Data from: Investigating reproducibility challenges in CD spectroscopy of FmocFF gels
Data files
May 01, 2026 version files 697 KB
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Figure_1_FmocFF_solution_CD_HT.csv
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Figure_2_FmocFF_HCl_gel_flat_windows_CD_HT.csv
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Figure_3_FmocFF_HCl_interfacial_gelation_CD_HT.csv
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Figure_4_FmocFF_GdL_gel_flat_windows_CD_HT.csv
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Figure_5_FmocFF_GdL_gel_in_situ_cuvette_CD_HT.csv
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Figure_6_FmocFF_CaCl2_gel_flat_windows_CD_HT.csv
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Figure_7_FmocFF_CaCl2_gel_in_situ_cuvette_CD_HT.csv
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Figure_8_FmocFF_5pct_DMSO_water_gel_CD_HT.csv
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Figure_9_FmocFF_10pct_20pct_DMSO_water_gel_CD_HT.csv
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Figure_S21_FmocFF_solution_path_length_comparison_CD_HT.csv
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Figure_S22_FmocFF_HCl_gel_0p01mm_cuvette_CD_HT.csv
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Figure_S23_FmocFF_optical_transmittance_summary.csv
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Figure_S24_FmocFF_GdL_gel_0p01mm_cuvette_CD_HT.csv
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Figure_S25_FmocFF_ATR_FTIR_solution_DCl_GdL.csv
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Figure_S26_FmocGG_GdL_gel_in_situ_cuvette_CD_HT.csv
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Figure_S27_FmocFF_CaCl2_gel_0p01mm_cuvette_CD_HT.csv
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Figure_S28_DMSO_water_background_HT_0p01mm_cuvette.csv
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Figure_S29_DMSO_water_background_HT_minimal_path.csv
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Figure_S30_FmocFF_DMSO_water_gel_0p01mm_cuvette_CD_HT.csv
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README.md
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Abstract
Fluorenylmethoxycarbonyl-diphenylalanine (FmocFF) is a widely studied low molecular weight gelator (LMWG) known for its ability to self-assemble into supramolecular gels with diverse applications. Despite extensive literature, the reported circular dichroism (CD) spectra of FmocFF are remarkably inconsistent, even under seemingly identical experimental conditions. In this study, we systematically investigate the factors contributing to this variability and propose a protocol for reproducible CD measurements of FmocFF in solution and gel states. We compare gelation triggers including HCl, glucono-δ-lactone (GdL), CaCl₂, and DMSO/water solvent-switch, and assess the impact of sample handling, loading techniques, and cuvette geometry on spectral features. Our findings demonstrate that sample homogeneity, cuvette path length, and high-tension (HT) signal monitoring are critical for reliable CD analysis of short peptide-based gels. This work highlights the importance of rigorous experimental practices in interpreting supramolecular chirality of dynamic, heterogeneous systems such as FmocFF gels.
Dataset DOI: 10.5061/dryad.7wm37pw72
Description of the data and file structure
All figures in the paper show circular dichroism spectra, high tension data, optical transmittance data, or ATR-FTIR data collected as described in the methods section of the main article and the electronic supplementary information. In the original submission, these data were stored in a single Excel workbook with 19 worksheets. In this revised submission, each worksheet has been exported as a separate CSV file, with one CSV file for each figure tab.
Files and variables
File: Figure_1_FmocFF_solution_CD_HT.csv
Description: all numerical data for Figure 1c and Figure 1d. This file contains CD spectra of FmocFF solution (5 mg mL-1, pH 10.5) collected from four independent samples and the corresponding HT profiles.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_2_FmocFF_HCl_gel_flat_windows_CD_HT.csv
Description: all numerical data for Figure 2a-d. This file contains CD spectra and corresponding HT profiles of HCl-triggered FmocFF gels mounted between two flat cuvette windows. Panels a-b are from four samples scooped from the same gel vial. Panels c-d are from four gels prepared in independent vials.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
- preparation type or panel identifier
File: Figure_3_FmocFF_HCl_interfacial_gelation_CD_HT.csv
Description: all numerical data for Figure 3a and Figure 3b. This file contains CD spectra and corresponding HT profiles of FmocFF gels formed inside a 0.01 mm cuvette by interfacial gelation with HCl.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_4_FmocFF_GdL_gel_flat_windows_CD_HT.csv
Description: all numerical data for Figure 4a-d. This file contains CD spectra and corresponding HT profiles of GdL-triggered FmocFF gels collected between two flat cuvette windows. Panels a-b are from four preparations from a single gel batch. Panels c-d are from four independently prepared gels.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
- preparation type or panel identifier
File: Figure_5_FmocFF_GdL_gel_in_situ_cuvette_CD_HT.csv
Description: all numerical data for Figure 5a and Figure 5b. This file contains CD spectra and corresponding HT profiles of FmocFF + GdL gels prepared directly in a 0.01 mm cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_6_FmocFF_CaCl2_gel_flat_windows_CD_HT.csv
Description: all numerical data for Figure 6a-d. This file contains CD spectra and corresponding HT profiles of CaCl2-triggered FmocFF gels collected between two flat cuvette windows. Panels a-b are from four samples prepared from a single gel batch. Panels c-d are from four independently prepared gels.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
- preparation type or panel identifier
File: Figure_7_FmocFF_CaCl2_gel_in_situ_cuvette_CD_HT.csv
Description: all numerical data for Figure 7a and Figure 7b. This file contains CD spectra and corresponding HT profiles of FmocFF + CaCl2 gels prepared in situ in a 0.01 mm cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_8_FmocFF_5pct_DMSO_water_gel_CD_HT.csv
Description: all numerical data for Figure 8a and Figure 8b. This file contains CD spectra and corresponding HT profiles of FmocFF gels prepared in 5% DMSO/water (v/v).
