Ferrous gluconate (C₁₂H₂₂FeO₁₄) is a commonly used organic iron supplement. Its molecular structure is formed by the coordination bonding of 2 gluconate anions (HOCH₂(CHOH)₄COO⁻) and 1 divalent iron ion (Fe²⁺), featuring both the characteristic functional groups of gluconic acid (hydroxyl group -OH, carboxyl group -COO⁻) and the coordination structure of Fe²⁺. Spectroscopic analysis is a core method for analyzing its molecular structure, identifying purity, and studying coordination environments. Among these techniques, Infrared Spectroscopy (IR) is used to identify functional groups and coordination modes, Ultraviolet-Visible Spectroscopy (UV-Vis) reflects the electronic transition properties of Fe²⁺, and Nuclear Magnetic Resonance Spectroscopy (NMR) analyzes the carbon-hydrogen bonding patterns and spatial configuration of the gluconate chain. Together, these three techniques reveal the molecular characteristics of ferrous gluconate from different dimensions.
I. Infrared Spectroscopy (IR) Characteristics: Functional Group Identification and Coordination Mode Analysis
IR spectroscopy detects the vibration frequencies of chemical bonds in molecules (wavenumber range: 4000–400 cm⁻¹) to identify characteristic functional groups in ferrous gluconate and determine the coordination mode between Fe²⁺ and gluconate anions. The key absorption peaks and their corresponding structural information are as follows:
(I) Characteristic Absorption Peaks of Gluconate Anion Functional Groups
As an organic ligand, the gluconate anion produces strong characteristic absorptions due to the vibrations of its hydroxyl (-OH), methylene (-CH₂-), methine (-CHOH-), and carboxyl (-COO⁻) groups, which serve as the primary basis for IR identification:
Hydroxyl (-OH) stretching vibration: The multiple hydroxyl groups (each gluconic acid molecule contains 5 -OH groups) in the gluconate chain generate a broad, intense absorption peak in the 3200–3600 cm⁻¹ range. Due to the easy formation of hydrogen bonds between hydroxyl groups, the absorption peak exhibits a "broad peak envelope" (full width at half maximum: ~300–400 cm⁻¹) with a central wavenumber of approximately 3400 cm⁻¹. This peak is a typical marker for gluconic acid compounds, distinguishing them from iron salts without hydroxyl groups (e.g., ferrous sulfate).
C-H stretching vibration: The C-H stretching vibrations of methylene (-CH₂-) and methine (-CH-) produce two weak, sharp absorption peaks at 2920 cm⁻¹ (asymmetric stretching) and 2850 cm⁻¹ (symmetric stretching), respectively. These peaks confirm the presence of saturated carbon-hydrogen chains in the molecule.
C-O stretching vibration: The ether bonds (-C-O-C-) and C-O bonds attached to hydroxyl groups in the gluconate chain generate strong absorptions in the 1050–1150 cm⁻¹ range. The absorption peak near 1100 cm⁻¹ corresponds to ether bond stretching, while the peak near 1030 cm⁻¹ corresponds to C-OH stretching. The overlap of these two peaks forms a broad, intense peak, which is a key characteristic of the gluconate backbone.
Bending vibration peaks: The in-plane bending vibration of hydroxyl groups produces a weak absorption peak at 1420–1450 cm⁻¹, and the bending vibration of methylene groups creates a peak near 1370 cm⁻¹, both serving as auxiliary identification criteria.
(II) Coordination-Characteristic Absorption Peaks of Carboxyl Groups (-COO⁻) and Fe²⁺
In ferrous gluconate, the carboxyl groups of gluconate anions exist in the form of negative ions (-COO⁻) and form coordinate bonds with Fe²⁺ via the oxygen atoms of the carboxyl groups. This coordination causes a significant shift in the vibration frequency of the carboxyl groups, which is critical for determining the coordination mode:
Difference between free and coordinated carboxyl groups: The C=O stretching vibration of free carboxyl groups (-COOH) produces a strong peak near 1720 cm⁻¹. In ferrous gluconate, however, carboxyl groups are dissociated into -COO⁻ and coordinated with Fe²⁺, leading to the splitting of their vibrations into two peaks: "asymmetric stretching" and "symmetric stretching." For uncoordinated -COO⁻, the asymmetric stretching peak appears at ~1580 cm⁻¹ and the symmetric stretching peak at ~1410 cm⁻¹.
