction mixture [[12]]. After the ether was removed, the reaction volume was increased by the addition of water to a final volume of 3.6 mL. About 4 mL of CHCl3 and CH3OH, and 0.063 mL of 12 M HCl were added to generate a 2:2:1.8 CHCl3:CH3OH:0.1 M HCl mixture. The tubes were capped and gently shaken to mix the phases. Caps were vented and the mixing repeated. Phases were resolved by centrifugation in a clinical centrifuge for 10 min. The upper phase was removed along with any interphase material. The lower phase was washed with 4 mL of pre-equilibrated acidic upper phase and the mixing, venting, and centrifugation described above repeated. The upper phase was removed and then the lower phase dried under N2.

NMR Analysis

Samples were dissolved in 1:1 CDCl3:CD3OD and analyzed in a Bruker Avance(DPX–300) 300 MHz NMR. All chemical shifts are given in parts per million (ppm) relative to tetramethylsilane. The resonance frequency for 1H was 300.13 MHz. All NMR data was analyzed using TopSpin 1.3.

Safety Considerations

All solvents, CHCl3, CH3OH, and diethylether, and concentrated HCl should be used in a fume hood and be disposed of accordingly. Students when handling solvents should wear appropriate personal protective equipment such as solvent resistant gloves and safety goggles. Any person with a medical implant should not come in close contact to the spectrometer's magnetic field.

Student Learning Outcomes

The goal of this module is to increase students' conceptual understanding of 1D and 2D-1H NMR, as well as give them the skills to interpret basic 2D NMR spectra of biological molecules.

Course Synopsis

The course, entitled “Biochemistry—Biol/Chem 272”, covers protein structure and function, carbohydrate and lipid structure, enzyme kinetics and catalytic mechanism, thermodynamics, central metabolism including electron transport, oxidative phosphorylation, and ATP synthesis. The lecture portion meets for three 50 min sections per week. The course has two pre-requisites—one semester of organic chemistry and introductory biology and is designed to be taken concurrently with the second semester of organic chemistry by students in their sophomore year though often juniors and seniors are enrolled. The laboratory section, held once a week for 4 hours, includes a protein purification module as well as a kinetic analysis of an enzyme. This module took the place of a module on bioinformatics and visualization of protein structure.

Student Demographics

Students were undergraduates at Vassar College, a primarily undergraduate institution located in the Mid-Hudson Valley region of NY. In Spring 2011, two lecture sections were offered of Biol/Chem 272 with a total of 52 students. Eighteen students in one of the laboratory sections participated in this NMR module. Ten were sophomores, 7 juniors, 1 senior; 10 were Biochemistry majors, 6 Biology majors, and 2 Neuroscience and Behavior majors. The other students enrolled in Biol/Chem 272 served as the control group for assessment purposes. A portion of those students (9 of 34) completed a voluntary assessment of their NMR knowledge. These students did not receive any additional NMR training through Biol/Chem 272. Instead of the NMR module they participated in several exercises on enzyme inhibition, enzyme structure, and bioinformatics.

Module Design

The 18-student class was divided into six groups of three students each. Each group was assigned a different GPL, PC, egg PC, PE, PG, PS, or PA. Their tasks were to enzymatically synthesize and purify the corresponding 2-acyl lyso GPL and characterize its structure using 2D-1H NMR. The work was divided into five separate sessions over six weeks as described below.

  • Laboratory no. 1—Introduction, review of NMR, and enzymatic synthesis of 2-acyl lyso GPLs.

    The first session started with an introduction to structure of the major GPLs found in biological membranes (Fig. 1). Because the research program of Dr. Garrett focuses on Escherichia coli GPL biosynthesis and function, the connection to her research was covered in this session. The 2-acyl lyso GPLs that the students would be synthesizing and structurally characterizing are not commercially available and are potential substrates for an acyltransferase being studied in the Garrett research lab [[11]]. This particularly excited students; for many it was their first involvement with original scientific research [[1]]. Following this overview, students digested their GPL using the R. arrhizus phospholipase A1 (Fig. 2) as described above. During the incubation period, the class continued with the NMR review, building on students' previous experience with 1H-NMR, introducing the foundations of multi-dimensional NMR and strategies for interpreting 2D-spectra.

  • Laboratory no. 2—Purification of 2-acyl lyso GPL.