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_9_FmocFF_10pct_20pct_DMSO_water_gel_CD_HT.csv
Description: all numerical data for Figure 9a-d. This file contains CD spectra and corresponding HT profiles of FmocFF gels prepared in 10% and 20% DMSO/water (v/v). Panels a-b correspond to 10% DMSO/water and panels c-d correspond to 20% DMSO/water.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
- DMSO content
- panel identifier
File: Figure_S21_FmocFF_solution_path_length_comparison_CD_HT.csv
Description: all numerical data for Figure S21. This file contains CD spectra and corresponding HT profiles of FmocFF solution recorded using 0.1 mm and 0.01 mm path length cuvettes.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- path length
File: Figure_S22_FmocFF_HCl_gel_0p01mm_cuvette_CD_HT.csv
Description: all numerical data for Figure S22. This file contains CD spectra and corresponding HT profiles of FmocFF + HCl gels loaded by scooping into a standard 0.01 mm demountable cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_S23_FmocFF_optical_transmittance_summary.csv
Description: all numerical data for Figure S23. This file contains percent transmittance values of FmocFF samples prepared under different conditions, measured at 800, 600, and 400 nm. The data are reported as mean values with the corresponding estimated standard deviations.
Variables
- sample condition
- wavelength (nm)
- transmittance (%)
- estimated standard deviation
File: Figure_S24_FmocFF_GdL_gel_0p01mm_cuvette_CD_HT.csv
Description: all numerical data for Figure S24. This file contains CD spectra and corresponding HT profiles of FmocFF + GdL gels collected in a 0.01 mm cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_S25_FmocFF_ATR_FTIR_solution_DCl_GdL.csv
Description: all numerical data for Figure S25a-b. This file contains ATR-FTIR spectra of FmocFF solution prepared in NaOD/D2O and gels prepared with DCl and GdL.
Variables
- wavenumber (cm-1)
- absorbance or intensity
- sample condition
- panel identifier
File: Figure_S26_FmocGG_GdL_gel_in_situ_cuvette_CD_HT.csv
Description: all numerical data for Figure S26. This file contains CD spectra and corresponding HT profiles of FmocGG + GdL gels prepared directly within a 0.01 mm cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_S27_FmocFF_CaCl2_gel_0p01mm_cuvette_CD_HT.csv
Description: all numerical data for Figure S27. This file contains CD spectra and corresponding HT profiles of FmocFF + CaCl2 gels collected in a 0.01 mm cuvette.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- sample identifier
File: Figure_S28_DMSO_water_background_HT_0p01mm_cuvette.csv
Description: all numerical data for Figure S28. This file contains HT voltage profiles of background DMSO/water mixtures at 5%, 10%, and 20% DMSO (v/v) recorded in a 0.01 mm path length cuvette.
Variables
- wavelength (nm)
- HT (V)
- DMSO content
File: Figure_S29_DMSO_water_background_HT_minimal_path.csv
Description: all numerical data for Figure S29. This file contains HT voltage profiles of background DMSO/water mixtures at 5%, 10%, and 20% DMSO (v/v) recorded at minimal path length.
Variables
- wavelength (nm)
- HT (V)
- DMSO content
File: Figure_S30_FmocFF_DMSO_water_gel_0p01mm_cuvette_CD_HT.csv
Description: all numerical data for Figure S30. This file contains CD spectra and corresponding HT profiles of FmocFF gels in DMSO/water collected in a 0.01 mm path length cuvette. Each condition was tested in duplicate.
Variables
- wavelength (nm)
- circular dichroism (mdeg)
- HT (V)
- DMSO content
- replicate identifier
Code/software
Excel, Origin, or similar software
Materials
FmocFF was prepared using a previously reported method.56 FmocGG, Sodium hydroxide (NaOH), hydrochloric acid (HCl), glucono-δ-lactone (GdL), anhydrous calcium chloride (CaCl2), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich or Fluorochem Ltd. Deionised water (resistivity > 15 MΩ·cm) was used in all experiments.