Peak shift caused by coordination: When -COO⁻ forms "monodentate coordination" (only one oxygen atom coordinated) with Fe²⁺, the asymmetric stretching peak shifts to lower wavenumbers (1560–1570 cm⁻¹), and the symmetric stretching peak shifts to higher wavenumbers (1420–1430 cm⁻¹). For "bidentate coordination" (both oxygen atoms coordinated), the asymmetric stretching peak shifts more significantly (1540–1550 cm⁻¹), and the symmetric stretching peak shifts to 1430–1440 cm⁻¹. In practical detection, the carboxyl asymmetric stretching peak of ferrous gluconate appears at ~1565 cm⁻¹ and the symmetric stretching peak at ~1425 cm⁻¹, confirming that monodentate coordination is dominant, with some uncoordinated -COO⁻ present.
Fe-O coordinate bond vibration: The coordinate bonds (Fe-O) formed between Fe²⁺ and the oxygen atoms of carboxyl/hydroxyl groups produce weak absorption peaks in the fingerprint region (400–600 cm⁻¹), with a central wavenumber of ~520 cm⁻¹. Due to their weak intensity and susceptibility to interference from other vibrations, these peaks require analysis using high-resolution IR spectroscopy (e.g., FT-IR) and deconvolution techniques, serving as direct evidence for confirming the coordination environment of Fe²⁺.
II. Ultraviolet-Visible Spectroscopy (UV-Vis) Characteristics: Electronic Transitions of Fe²⁺ and Reflection of Coordination Environment
The UV-Vis absorption of ferrous gluconate primarily originates from the d-d electronic transitions of Fe²⁺ and ligand-to-metal charge transfer (LMCT) transitions between the ligand (gluconate anion) and the metal ion. The position, intensity, and shape of absorption peaks reflect the coordination environment of Fe²⁺ (e.g., coordination number, ligand type) and its oxidation state (whether Fe²⁺ is oxidized to Fe³⁺). The characteristic absorption ranges are as follows:
(I) UV Region (200–380 nm): Ligand-to-Metal Charge Transfer (LMCT) Transitions
Absorption peaks in the UV region are mainly generated by LMCT transitions, where electrons are transferred from the oxygen atoms (with lone pairs) of gluconate anions to the empty d orbitals of Fe²⁺. These are intense absorption peaks (molar absorptivity ε > 1000 L·mol⁻¹·cm⁻¹), reflecting the binding strength between the ligand and the metal:
Strong absorption peak in the 220–240 nm range: This peak corresponds to the transition of lone-pair electrons from the carboxyl oxygen (O=C-O⁻) of the gluconate anion to the empty 4s orbital of Fe²⁺, with a central wavelength of ~230 nm and ε ≈ 1800 L·mol⁻¹·cm⁻¹. The peak intensity is positively correlated with the number of coordinated carboxyl groups—the more carboxyl groups coordinated, the higher the peak intensity.
Weak absorption peak in the 280–320 nm range: This peak arises from the transition of lone-pair electrons from the hydroxyl oxygen (-OH) of the gluconate anion to the empty 3d orbital of Fe²⁺, with a central wavelength of ~300 nm and ε ≈ 300 L·mol⁻¹·cm⁻¹. Due to the lower electron density of hydroxyl oxygen compared to carboxyl oxygen, the transition probability is low, resulting in weak peak intensity. If Fe³⁺ (oxidative impurity) is present in ferrous gluconate, an additional absorption peak appears in the 350–400 nm range (ε ≈ 500 L·mol⁻¹·cm⁻¹), which can be used to determine the oxidation degree of the product. No obvious absorption should be observed in this range for pure samples.
