Calibration Method for Ultra-micro Spectrophotometer
Release time：2025-06-09
The following is a detailed analysis covering core steps, technical key points, and precautions:
Ⅰ. Instrument Self-Calibration Function
1. Principle and Purpose
Ultramicro spectrophotometers typically have built-in self-calibration programs that initialize and calibrate the optical system using the instrument's standard parameters (such as dark current correction and light source intensity compensation).
Self-calibration quickly corrects deviations caused by electronic drift, light source aging, etc., serving as a fundamental step in daily maintenance.
Self-calibration quickly corrects deviations caused by electronic drift, light source aging, etc., serving as a fundamental step in daily maintenance.
2. Operation Steps
(1) Access the instrument menu and select the "Self-Calibration" or "Performance Verification" mode;
(2) Perform blank measurement as prompted (using air or pure water as the reference);
(3) The instrument automatically adjusts to factory-preset parameters and generates a calibration report.
(2) Perform blank measurement as prompted (using air or pure water as the reference);
(3) The instrument automatically adjusts to factory-preset parameters and generates a calibration report.
3. Limitations
Self-calibration cannot correct wavelength drift or physical changes in the optical path (such as fiber displacement), requiring regular verification with other methods.
Ⅱ. External Standard Solution Calibration
1. Application Scenarios
When the instrument is used for quantitative analysis (e.g., nucleic acid or protein concentration determination), absorbance accuracy must be verified with standard solutions.
Common standard materials include:
(1) Horseradish Peroxidase (HRP) buffer: used for calibration in the ultraviolet region (230 nm, 260 nm, 280 nm);
(2) Neutral density filters: to verify the absorbance linear range (0–4 OD);
(3) NIST-traceable standard solutions (e.g., potassium dichromate solution): for multi-wavelength cross-validation.
Common standard materials include:
(1) Horseradish Peroxidase (HRP) buffer: used for calibration in the ultraviolet region (230 nm, 260 nm, 280 nm);
(2) Neutral density filters: to verify the absorbance linear range (0–4 OD);
(3) NIST-traceable standard solutions (e.g., potassium dichromate solution): for multi-wavelength cross-validation.
2. Operation Key Points
(1) Liquid column formation: Ultramicro instruments rely on a base detection mode. Use a pipette to accurately dispense 0.3–2 μL of the standard solution to form a stable liquid column (light path: 0.05–0.5 mm);
(2) Multi-point calibration: Measure standard samples at different concentrations at the target wavelength (e.g., 260 nm for RNA), plot a standard curve, and calculate the instrument's linear correlation coefficient (R² > 0.995 is qualified);
(3) Blank control: Use the same batch of solvent (e.g., ultrapure water) as the reference to avoid interference from solvent impurities.
(2) Multi-point calibration: Measure standard samples at different concentrations at the target wavelength (e.g., 260 nm for RNA), plot a standard curve, and calculate the instrument's linear correlation coefficient (R² > 0.995 is qualified);
(3) Blank control: Use the same batch of solvent (e.g., ultrapure water) as the reference to avoid interference from solvent impurities.
Ⅲ. Wavelength Calibration
1. Necessity
Wavelength accuracy directly affects qualitative analysis (e.g., identification of characteristic absorption peaks).
The xenon lamp or LED light source of ultramicro instruments may experience wavelength drift, requiring regular calibration.
The xenon lamp or LED light source of ultramicro instruments may experience wavelength drift, requiring regular calibration.
2. Calibration Methods
(1) Mercury lamp characteristic spectral line method: Use the sharp absorption peaks of the mercury lamp at 253.6 nm, 296.7 nm, etc., and compare the wavelength displayed by the instrument with the actual peak value (deviation should be < ±1 nm);
(2) Holmium oxide solution method: Perform multi-point calibration using multiple characteristic absorption peaks of holmium glass in the visible region (e.g., 486 nm, 536 nm);
(3) Software calibration: Some models support automatic scanning of standard filters to correct wavelength drift through algorithms.
(2) Holmium oxide solution method: Perform multi-point calibration using multiple characteristic absorption peaks of holmium glass in the visible region (e.g., 486 nm, 536 nm);
(3) Software calibration: Some models support automatic scanning of standard filters to correct wavelength drift through algorithms.
Ⅳ. Stability and Repeatability Testing
1. Short-Term Stability
(1) Continuously measure the same standard sample (e.g., 1 mg/mL BSA solution) at least 6 times, and record the absorbance values (error at 280 nm should be < ±0.005 OD);
(2) Check baseline noise (typically required to be < 0.002 OD) to evaluate optical path stability.
(2) Check baseline noise (typically required to be < 0.002 OD) to evaluate optical path stability.
2. Long-Term Stability
(1) Perform a full-wavelength range (200–1000 nm) baseline scan monthly to observe stray light and wavelength repeatability;
(2) Use standard solutions for periodic verification (e.g., once a week) and record deviation trends.
(2) Use standard solutions for periodic verification (e.g., once a week) and record deviation trends.
Ⅴ. Special Precautions
1. Base Cleaning and Maintenance
(1) Wipe the base with lens paper after each use to prevent residual protein or salt crystals from affecting liquid column formation;
(2) Regularly inspect the optical fiber probe for wear to avoid light loss.
(2) Regularly inspect the optical fiber probe for wear to avoid light loss.
2. Environmental Control
(1) Maintain laboratory temperature at 20–25°C and humidity < 60% to prevent condensation from interfering with the optical system;
(2) Avoid strong electromagnetic fields (e.g., centrifuges, microwave ovens) to prevent signal noise.
(2) Avoid strong electromagnetic fields (e.g., centrifuges, microwave ovens) to prevent signal noise.
3. Traceability and Compliance
(1) Calibration standard materials must have NIST or ISO certification to ensure the reliability of quantity transfer;
(2) For clinical diagnosis or quality inspection, comply with industry standards (e.g., CLSI standards or Pharmacopoeia requirements).
(2) For clinical diagnosis or quality inspection, comply with industry standards (e.g., CLSI standards or Pharmacopoeia requirements).
Key Technical Terms Note:
- OD: Optical Density
- NIST: National Institute of Standards and Technology
- CLSI: Clinical and Laboratory Standards Institute