Preparation of FmocFF solutions and hydrogels
High pH FmocFF solution
A 5 mg·mL−1 stock solution of FmocFF was prepared by stirring the dipeptide in water containing 0.95 equivalents of NaOH (~16 h) at room temperature. The pH of the solution was then adjusted to 10.5 using 1 M NaOH. This solution forms a viscous micellar fluid and was used immediately for CD experiments to minimise hydrolysis.
HCl-triggered gels
1 mL of FmocFF solution (5 mg·mL–1, pH 10.5) was added to 30 μL of 0.1 M HCl and gently shaken by hand. The mixture gelled immediately, reaching a final pH of ~2-3. For uniform gel preparation in cuvettes, the high pH FmocFF solution was first spread on the cuvette surface and immersed in 100 mL 0.1 M HCl solution in a beaker. After gelation, excess HCl solution was wiped before closing the cuvette.
GdL-triggered gels
1 mL of FmocFF solution (5 mg·mL−1, pH 10.5) was added to 8 mg of solid GdL. The mixture was vortexed and left undisturbed at room temperature for ~1-2 hours to allow gradual pH reduction and gelation. For in situ gelation in the cuvette, the mixture was immediately loaded into the cuvette after mixing, sealed with parafilm, and incubated for 2 hours before data collection.
CaCl₂-triggered gels
1 mL of FmocFF solution (5 mg·mL−1, pH 10.5) was added to 28 μL of 1.0 M CaCl2 solution to obtain a FmocFF:CaCl₂ at 1:3 molar ratio, and gel formed within minutes. However, due to the localised nature of the addition, the resulting gel displayed visible heterogeneity. To overcome this, samples were gently shaken and left to equilibrate for one hour. The uniform gel in the cuvette was prepared in the same way as described for HCl, by immersing the FmocFF solution in 100 mL of 50 mM CaCl2 solution.
Solvent-switch gels (DMSO/water)
FmocFF was dissolved in DMSO at concentrations of 25, 50, or 100 mg·mL−1. Appropriate volumes of these solutions were then added dropwise into water to yield final FmocFF concentration of 5 mg·mL−1 and final DMSO content of 5%, 10%, or 20% (v/v). The resulting gels were allowed to equilibrate for 1 hour prior to CD measurements.
FmocGG gels
FmocGG (10 mg) was placed in a 7 mL sterilin vial, then 1.72 mL DI water and 275 μL of 0.1 M NaOH were added sequentially. The mixture was stirred overnight, after which the pH was adjusted to 10.5 with 1 M NaOH. To this, 16 mg GdL was added, the mixture was vortexed briefly, and it was left undisturbed to gel (~2 h).
ATR-FTIR
Infrared spectra were recorded on an Agilent Cary 630 FTIR spectrometer using an ATR attachment. Spectra (4000–600 cm−1) were acquired at 1 cm−1 resolution, averaging 64 scans for both the D₂O background and each sample. The basic aqueous solution was prepared as mentioned above, but using D2O and NaOD instead of NaOH/H2O. The pD of the solution was adjusted to 10.9 (pD = pH + 0.4), and a drop of the solution was placed on the spectrometer. Gels were prepared with DCl (0.1 M in D2O) and with GdL, and scooped with a spatula. In each case, a D2O background was subtracted. Baselines were corrected in OriginPro 2018.
CD sample loading
For the FmocFF high-pH solution, the sample was pipetted using a pasteur pipette, and a drop was placed onto the spacer window of a demountable cuvette with a path length of either 0.1 mm or 0.01 mm. Taking advantage of the sample's fluidity, the drop was spread as evenly as possible across the spacer. The cuvette was then sealed with a flat quartz window. Any excess liquid that spilled out was carefully wiped off before loading the cuvette into the CD spectrometer.
For gel samples prepared in the 0.01 mm cuvette, a small amount of gel was gently scooped and placed onto the spacer window. The gel was carefully spread using a spatula to maximise surface coverage. The cuvette was then sealed with a flat window, and any excess gel was wiped off.
Similarly, for samples measured using the minimal path length setup, the gel was first scooped onto a flat window of a cuvette and gently spread with a spatula. This was then covered with another flat window from a second cuvette to form the cell.
CD measurements
CD spectra were acquired using a Chirascan CD spectrometer (Applied Photophysics) at 25 °C, over a wavelength range of 180–400 nm. Data were recorded with a step size and bandwidth of 1 nm. Each spectrum was an average of at least three accumulations. The contributions from the appropriate backgrounds were subtracted, and data were smoothed using a Savitzky-Golay 10-point window in OriginPro 2018.