(II) Visible Region (380–800 nm): d-d Electronic Transitions of Fe²⁺
Fe²⁺ has an electron configuration of [Ar] 3d⁶. Under the influence of the coordination field, the 3d orbitals undergo energy splitting, and electrons transition between different energy levels (d-d transitions), producing absorption peaks in the visible region. Since d-d transitions are spin-forbidden, the absorption peaks are weak (ε < 100 L·mol⁻¹·cm⁻¹), but their positions are sensitive to the coordination environment, making them critical for analyzing the coordination number and geometric configuration of Fe²⁺:
Broad absorption peak in the 450–500 nm range: In ferrous gluconate, Fe²⁺ has a coordination number of 6 (octahedral coordination geometry), coordinating with 2 carboxyl oxygen atoms, 2 hydroxyl oxygen atoms, and 2 water molecules (crystalline or solvent water). The 3d orbitals split into two sets of energy levels (t₂g and eg), and d-d transitions mainly occur between the t₂g and eg orbitals, producing a broad peak with a central wavelength of ~480 nm and ε ≈ 50 L·mol⁻¹·cm⁻¹. The broad shape of the peak is due to slight distortion of the octahedral coordination, leading to incomplete energy splitting.
Weak shoulder peak in the 600–700 nm range: This peak is a secondary peak of d-d transitions, resulting from further splitting of the 3d orbitals of Fe²⁺ (e.g., Jahn-Teller distortion), with a central wavelength of ~650 nm and ε ≈ 20 L·mol⁻¹·cm⁻¹. It can only be observed in high-concentration solutions (e.g., >10⁻³ mol/L) and serves as an auxiliary criterion for confirming the octahedral coordination geometry.
Effect of oxidation state changes: If Fe²⁺ is oxidized to Fe³⁺, the visible-region absorption changes significantly. The d-d transition peaks of Fe³⁺ appear at 400–500 nm (ε ≈ 100 L·mol⁻¹·cm⁻¹) and 600–700 nm (ε ≈ 80 L·mol⁻¹·cm⁻¹), with higher intensity than those of Fe²⁺. Meanwhile, the solution color changes from the pale yellow-green of ferrous gluconate to the yellow-brown of Fe³⁺ compounds, allowing rapid assessment of product stability (whether oxidation and deterioration have occurred) via visible-region spectroscopy.
III. Nuclear Magnetic Resonance (NMR) Spectroscopy Characteristics: Analysis of the Structure and Configuration of the Gluconate Chain
Nuclear Magnetic Resonance (¹H NMR, ¹³C NMR) spectroscopy analyzes the hydrogen (¹H) and carbon (¹³C) atoms in ferrous gluconate by detecting their chemical shifts (δ), coupling constants (J), and peak integration areas. It is a core method for the detailed analysis of the molecular structure, enabling the elucidation of the carbon-hydrogen bonding pattern of the gluconate chain, its spatial configuration (e.g., the pyranose ring structure of gluconic acid), and the influence of Fe²⁺ on surrounding nuclei. (Note: Fe²⁺ is a paramagnetic ion that accelerates the relaxation of adjacent nuclei, leading to broadened NMR peaks. Testing at low temperatures (e.g., 273 K) is required to improve peak shape.)
(I) ¹H NMR Spectroscopy (Solvent: Deuterated Water D₂O, Internal Standard: TMS)
¹H NMR primarily reflects the chemical environment of different hydrogen atoms in the gluconate chain. The key peaks and their corresponding structures are as follows:
Hydroxyl hydrogen (-OH): The 5 hydroxyl hydrogen atoms in the gluconate chain (one -OH in each -CHOH- group) typically do not show peaks at room temperature due to exchange with deuterium (D) in D₂O. When tested at low temperatures (253 K), 5 weak, broad peaks can be observed in the δ 4.5–5.5 ppm range. Each peak corresponds to one -OH hydrogen, with a peak integration area ratio of 1:1:1:1:1, confirming that the gluconate chain contains 5 hydroxyl groups.
