Overview
Anti-counterfeiting fluorescent ink is a functional material widely used in licenses, bills, packaging and other fields, and its key feature is that it produces visible fluorescence under specific wavelength excitation. Fluorescence spectrophotometers can accurately determine the excitation and emission spectra of inks, providing a reliable basis for quality control and formulation optimization. This article systematically introduces the operation process, parameter setting and data processing points of the instrument in the spectroscopy of anti-counterfeiting fluorescent inks, helping laboratory personnel to obtain stable and reproducible measurement results.
Instrument preparation
Before measurement, it is necessary to ensure that the fluorescence spectrophotometer is in a stable working state. First, the light source and electronic system are turned on to warm up for at least 30 minutes to achieve thermal equilibrium between the xenon lamp and the detector. Wavelength calibration and intensity calibration were performed using standard fluorescent substances such as quinine sulfate solution. Calibration frequency is recommended once a week, or when the instrument is turned off and re-enabled. The calibration solution was loaded into a quartz cuvette with a 1 cm optical path, and the excitation and emission peak positions were recorded, and the deviation should be within ±1 nm. Check the consistency of the slit width, usually with an excitation and emission slit set to 5 nm or 10 nm, which increases signal strength but reduces resolution.
Sample preparation
The anti-counterfeiting fluorescent ink sample should be evenly dispersed on the transparent carrier. Common methods include: diluting the ink with an inert diluent (such as cyclohexane) to a suitable concentration, then coating it on the surface of the quartz sheet, and drying it to form a film; Or press the ink sample directly into the solid sample cell. The sample concentration is adjusted to a stable fluorescence signal with no self-absorption effect, and the dilution factor is generally 100 to 1000 times. Make sure the sample surface is perpendicular to the excitation path to avoid scattered light entering the detector. For powdered inks, a special solid sample holder is used to fill the powder into a quartz cup to compact it with a flat surface.
Excitation spectroscopy assay method
The goal of excitation spectroscopy is to find the optimal excitation wavelength that will cause the ink to fluoresce the strongest. The procedure is as follows: Fix the emission wavelength over the typical emission range of the ink (e.g., 530 nm) and set the excitation wavelength scan range from 250 nm to 500 nm, in steps of 1 nm or 2 nm. Choose a medium scan speed, such as 200 nm/min, to balance resolution and measurement speed. The fluctuation curve of fluorescence intensity with excitation wavelength was recorded, and the highest peak corresponded to the peak wavelength of the excitation spectrum. Note the deduction of blank substrate signals (blank quartz sheets without ink). Excitation spectra typically show 1 to 3 distinct absorption bands, and the main peak position can be used for subsequent emission spectroscopy.
Emission spectroscopy assay
The emission spectrum reflects the fluorescence distribution produced by the ink at a fixed excitation wavelength. Set the excitation wavelength to the peak wavelength obtained in the excitation spectrum (e.g., 365 nm), and set the emission wavelength sweep range from +10 nm to 700 nm in 1 nm steps. Reduce integration time to 0.1 or 0.2 seconds to increase scan rate. The emission spectrum often presents one or two fluorescence peaks, and the peak position and half-peak width are used to identify fluorescent dye species. The fluorescence emission main peak wavelength, secondary peak wavelength, relative value of fluorescence intensity and peak shape symmetry of the ink were recorded. Peak drift or multi-peaking may indicate batch differences or aging issues in the ink formulation.
Data processing
Extract key parameters from spectral data. First, the peak wavelength and peak intensity of the excitation and emission spectra are identified. The difference between the fluorescence intensity value and the blank substrate intensity value is calculated as the net fluorescence intensity. Determine the half-peak full width (FWHM) of the excitation and emission spectra, which reflects fluorescence uniformity. Comparing the differences in spectral characteristics of different batches of inks, it is recommended to repeat the measurement at least 3 times to obtain the average. Draw a table to record the following core parameters:
| Parameter categories | Specific values (example) |
| Excitation peak wavelength | 365 nm |
| Excites peak intensity | 4650 Count |
| Emission peak wavelength | 530 nm |
| Peak emission intensity | 8720 Count |
| Launch half-peak full width | 55 nm |
For quantitative analysis, the working curve of fluorescence intensity versus ink concentration can be established. The emission spectra of the known concentration ink dilution series were determined from low concentration to high concentration, and a linear regression line was drawn for intensity to concentration, and the correlation coefficient R² should be greater than 0.995. The fluorescence intensity of the measured unknown sample was calculated by substituting the regression equation.
Spectral interference and correction strategies
Common interferences in fluorescence spectrophotometer assays include Raman scattering, Rayleigh scattering, and fluorescence quenching. Rayleigh scattering occurs at the excitation wavelength and can be suppressed by adding a cut-off filter to the front end of the emitting monochromator. Raman scattering behaves as weaker peaks with an excitation wavelength redshift of about 10-50 nm (e.g., Raman peaks of about 410 nm for water at 365 nm excitation), which can be corrected by subtracting the blank solution spectrum. Fluorescence quenching can be caused by oxygen, heavy metal ions, or high concentrations of the sample itself, and it is necessary to maintain a stable environment and control the sample concentration.
Another common interference is the second-order diffraction effect. When the spacing between the excitation spectrum and the emission spectrum is too large, long-wave detection may capture the second harmonics of the excitation light. The use of a long-pass filter or adjustment of the slit width can effectively reduce this phenomenon. The method of checking for interference is to scan the same wavelength range and record the background signal under unreleased samples.
Typical applications:
The excitation spectrum of anti-counterfeiting fluorescent inks is typically located in the ultraviolet region (300-400 nm), and the emission spectrum ranges from visible blue to green (450-550 nm). There may be a deviation of ±5 nm in the spectral position of inks from different manufacturers, and it is recommended to establish spectral files for each batch of products. For multi-component anti-counterfeiting inks (such as mixing two fluorescent dyes), multiple peaks may appear in the excitation spectrum, and each excitation peak should be set to the corresponding emission spectrum for excitation wavelength determination to determine the independent fluorescence characteristics of each dye.
Avoid photobleaching effect during operation: too long irradiation time can lead to attenuation of fluorescence intensity, so the time of a single scan should be controlled within 1 minute, and repeated continuous measurements of the same sample should not be repeatedly measured. The sample chamber was shaded and the temperature was stable at 22±2 °C. Wipe the cuvette with analytical grade ethanol immediately after measurement to avoid residual ink contamination of subsequent samples. Regularly use purified water to verify system baseline drift, recalibration is required if drift exceeds 2%.
Quote reference
1. National Standardization Administration of China. Verification procedure of fluorescence spectrophotometer. 2018 Revision, Section on Calibration Methods for Wavelength Accuracy and Spectral Bandwidth.
2. Advances in fluorescence spectroscopy. Analytical Testing Technology and Instruments, No. 4, 2020, Discussion on Excitation-Emission Matrix Method for Anti-counterfeiting Ink Fluorescence Detection.
3. Properties and applications of organic fluorescent dyes. Functional Materials, Volume 28, 2021, Recommendations for Photostability Testing of Ink Plates.