Essential Basic Knowledge for Using a Spectrophotometer

Figure 1:Working Principle Of An Double Beam Spectrophotometer.Image from SMACGIWORLD

Principle

1. Selective Absorption of Light by Substances

When a beam of light strikes a substance, the interaction between the light and the substance results in reflection, scattering, absorption, or transmission. If the illuminated substance is a homogeneous solution, the scattering of light can be neglected.

• Generation of Solution Color

When a beam of white light passes through a colored solution, some wavelengths of light are absorbed by the solution while others are transmitted through it. The transmitted or reflected light stimulates the human eye, creating the perception of color. Visible light is defined as the light that can be perceived by the human eye. Different wavelengths of light in the visible spectrum exhibit different colors, thus the color of the solution is determined by the wavelength of the transmitted light. The transmitted light and absorbed light together form white light, making these two lights complementary colors.

• Nature of Light Absorption

When light shines on a substance or its solution, the molecules, atoms, or ions of the substance interact with photons. The energy of the photons is transferred to the molecules, atoms, or ions, causing them to transition from the lowest energy state (ground state) to a higher energy state (excited state). This process is called the absorption of light by the substance. The excited particles return to the ground state within approximately (10^-8) seconds and release energy in the form of heat or fluorescence. Molecules, atoms, or ions have discrete quantized energy levels and will only absorb light if the energy of the photons (hν) matches the energy difference between the ground state and the excited state. Due to structural differences, different particles have different quantized energy levels, resulting in selective absorption of light.

• Absorption Curve

The absorption curve, also known as the absorption spectrum, describes the substance's ability to absorb light of different wavelengths. By passing light of various wavelengths through a solution of fixed concentration and thickness and measuring the absorbance at each wavelength, a graph can be plotted with wavelength on the x-axis and absorbance on the y-axis, resulting in the absorption curve.

The absorbance of the same substance at different concentrations increases near the absorption peak as concentration increases, while the maximum absorption wavelength remains constant. Measuring absorbance at the maximum absorption wavelength provides the highest sensitivity. Therefore, the absorption curve is a crucial reference for selecting the measurement wavelength in spectrophotometry.

2. Basic Laws of Light Absorption

   • The Lambert-Beer Law:

   When a parallel beam of monochromatic light passes through a colored solution with a path length of (b), the solute absorbs some of the light's energy, reducing the light's intensity. The greater the concentration of the solution, the thicker the liquid layer, and the stronger the incident light, the more light is absorbed, and the greater the reduction in light intensity. This law, derived from experimental observation, underpins quantitative analysis in ultraviolet-visible spectrophotometry and other absorbance-based methods. It applies not only to solutions but also to other homogeneous, non-scattering absorbing substances.

   The physical significance of this is: when a parallel beam of monochromatic light passes through a solution of a single, homogeneous, non-scattering absorbing substance, the absorbance of the solution is directly proportional to the product of the solution's concentration and the path length of the light.

   In the equation, ε is a characteristic constant of the absorbing substance at a specific wavelength and solvent, numerically equal to the absorbance of a 1 mol/L solution of the absorbing substance in a 1 cm path length. ε is a measure of the absorbing ability of the substance; the larger the ε value, the higher the sensitivity of the method. When calculating ε from experimental results, the total concentration of the substance being measured is often used instead of the concentration of the absorbing substance, representing the apparent molar absorptivity. In a multi-component system, if there is no interaction between the various absorbing substances, the total absorbance of the system equals the sum of the absorbances of the individual components, indicating that absorbance is additive.

Main Components

Despite the variety of types and models of spectrophotometers, they all consist of the same basic components: a light source, a monochromator, an absorption cell, and a detection system.

1. Light Source

When measuring absorbance, the light source must emit a continuous spectrum within the required wavelength range, have sufficient light intensity, and maintain stability over a certain period.