Methylene hydrogen (-CH₂OH): In the terminal -CH₂OH group of the gluconate chain, the two hydrogen atoms (Hₐ, Hᵦ) have slightly different chemical environments due to the influence of the adjacent hydroxyl group. They produce two coupled peaks (doublets or multiplets) in the δ 3.6–3.8 ppm range, with a coupling constant J ≈ 12 Hz and a peak integration area ratio of 2 (corresponding to 2 H atoms). This is a characteristic peak of the gluconate chain terminal.
Methine hydrogen (-CHOH-): In the 4 -CHOH- groups (at the C₂–C₅ positions) of the gluconate chain, each methine hydrogen (-CH-) has a different chemical shift due to its position in the carbon chain. The -CH- hydrogen at the C₂ position appears in the δ 4.2–4.4 ppm range, while those at the C₃–C₅ positions appear in the δ 3.8–4.1 ppm range, resulting in a total of 4 peaks with a peak integration area ratio of 1:1:1:1 (corresponding to 4 H atoms). The coupling constant J ≈ 8 Hz for these peaks confirms that the gluconate chain adopts a "chair conformation" (pyranose ring structure) with cis coupling between adjacent hydrogen atoms.
Influence of Fe²⁺ on hydrogen nuclei: The paramagnetism of Fe²⁺ causes a slight shift in the chemical shift of surrounding hydrogen nuclei (e.g., a 0.1–0.2 ppm downfield shift in the δ of -CHOH- hydrogen) and an increase in peak width (full width at half maximum: ~10–20 Hz). The degree of shift is positively correlated with the distance from the hydrogen nucleus to Fe²⁺—hydrogen atoms adjacent to the carboxyl group near Fe²⁺ (e.g., -CH₂- at the C₆ position) show a more significant shift, allowing indirect determination of the coordination position of Fe²⁺.
(II) ¹³C NMR Spectroscopy (Solvent: Deuterated Water D₂O, Internal Standard: TMS)
¹³C NMR reflects the chemical environment of carbon atoms in the gluconate chain, clarifying the bonding sequence of the carbon chain and the substitution positions of functional groups. The key peaks and their corresponding structures are as follows:
Carboxyl carbon (-COO⁻): The carboxyl carbon of the gluconate anion (at the C₁ position) experiences reduced electron density due to coordination with Fe²⁺, leading to a downfield shift in its chemical shift. A strong peak is observed in the δ 175–180 ppm range (the intensity of ¹³C NMR peaks is positively correlated with the number of carbons; the carboxyl carbon is a single carbon, so the peak intensity is moderate). The uncoordinated carboxyl carbon has a δ of ~178 ppm, while the coordinated carboxyl carbon has a δ of ~176 ppm. Two peaks (δ 176.5 ppm and δ 177.8 ppm) appear in this range for ferrous gluconate, confirming the presence of both coordinated and uncoordinated carboxyl carbons—consistent with the conclusions from IR spectroscopy.
Hydroxyl-substituted carbon (-CHOH-): The -CHOH- carbons in the gluconate chain (at the C₂–C₅ positions, substituted with hydroxyl groups) show a downfield shift in chemical shift due to the electronegativity of the hydroxyl group. The carbon at the C₂ position (adjacent to the carboxyl group) appears in the δ 75–78 ppm range, while those at the C₃–C₅ positions appear in the δ 70–75 ppm range, resulting in 4 broad peaks (affected by the paramagnetism of Fe²⁺). This confirms that there are 4 hydroxyl-substituted carbons in the carbon chain.
Methylene carbon (-CH₂OH): The terminal -CH₂OH carbon of the gluconate chain (at the C₆ position) has a chemical shift in the δ 62–65 ppm range. Due to its distance from the carboxyl group and Fe²⁺, it has high electron density, resulting in a relatively sharp peak—this is a characteristic peak of the gluconate chain terminal.