    Students purified their lyso-GPL product by extraction and analyzed the product using TLC, a technique learned in organic chemistry but here applied to a biochemical analysis. TLCs were spotted with a portion of un-digested GPL as well as a sample of the digestion mixture as described above. During the time that TLC plates were developing, students did an exercise matching molecules related to GPL structure with theoretical 1H-NMR spectra generated with ChemBioDraw Ultra (CambridgeSoft). The structures and predicted 1H-NMR spectra for serine, ethanolamine, and saturated and unsaturated fatty acids, for example, were given to students to match. This activity gave students a chance to practice NMR interpretation skills learned in organic chemistry.

  • Laboratory no. 3—Preparation for NMR.

    To effectively analyze the 2-acyl lyso GPL with NMR, the sample must be fully soluble in a deuterated solvent. Students investigated which combination of CHCl3, CH3OH, and water was the most effective at dissolving their lyso-GPL for NMR analysis with no aggregation.

  • Laboratory no. 4—NMR of purified lyso-GPLs.

    Over two weeks, each group did an overnight collection of 2D-1H-NMR data. Each student obtained hands-on experience with the NMR spectrometer through this module.

  • Laboratory no. 5—Group data analysis

    Students gathered to interpret their NMR spectra attempting to assign each of the cross peaks in their 2D-1H-NMR spectra.

Figure 1.

Structures of the major GPLs. The structures of PA, PE, PG, PS, and PC are shown with a 16:0 (number of carbons on the acyl chain:number of double of bonds), and 18:1Δ9 acyl chains. The acyl chain composition will vary depending on the source of the GPL. Egg PC, for example, has a variety of acyl chains, including 18:2 (most likely linoleate, 18:2Δ9,12).

Figure 2.

Reaction used to generate 2-acyl lyso GPLs. The Rhizopus arrhizus phospholipase A1 was used to enzymatically deacylate the sn-1 position as is shown for the generation of 2-acyl lyso PE from PE.

Final Report

Each student wrote a manuscript-like paper detailing the methods and results of the module. All spectra were shared among the groups including the spectra obtained from 1-acyl and di-acyl GPLs for comparison. Through comparing these spectra, students learned how NMR can distinguish between 1-acyl lyso-, 2-acyl lyso-, and di-acyl GPLs.

Course Material Access

Sample spectra, detailed protocols, and lecture material can be requested by contacting


Synthesis and purification of lyso GPLs

Students successfully synthesized the 2-acyl lyso GPLs as shown in Fig. 3, for 2-acyl lysoPE. The relative migration of the different GPL/2-acyl lyso GPLs differed depending on the GPL headgroup, the lyso-GPL consistently migrates more slowly on the TLC plate. Rf's for common GPLs and lyso GPLs are available from the Avanti Polar Lipids website ( 2-acyl lyso GPLs are subject to acyl chain migration [[13]]. To increase stability, the 2-acyl lyso GPLs were stored dry at −20 °C.

Figure 3.

Thin layer chromatography of de-acylation reaction products. The 2-acyl lyso PE digestion product, PE, and a 1-acyl lyso PE standard are displayed on HPTLC as described in the Methods section. The migration of PE, lyso PE, and the free fatty acid released by the enzyme are indicated. Both 1-acyl lyso PE and 2-acyl lyso PE nearly co-migrate on the TLC plate. Occasionally, a minor, rapidly migrating spot is seen near the solvent front. It has not been definitively identified but is likely a fatty acid oxidation product. The origin, where the sample was initially spotted, and the position of the solvent front after the solvent moved up the plate, are indicated.

NMR Analysis of Lyso GPLs

GPLs, and lyso GPLs are notoriously difficult to fully dissolve due to their amphipathic nature. Each group assessed visually which solvent combination of CHCl3, CH3OH, and water best dissolved their lyso GPL product. They were looking for crystal clear solutions, with no visual opalescence or floating particles of dried lipid. Most groups found that 1:1 CHCl3:CH3OH was most effective at solubilizing these lipids.

Each group signed up for a late-afternoon slot, outside of class time, to set up their overnight NMR experiment. At that session, which took about 45 min, students dissolved their sample in deuterated solvents and transferred them to a 5 mm NMR tube. Each group then brought their sample to the NMR and was presented a brief safety overview and introduction to the instrument. For most students this was their first opportunity to interact with high-end scientific instrumentation. They placed their sample in the instrument and then followed a set protocol, with faculty guidance, to shim and lock the NMR. A brief 1H-spectra (∼5 min) was obtained and then an 18-hour 1H-COSY was collected. The next morning, one student from the group came and removed the sample, with faculty guidance, and then plotted the 2D spectrum.

