1、 Instrument performance itself: the "basic hardware" of accuracy

The core components and design of the instrument directly determine the measurement lower limit and are the fundamental factors affecting accuracy, mainly including the following aspects:

The core principle of the optical system precision chlorophyll analyzer is the "absorption/transmission difference of specific wavelength light" (chlorophyll has strong absorption of 660nm red light and weak absorption of 940nm near-infrared light, and the content is calculated by the ratio of the two). The deviation of the optical system will directly cause data distortion:

Light source stability: If the LED light source (660nm/940nm) has unstable luminous intensity (such as aging, voltage fluctuations), it will cause the light signal reference of the two measurements to be different, resulting in the problem of "large differences in multiple measurements of the same sample";

Filter purity: If the filter cannot accurately filter out stray light (such as mixing with other wavelengths of light), it will interfere with the light signal of the target wavelength, resulting in absorption value calculation deviation (for example, if stray light is mixed into the 660nm channel, it will overestimate the absorption of chlorophyll and make the result biased);

Detector sensitivity: If the sensitivity of a photodetector (such as a photodiode) is low or there is a "dark current" (weak current in the absence of light), it will not be able to accurately capture weak transmitted light signals, especially for samples with extremely low chlorophyll content (such as yellowed leaves), and the error will be significantly amplified.

The calibration status instrument needs to establish a measurement benchmark through a "standard calibration piece" (a simulated sample with known chlorophyll content). If the calibration is not timely or the calibration method is incorrect, it will cause overall data deviation:

Unregulated calibration: After long-term use, light source attenuation and detector aging can cause systematic overestimation or underestimation of measurement values (such as a sample with an original chlorophyll content of 50SPAD, which may display as 45 or 55 without calibration);

Calibration operation error: If there are stains on the surface of the calibration piece or it is not fully attached to the measurement window, it will cause the calibration reference to be incorrect, and all subsequent sample measurements will be affected.

Structural design defects

Measurement of window sealing: If the window (usually quartz glass) is not in close contact with the sample, it can cause ambient light (such as natural light, light) to enter the measurement area and interfere with the light signal (such as strong light entering will cause the transmitted light signal to be biased and the calculated chlorophyll content to be lower);

Consistency of sample clamping pressure: Some instruments measure by clamping blades. If the clamping pressure is uneven (such as spring aging), it will result in different degrees of compression of blade thickness - blade thickness affects the transmittance of light (thicker blades transmit less light). Inconsistent pressure will cause large differences in the transmitted light signal of "samples of the same thickness", thereby increasing the error.

2、 Sample state: "Variable source" of precision

The inherent characteristics of plant samples can lead to "non chlorophyll related differences" in light signals, which interfere with measurement results, mainly including:

Physical characteristics of leaves

Thickness and uniformity: The chlorophyll analyzer defaults to "uniform leaf thickness and no impurities". If there is uneven thickness (such as thin leaf edges, thick middle), wrinkles, or wormholes in the leaves, it will cause abnormal local transmission of light (such as direct light penetration at the wormhole, with an absorption value of 0, making the measured value at that point much lower than the actual value);

Surface attachments: Dust, dew, and pesticide residues on the surface of leaves can reflect or absorb some light signals (such as red light reflected by dust, resulting in a decrease in the transmitted light received by the detector and a misjudgment of high chlorophyll content), especially when the sample is not cleaned after field measurement, the error is more significant.

Leaf physiological state

Uneven distribution of chlorophyll: In some plants (such as corn), the chlorophyll content near the midrib is higher than that near the edge of the leaf. If the midrib is not avoided during measurement or the measurement position is different each time, it can lead to "large differences in multiple measurements of the same leaf";

Freshness of samples: If the harvested leaves are left for too long (such as more than 30 minutes), it will cause changes in cell structure due to water loss, affecting light transmittance (such as increased light scattering caused by curled leaves), and chlorophyll may be slightly degraded, resulting in measured values lower than the actual live state.

The leaf structure (such as stratum corneum thickness and pigment composition) of different plant varieties varies depending on their variety and growth stage. For example, succulent plants have a thick stratum corneum that absorbs additional red light. If the instrument does not adjust parameters for this variety (such as correcting the light absorption coefficient of the stratum corneum), it will result in systematically higher measurement values; In addition, the pigment composition of young and old leaves of the same plant is different (the proportion of carotenoids in old leaves may increase), and carotenoids have a small absorption of 660nm light, which can interfere with the calculation of chlorophyll.

3、 Operation method: "Human control factor" of accuracy

Correct operation is the key to avoiding "non systematic errors", and common operational errors have an impact on accuracy, including:

Improper selection of measurement location did not follow the principle of "avoiding the midrib and selecting a uniform area in the middle of the leaf": for example, aligning the probe with the midrib of the leaf during measurement (the midrib has no chlorophyll, mainly contains cellulose, and transmits light intensity) can result in measurement values that are much lower than the actual values (such as an actual 50SPAD, the measurement value may only be 20); Or there may be significant differences in measurement positions each time (such as measuring the middle or edge once), resulting in poor accuracy in repeated measurements.

Sample placement and probe pressing method

The sample does not fully cover the measurement window: If the leaf area is small (such as seedling leaves) and the window is not completely blocked, it will cause ambient light to enter, resulting in a larger transmitted light signal and a lower calculated chlorophyll value;

Uneven pressure of the probe: For some manual pressing instruments, if the pressure is too light (due to loose contact between the window and the blade, light leakage) or too heavy (due to blade crushing, cell fluid leakage, and changes in light transmission), it can cause abnormal light signals.

Data recording and processing errors

Not conducting repeated measurements: Single measurements are easily affected by accidental factors (such as small wormholes at a certain point on the leaf). If the standard of "taking the average of three repeated measurements of the same sample" is not followed, it will result in large random errors in the data;

Outliers not excluded: For example, if the results of three measurements are 48, 50, and 22 (22 is an outlier due to misalignment with the midrib during measurement), taking the average directly (40) will significantly deviate from the actual value (about 49).