Confirmation of carbon chain bonding sequence: Through ¹³C-¹H Heteronuclear Multiple Bond Correlation (HMBC) spectroscopy, the coupling relationships between adjacent carbon and hydrogen atoms can be observed (e.g., coupling between C₁ and the hydrogen of C₂, and between C₂ and the hydrogen of C₃). This confirms that the gluconate chain has a linear structure: "C₁ (-COO⁻)-C₂ (-CHOH-)-C₃ (-CHOH-)-C₄ (-CHOH-)-C₅ (-CHOH-)-C₆ (-CH₂OH)" with no branched or cyclized structures, further verifying the correctness of the molecular structure.
IV. Synergistic Application and Analytical Value of the Three Spectroscopic Techniques
IR, UV-Vis, and NMR spectroscopy complement each other from different dimensions, collectively building a comprehensive understanding of the molecular structure and properties of ferrous gluconate. Their synergistic application value is mainly reflected in the following three aspects:
(I) Structural Confirmation and Purity Identification
IR spectroscopy confirms functional groups (-OH, -COO⁻, Fe-O) and coordination modes; UV-Vis spectroscopy verifies the coordination environment and oxidation state of Fe²⁺; NMR spectroscopy elucidates the carbon-hydrogen bonding pattern and configuration of the gluconate chain. Together, they fully confirm the molecular structure of ferrous gluconate and exclude interference from impurities (e.g., gluconic acid, ferrous sulfate).
For example, if the product contains unreacted gluconic acid, IR spectroscopy will show a C=O stretching peak of free carboxyl groups (-COOH) at 1720 cm⁻¹, ¹H NMR will show a carboxyl hydrogen peak at δ 12.0 ppm, and UV spectroscopy will show no Fe-O-related absorption—allowing rapid purity assessment. If Fe³⁺ impurities are present, additional absorption peaks will appear in the visible region, enabling quantitative analysis of the oxidation degree.
(II) Study of Coordination Environment and Stability
The Fe-O vibration peaks and carboxyl peak shifts in IR spectroscopy reflect the coordination strength of Fe²⁺; the d-d transition peak positions in UV-Vis spectroscopy indicate changes in the coordination number; the chemical shift shifts of hydrogen nuclei in NMR spectroscopy monitor the interaction between Fe²⁺ and ligands. Together, they enable the study of the influence of environmental factors (e.g., temperature, pH) on the coordination environment (e.g., whether Fe-O bonds break at high temperatures).
For example, under acidic conditions (pH < 3), the asymmetric stretching peak of the carboxyl group in ferrous gluconate shifts to a higher wavenumber (from 1570 to 1575 cm⁻¹) in IR spectroscopy, the d-d transition peak shifts to a shorter wavelength (from 480 to 470 nm) in UV-Vis spectroscopy, and the chemical shift of -CHOH- hydrogen shifts to an upfield position (from 4.0 to 3.9 ppm) in ¹H NMR. These changes confirm that acidic conditions weaken the coordination between Fe²⁺ and carboxyl groups, altering the coordination environment.
(III) Production Process and Quality Control
IR spectroscopy enables rapid screening of raw material purity during production (e.g., verifying the quality of gluconic acid); UV-Vis spectroscopy allows real-time monitoring of Fe²⁺ oxidation (preventing oxidative deterioration during production); NMR spectroscopy is used for structural consistency testing of finished products (ensuring uniform structure across batches). Together, they form a full-process quality control system covering "raw materials → production → finished products," ensuring product safety and efficacy.
The spectroscopic characteristics of ferrous gluconate are a direct reflection of its molecular structure and chemical environment: IR spectroscopy identifies coordination modes and characteristic groups through functional group vibrations; UV-Vis spectroscopy reflects the coordination state and oxidation degree of Fe²⁺ through electronic transitions; NMR spectroscopy elucidates the detailed structure and carbon-hydrogen bonding of the gluconate chain through nuclear spin signals. The synergistic application of these three techniques not only enables structural confirmation and purity identification of ferrous gluconate but also supports in-depth studies of its coordination environment and stability. It provides key data for production quality control, dosage form design (e.g., tablets, oral solutions), and bioavailability research, making it a core analytical tool for this class of organic iron compounds.