Figure 4, panel a shows the 2D-1H-NMR of 2-acyl lyso PE collected by students during this module. As indicated in Table 1, each of the cross peaks generated from the coupling of protons on adjacent carbons can be assigned (as indicated in Table 1 and Figs. 4 and 5) and are consistent with the 2-acyl lyso PE structure. Figure 4 (panels b and c) also includes the 2D-1H-NMR spectra of commercially available 1-acyl lyso PE and di-acyl PE.

Figure 4.

Examples of 2D-1H-NMR spectra. The 1H-COSY spectra for 2-acyl lyso PE (panel a), 1-acyl lyso PE (panel b), and PE (panel c) are shown. The top panel shows the full spectra. The middle panel shows an expansion of the low ppm region. The bottom panel shows an expansion of the high ppm region. The letters shown on the spectra indicate the couplings between the protons as detailed in Table 1. A larger version of each spectrum is provided in Supporting Information.

Figure 5.

Structures highlighting protons whose coupling generates the major cross peaks in the 2D-1H-NMR spectra. Panel (a) shows the acyl chain. R indicates the remainder of the GPL. The protons are labeled to correlate with Table 1. The value for “n” depends on the acyl chain length. Panel (b) shows the glycerol portion of the GPL for the 2-acyl lyso GPL, 1-acyl lyso GPL, and di-acyl GPL. R1 indicates an acyl chain, and R2 indicates the phosphate and head-group portion of the GPL. The numbering of the glycerols is indicated to correlate with Table 1. Stereospecific numbering (sn) is used. The hydrogen atoms attached to the sn-1 carbon are referred to as the sn-1a and sn-1b in Table 1. It is difficult to identify which of the sn-1 hydrogen atoms is the sn-1a versus sn-1b therefore they have not been specifically labeled in this figure. Panel (c) shows the ethanolamine head group. R3 is the phosphatidyl portion of the GPL. The * indicates the —PO4—CH2— and the indicates the —CH2—NH2.

Table 1. 1H-COSY couplings observed for the 1-acyl, 2-acyl, and di-acyl PE
 CouplingsCross peak
Proton TypeProton type
Acyl Chainω-CH3ω-1 CH2a
 —CH2—CH CH—(CH2)nc

The cross peaks generated from the acyl-chains are similar among the spectra shown and the GPL analyzed. Couplings between all of the hydrogen atoms of the acyl chain are easily interpreted by students with only a little guidance by faculty. The NMR spectra of GPLs isolated from natural products, such as egg PC show a cross peak from the coupling of protons attached to a carbon between two double bonds (indicated by d in Table 1 and Fig. 4c). Depending on the source of the GPL starting material this coupling may not appear.

Cross peaks generated from coupling of protons attached to the glycerol distinguish among the 2-acyl lyso-, 1-acyl lyso-, and di-acyl GPLs. In a di-acyl GPL the protons on the sn-1 carbon are non-equivalent. These protons, referred to as sn-1a and sn-1b in Table 1 yield different chemical shifts likely due to the different physical orientation that the glycerol backbone can obtain when an acyl chain is attached to the sn-1 and sn-2 position [[13]]. This leads to three distinct couplings to the sn-2 1H (chemical shift ∼5.2 ppm, Fig. 4c, marked I, II and III). When the acyl chain is removed from the sn-1 position the sn-1 H's become more equivalent, likely due to the free rotation of the terminal CH2OH, yielding a single cross peak as indicated in Fig. 4a (marked I, II, and III). In the 1-acyl lyso PE, the lack of an acyl chain on the sn-2 carbon causes the chemical shift of the sn-2 H to move up field to ∼4.2 ppm (Fig. 4b, marked I, II, and III). Unfortunately this is also the region of the spectrum that the other H's attached to the glycerol resonate making assignment of those cross peaks more challenging.

The cross peaks generated from the coupling of protons attached to the head group is also challenging. For PE, the methylene protons (Fig. 5) are identifiable as indicated (Fig. 4, marked with * and Table 1). The NH2 protons are likely resonating similarly to the other headgroup protons leading to a cross peak on the axis, that is difficult to resolve in these spectra.

