Investigating reproducibility challenges in CD spectroscopy of FmocFF gels

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..., 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 firs..., # Data from: Investigating reproducibility challenges in CD spectroscopy of FmocFF gels Dataset DOI: [10.5061/dryad.7wm37pw72](https://doi.org/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) * circula..., ,芴甲氧羰基二苯丙氨酸（Fluorenylmethoxycarbonyl-diphenylalanine，简称FmocFF）是一类被广泛研究的低分子量凝胶因子（low molecular weight gelator, LMWG），因其可自组装形成超分子凝胶且具备多样应用场景而广受关注。尽管已有大量相关文献报道，但即便在实验条件看似一致的情况下，所报道的FmocFF圆二色性（circular dichroism, CD）光谱仍存在显著不一致性。本研究系统探究了导致该差异的影响因素，并提出了可重复获取溶液态与凝胶态FmocFF CD光谱的实验方案。我们对比了包括盐酸（HCl）、葡萄糖酸δ-内酯（glucono-δ-lactone, GdL）、氯化钙（CaCl₂）以及二甲基亚砜/水溶剂切换在内的凝胶触发方式，并评估了样品处理、上样技术以及比色皿几何形状对光谱特征的影响。研究结果表明，样品均一性、比色皿光程以及高张力（high-tension, HT）信号监测对于短肽基凝胶的可靠CD分析至关重要。本工作强调了严谨实验操作的重要性…… 材料 FmocFF的合成采用已报道的方法56。芴甲氧羰基甘氨酸二肽（FmocGG）、氢氧化钠（NaOH）、盐酸（HCl）、葡萄糖酸δ-内酯（GdL）、无水氯化钙（CaCl₂）以及二甲基亚砜（dimethyl sulfoxide, DMSO）均购自Sigma-Aldrich或Fluorochem Ltd.。所有实验均使用电阻率大于15 MΩ·cm的去离子水。 FmocFF溶液与水凝胶的制备 高pH值FmocFF溶液 将二肽FmocFF与含0.95当量NaOH的水溶液混合，于室温下搅拌约16小时，制备得到浓度为5 mg·mL⁻¹的储备液。随后使用1 M NaOH将溶液pH值调节至10.5。该溶液呈粘性胶态流体，需立即用于CD实验以降低水解风险。 盐酸触发型凝胶 取1 mL浓度为5 mg·mL⁻¹、pH 10.5的FmocFF溶液，加入30 μL 0.1 M HCl，手动轻柔振荡混匀。混合物会立即凝胶，最终pH值约为2~3。若需在比色皿中制备均匀凝胶，高pH FmocFF溶液需先…… # 数据来源：探究FmocFF凝胶圆二色光谱的可重复性挑战 数据集DOI：[10.5061/dryad.7wm37pw72](https://doi.org/10.5061/dryad.7wm37pw72) ## 数据与文件结构说明 本文所有图表均包含圆二色性光谱、高张力信号数据、透光率数据或衰减全反射傅里叶变换红外光谱（ATR-FTIR）数据，数据采集方法详见正文方法部分与电子补充材料。在初始投稿阶段，所有数据存储于单个包含19个工作表的Excel工作簿中。在本次修订版投稿中，每个工作表均被导出为独立的CSV文件，对应原投稿中每个图表工作表。 ### 文件与变量 #### 文件：Figure_1_FmocFF_solution_CD_HT.csv **描述**：对应图1c与图1d的全部数值数据。该文件包含4组独立样品的FmocFF溶液（5 mg·mL⁻¹，pH 10.5）CD光谱及其对应的高张力信号曲线。 ##### 变量 * 波长（nm） * 圆二色性信号（circula..., , ）