Applications | ATF4500 3D Fluorescence Fingerprinting of Birch Sap

A bottle of birch sap, moving from a niche beverage in the forests of Northeast China to mass-market shelves, can vary in price by several times. The ratios of active components such as amino acids, polyphenols, and flavonoids naturally change with origin, brand, and harvest period—and the room for dilution with water or passing off inferior products hides within these variations. To verify this bottle of "water" clearly, the conventional approach is to separate and purify the components and then run each item on instruments—one sample can take a whole day. This time, our application engineers used the ATF4500 three-dimensional fluorescence spectrophotometer to directly test a pure water reference and several birch sap samples. What we wanted to see was another path: without any pretreatment, directly reading the overall fluorescence fingerprint—can it work for birch sap?

01  Where Quality Evaluation of Birch Sap Gets Stuck

Birch sap is a product that "speaks through its raw material." The spring sap collection period lasts only a few weeks, the freshness window is short, and active components fluctuate with origin, tree age, and harvest time. To clarify quality, there are three main bottlenecks:

★ Long process: Amino acids, polyphenols, and sugars must be measured separately. Samples are first separated and purified, then run item by item—from sampling to results often takes days.

★ High cost: Chromatographic methods provide reliable data, but equipment, consumables, and dedicated personnel cannot be avoided—making batch-by-batch testing unsustainable.

★ Only one number: Meeting physicochemical specifications does not mean the composition is the same. Two batches of sap that both "meet standards"—the diluted batch may not reveal itself in total quantity indicators.

Put these three together, and the demand is actually quite simple: no pretreatment, fast results, and information that reflects the overall composition rather than a single isolated number.

02  Principle: Why Birch Sap Naturally Has a "Fluorescence Fingerprint"

1. Where Fluorescence Comes From

A molecule absorbs excitation light and transitions to an excited state; it first loses some energy through vibrational relaxation (on the picosecond scale), then returns to the ground state by emitting a photon. The emitted photon has lower energy than the excitation photon, so the emission wavelength is longer than the excitation wavelength—this is the Stokes shift. Peak position and intensity are determined by the molecule's conjugated structure and substituents, and are also affected by the polarity, pH, temperature, and quenchers of the microenvironment. Therefore, in a cup of a complex natural beverage, fluorescence is rarely the signal of just one substance—it is often emitted together by many molecules with conjugated structures.

2. Who Is Emitting Light in Birch Sap?

Tyrosine and tryptophan among free amino acids have typical "protein-like" fluorescence in the ultraviolet region; polyphenols, flavonoids and their glycosides, as well as oxidative condensation products of sugars and polyphenols, each have characteristic peaks from the ultraviolet to visible region. These components require no derivatization, no labeling, and no color development—they can be measured directly upon sampling. Their proportions change with origin, harvest period, and processing technology, and the fluorescence fingerprint changes accordingly.

3. What Makes 3D Fluorescence "3D"?

Conventional fluorescence analysis fixes one excitation wavelength and scans only one emission curve—seeing only one slice of the sample. In birch sap, fluorescent components overlap, pressing against each other on a single curve, leaving very little usable information. Three-dimensional fluorescence (EEM) continuously changes the excitation wavelength in steps, collects emission spectra one by one, and finally obtains a data matrix of excitation wavelength × emission wavelength × fluorescence intensity, plotted as contour maps. One scan captures the fluorescence information of the entire system. Interpretation looks at three things:

★ Peak position (λex/λem): roughly determine which class of components it belongs to.

★ Number and shape of peaks: see how complex the system is and how the peaks overlap.

★ Intensity distribution: under low concentration and low absorbance conditions, positively correlated with concentration—allowing semi-quantitative comparison.

4. First Identify Two Lines That Are "Not Fluorescence"

The bright band along the diagonal on the spectrum is first-order Rayleigh scattering: excitation light scattered by solvent molecules directly enters the detection optical path, with emission wavelength almost the same as excitation wavelength. Where the emission wavelength reaches twice the excitation wavelength, there is also a second-order scattering band, appearing in the lower right of the plot. Neither is fluorescence and must be stripped away during interpretation. This is also why a pure water reference was included in the case: the blank system has only scattering and no fluorescence peaks—anything extra on the sample plot is the sample's own signal. In addition, for samples with dark color and high concentration, pay attention to the inner filter effect—dilute before measurement so that peak intensities are reliable.