Derivatives of this Module

This laboratory module and its data may be used in a variety of ways to supplement biochemistry teaching. Here we digested commercially available GPLs to generate 2-acyl lyso GPLs that are not commercially available. One could eliminate the digestion and purification steps and do NMR analysis of di-acyl GPLs as compared to 1-acyl lyso GPLs. In addition, some students could enzymatically generate 1-acyl lyso GPLs using commercially available PLA2 to compare to the 2-acyl lyso GPL generated using the Rhizopus lipase. Even at institutions that do not possess high field NMR instruments, the data generated in this module could be used on its own as part of an in-class exercise to give students exposure to macromolecular structure determination. The module could also be modified for upper-level chemistry labs by adding a reverse-phase purification step [[14]] to separate the 2-acyl lyso GPL from the free fatty acid or by discussing in greater detail concepts of NMR coupling.

Implementing this Module

NMR expertise is not required for implementing this module. An instrument manager or colleague can help to familiarize instructors interested in implementing this or similar modules with the fundamentals of NMR and setting up the instrument for the data collection. Step-by-step instructions for how to insert the sample into the machine, lock, shim, and set up the data collection can be developed in collaboration with an instrument manager or colleague more familiar with NMR. The 2D-NMR data collected can be interpreted without extensive experience with NMR or knowledge of coupling constants. An instructor that is willing to learn the fundamentals of NMR and how to run an NMR, at a basic level, will be able to implement this module with their students.

Assessment of Module Effectiveness

Students participating in the module were given an anonymous pretest on the fundamentals of nuclear magnetic resonance analysis and techniques. This 28-question assessment included five questions asking students to rate their level of confidence [[15]] with regard to several specific aspects of working with and understanding NMR analysis. The pretest was given at the beginning of the first laboratory session. Students, those who completed the lab module and those in the control group, completed the post-test at the end of the semester outside of class.

Students' scores on the assessment increased on average by 30 percentage points after completing the module; these students' scores were also 22 points higher on average than the control group of students who did not participate in the lab module but had otherwise completed the same biochemistry course (Fig. 6). These results were statistically significant. When comparing participants before (pretest) and after (post-test) participating in the lab module and when comparing to non-participants (control), the t-tests (both 1.7) and p values (both <10−5) indicate that participation in the module contributed to improved scores on the non-confidence questions of the assessment.

Figure 6.

NMR knowledge increases for students participating in the lab module. The average percent correct of the 23 non-confidence questions on the assessment are given for the control group (students who did not participate in the lab module, n = 9), compared to the average scores of the students who did participate before (pretest, n = 18) and after (post-test, n = 13) participating in the lab module. Error bars represent the standard deviation. A table is included in the Supporting Information summarizing the statistical data.

For the confidence-related questions, their responses were scored, 1–4 where least confident was scored 1 and most confident was scored 4. The sum of the confidence scores on these five questions for the pre- and post-test as well as the control group is presented in Fig. 7. Overall, the results indicated that students who had completed the module were at least 62% more confident than before they were exposed to the new module and more confident than the students who did not participate (Fig. 7). In addition, there was a correlation (r2 = 0.42) between how well the students felt they understood the material and their actual performance on the assessment. While this correlation is not particularly strong, it does indicate that student confidence was positively impacted by participation in the lab module. This increased confidence from participation in this module may make them more likely to apply NMR in other advanced courses or in independent research endeavors.

Figure 7.

Confidence in NMR skills increases for students participating in the lab module. The confidence of each student was assessed with five questions on the assessment. For each question confidence was scored for each question; 1–4 for least confidence to most confidence and the score summed to yield the confidence score for each student. The average confidence for the non-participants (control) and the participants before (pretest) and after (post-test) the lab experience is presented in the graph. Error bars represent the standard deviation.

Based on these results, the laboratory module was very effective at providing students with a more extensive understanding of the underlying concepts of NMR as a tool for structural determination. It also provided students with hands-on experience with, and confidence in their abilities to use, NMR structural determination techniques. Given the fundamental role of structural determination in modern biochemical analysis, the experience students gained working with 2D-1H-NMR for the determination of the structure of small biomolecules is valuable and applicable to future learning and laboratory work.


The development of this module was supported by funds from a Howard Hughes Medical Institute College Grant to Vassar College. Thanks to Karen Wovkulich for assistance with NMR runs.