Multifaceted Spectrophotometric Analysis of Vitamins and Vitamin-like Substances

Significance of the Topic

Vitamins and vitamin-like compounds play essential roles in human health, yet they cannot all be synthesized by the body and must be obtained through diet or supplements. Reliable analytical methods are needed to identify, quantify, and characterize these molecules in complex matrices such as food, pharmaceuticals, and biological fluids. Combining multiple spectrophotometric techniques enhances confidence in identification, provides complementary information on molecular structure and behavior, and supports routine quality control and research applications.

Objectives and Study Overview

This study evaluates a suite of spectrophotometric instruments for comprehensive analysis of water- and fat-soluble vitamins and related compounds. Key goals include:

• Determining UV-visible absorption characteristics across solvents for a panel of vitamins.
• Assessing fluorescent properties of selected compounds under varied excitation wavelengths.
• Acquiring infrared and Raman spectra for solid and powdered samples using FTIR and Raman microscopy.
• Comparing data with library references to demonstrate identification capability.

Methodology and Instrumentation

Sample Preparation:

• Water-soluble vitamins were dissolved in purified water or 0.1 M NaOH.
• Fat-soluble vitamins were dissolved in ethanol or chloroform.
• Concentrations were adjusted to achieve absorbance ≤1 Abs at target wavelengths; fluorescence samples were further diluted.

Measurement Techniques:

• UV-Visible Spectrophotometry (200–800 nm) for absorption peak determination and quantification.
• Spectrofluorophotometry for detection of emission maxima under specific excitation wavelengths.
• Fourier Transform Infrared Spectroscopy (FTIR, ATR mode) for functional group analysis in solids and powders.
• Infrared Raman Microscopy for complementary vibrational data using 532 nm and 785 nm excitation.

Main Results and Discussion

UV-Visible Absorption:

• Distinct absorption peaks were observed for each vitamin: e.g., vitamin A at 332 nm, B2 at multiple bands (270/356/449 nm), and vitamin K at 248/270/330 nm.
• Vitamin B12 and nicotinic acid showed peaks outside the traditional UV range, highlighting the need for broader spectral coverage.

Fluorescence Characteristics:

• Fluorescent signals varied by compound and excitation: riboflavin emitted around 440 nm when excited at 356 nm, while folic acid showed emissions at 350 nm and 458 nm.
• Some vitamins (A, E, K) exhibited unstable fluorescence intensities, suggesting photobleaching or structural changes under irradiation.

Infrared and Raman Spectra:

• FTIR spectra provided clear identification of characteristic functional groups, and ATR sampling enabled direct measurement of powders and films.
• Raman information complemented FTIR, though strong fluorescence in some powders (e.g., vitamin B2) increased baseline noise.
• Most spectra matched library data, facilitating rapid qualitative analysis.

Benefits and Practical Applications

Combining UV-visible, fluorescence, FTIR, and Raman techniques delivers a robust workflow for:

• Simultaneous screening of multiple vitamins in food and nutraceutical formulations.
• Confirmation of compound identity in quality control and regulatory compliance.
• Detection of trace or minor components based on fluorescence sensitivity.
• Non-destructive analysis of solid and powdered samples without extensive preparation.

Future Trends and Applications

Advances likely to enhance vitamin analysis include:

• Integration of chemometric tools and machine learning to automate spectral interpretation and quantification.
• Development of portable, field-deployable spectroscopic systems for on-site testing in agriculture and food production.
• Expansion of spectral libraries with higher resolution and broader compound coverage.
• Combination with hyphenated techniques (e.g., LC-MS) for increased specificity in complex matrices.

Conclusion

This comparative study demonstrates that a multifaceted spectrophotometric approach offers comprehensive insight into the optical and structural properties of vitamins and related substances. UV-vis methods enable rapid quantification, fluorescence highlights trace detection, and FTIR/Raman provide definitive structural identifiers. Implementing this integrated platform supports efficient, reliable analysis across research, industrial, and quality control settings.

Instrumentation

• UV-2600i Plus (UV-Vis absorption, 200–800 nm)
• RF-6000 Spectrofluorophotometer (UV-visible excitation and emission scanning)
• IRSpirit-TX with QATR-S ATR accessory (FTIR, 4 cm⁻¹ resolution)
• IRTracer-100 + AIRsight Raman Microscope (532 nm & 785 nm excitation)

References

1. Analytical Solutions for Food Development. Shimadzu Application News.
2. Difference in Quantifiable Concentration Ranges of UV-Vis Spectrophotometer and Fluorescence Spectrophotometer. Application News No. A615.