03  ATF4500 Three-Dimensional Fluorescence Spectrophotometer

The instrument used in this test is the Optosky ATF4500 three-dimensional fluorescence spectrophotometer. The light source is a 150 W long-life xenon lamp with a nominal life of about 3,000 hours, extending the lamp replacement cycle and reducing the burden of consumables and maintenance. The detector is a wide-range high-sensitivity photomultiplier tube (PMT), and the sample compartment is large enough to accommodate both liquid and solid samples. Optional accessories include a solid sample holder, micro sample cell, polarization accessory, water bath thermostatic holder, low-temperature testing accessory, and integrating sphere assembly. The software connects via USB for spectrum acquisition and data processing; the interface is not complicated, and new users can basically run samples independently after one round of training.

ATF4500 / ATF4500-17 Performance Parameters

04  Real Testing: From Blank Reference to Two Samples

Instrument: ATF4500 three-dimensional fluorescence spectrophotometer

Samples: Pure water reference + multiple birch sap samples (two shown in this article)

Method: No extraction, separation, or other pretreatment—direct acquisition of 3D fluorescence spectra and contour maps.

First, look at the reference. The pure water spectrum shows only two scattering bands: first-order Rayleigh scattering on the diagonal, and second-order scattering at an emission wavelength approximately twice the excitation wavelength. No fluorescence peaks can be found in the entire plot. The system is clean—any extra signal on the sample plots can only come from the samples themselves.

Pure water reference 3D fluorescence spectrum (only first/second-order scattering bands visible)

Now look at the two sap samples. Fluorescence mainly falls in two regions: the main peak is at excitation approximately 250–350 nm and emission approximately 380–480 nm, originating from polyphenols, flavonoids, and their oxidation products. Above and to the right of the main peak, between excitation approximately 420–490 nm and emission approximately 480–560 nm, there is another weaker fluorescence region, corresponding to visible humic substances, flavonoids, phenolic acids, and oxidized tannins. On the shorter excitation wavelength side, protein-like fluorescence can also be seen, along with signals from polyphenol oxides and polyphenol polymers. The difference lies in intensity and composition: Sample 1 has a full main peak, with clear signals in both the protein-like and polyphenol polymer regions; Sample 2 has similar peak positions but noticeably lower intensity, with the polyphenol polymer region essentially flat.
 

Sample 1 birch sap 3D fluorescence spectrum (with main fluorescence region annotations)
 

Sample 2 birch sap 3D fluorescence spectrum

Switching to contour maps, the magnitude difference is directly visible in the readings: the scattering bands of the pure water reference are around 3 × 10⁻³ (a.u.), while the main peaks of Sample 1 and Sample 2 are approximately 3.9 × 10⁻⁴ and 2.1 × 10⁻⁴, respectively—7–12 times higher than the blank, and the two samples differ by about 1.8 times. The normal fluctuation between parallel samples cannot explain such a large difference. Two bottles of sap that look indistinguishable side by side actually differ considerably in composition.
 

Pure water reference 3D fluorescence contour map

Sample 1 3D fluorescence contour map

Sample 2 3D fluorescence contour map.

05  Putting the Fingerprint to Use: Three Steps for Batch Monitoring

Being able to see differences in spectra and using them for quality control are two different things. To implement, the maps must be turned into actionable rules—generally in three steps:

★ Step 1: Build a fingerprint library. Continuously sample qualified batches and record the positions, numbers, and intensity ratios of main and secondary peaks to form a fingerprint interval for a brand or origin.

★ Step 2: Set criteria. Use characteristic parameters such as main peak intensity and protein-like/humic-like intensity ratio to set thresholds. After scanning a new batch, compare it with the library fingerprint; if it exceeds the threshold, mark it as abnormal.

★ Step 3: Tiered handling. Send marked abnormal samples for HPLC or mass spectrometry confirmation—expensive methods are used only on a small number of suspicious samples.

Once enough fingerprint library samples are accumulated, parallel factor analysis (PARAFAC) can be used to mathematically separate overlapping peaks for component resolution, further estimating the proportion of dilution or adulteration. Screening first, then testing—this is how testing costs can be kept under control.

06   Final Remarks

Whether the sap is good or not cannot be seen on the label, but it can be seen on the spectrum. Three-dimensional fluorescence cannot provide confirmation—that still belongs to chromatography and mass spectrometry. Its value lies in screening: one map takes a few minutes, first picking out the batches that deserve close attention. Beyond birch sap, the ATF4500 is also used in environmental water quality, biomedicine, materials, petrochemicals, agricultural soils, and other fields.

For more information, please contact:

Email: [email protected]

Web: www.optosky.net