For measurements in the visible light range, tungsten filament lamps are typically used. When heated to incandescence, the tungsten filament emits a continuous spectrum with wavelengths between 320 and 2500 nm. The working temperature of a tungsten filament lamp is generally between 2600 and 2870K, with a melting point of 3680K. The temperature of the tungsten filament is determined by the power supply voltage, and even slight fluctuations in the power supply voltage can significantly change the light intensity of the tungsten lamp. Therefore, a stabilized power supply must be used. For measurements in the ultraviolet range, hydrogen or deuterium lamps are commonly used as light sources, producing continuous spectra with wavelengths between 180 and 375 nm. Ideally, the light source should provide continuous radiation covering the entire UV-visible range, with relatively high intensity and minimal energy variation with wavelength. However, this is difficult to achieve in practice. Deuterium lamps have radiation intensities 2-3 times higher than hydrogen lamps and have a longer lifespan. Xenon lamps generally have higher intensity than hydrogen lamps but are less stable, with a wavelength range of 180-1000 nm, and are often used as excitation sources in fluorescence spectrophotometers.

2. Monochromator

A monochromator is a device that separates the composite light emitted by the light source into monochromatic light.

It generally consists of five parts: the entrance slit, collimator (usually a lens or concave mirror that makes the incident light into a parallel beam), dispersive element, focusing optics (usually a lens or concave mirror that projects the dispersed monochromatic light onto the exit slit), and the exit slit.

The dispersive element is the core component of the monochromator, with common dispersive elements being prisms or diffraction gratings.

Prisms are made of glass or quartz. Glass prisms have strong dispersive abilities but absorb UV light, making them suitable for analyses within the 350-820 nm wavelength range. Quartz prisms must be used in the UV range.

Diffraction gratings are characterized by uniform dispersion, linearity, and wide operational wavelength ranges, facilitating automated photometric measurements.

3. Absorption Cell

Also known as a cuvette, the absorption cell is a container for holding the sample solution, featuring two parallel, light-transmitting planes of precise thickness.

Glass absorption cells typically have a path length of 1 cm, though they can range from 0.1 to 10 cm.

Due to potential thickness variations and non-transparent materials in the absorption cell, compatibility tests should be conducted for quantitative analyses, marking the placement direction post-test. Quartz is used for measurements in the UV range.

4. Detection System

The detection system includes the detector and the display recording device.

A detector is an optoelectronic device that converts light intensity into an electrical signal for display.

Common detectors include photocells, photomultiplier tubes, and photodiode array detectors.

Photocells produce relatively large photocurrents without amplification and are used in basic spectrophotometers, though they suffer from severe fatigue effects.

Photomultiplier tubes amplify the photocurrent through secondary electron emission, with amplification factors reaching up to 10^8, making them widely used.

Photodiode array detectors enable simultaneous detection of all wavelengths, offering rapid scanning speeds that can complete scans of the 190-800 nm range within 0.1 seconds.

Display recording devices include amplifiers and result display units. Digital readout devices were adopted in the 1970s. Modern instruments feature microprocessors or external microcomputers for operational control and data processing, with screens, printers, and plotters for display and output.

Selection of Measurement Conditions

Selection of Colorimetric Reactions and Conditions

For colorimetric or photometric analysis, the target component must first be converted into a colored compound for measurement.

The reaction that converts the target component into a colored compound is called a colorimetric reaction, and the reagent forming the colored compound with the target component is called a color reagent.

• Selection of Colorimetric Reactions

Colorimetric reactions are classified into complexation reactions and redox reactions, with complexation reactions being the most common.

Principles for selection include:

• Choosing sensitive colorimetric reactions. The molar absorptivity (ε) is an important indicator of reaction sensitivity, with higher ε values indicating more sensitive reactions. Generally, ε values of 10^4-10^5 denote high sensitivity.

• Selecting color reagents with good selectivity, reacting only with the target component or a few components.

• Ensuring the color reagent has no significant absorption at the measurement wavelength. The contrast between two colored substances, or the difference in their maximum absorption wavelengths, should ideally be over 60 nm.

• The resulting colored compounds must have stable and consistent compositions.

• Selection of Colorimetric Conditions

Absorbance measurements require equilibrium in the colorimetric reaction, necessitating the control of conditions to ensure complete and stable reactions. Equilibrium constants, excess color reagent, and reaction time and temperature must be optimized to avoid side reactions and ensure accurate results.

Elimination of Interference

1. Types of Interference

In photometric analysis, coexisting ions can interfere by either being colored themselves or by forming colored compounds with the color reagent.

• Colored interfering ions.

• Interfering ions reacting with the color reagent to form stable complexes, either colored or colorless, which can deplete the color reagent or directly interfere with the measurement.

• Interfering ions reacting with the target ions to form complexes or precipitates, affecting the measurement.

2. Methods to Eliminate Interference

• Control solution acidity.

• Add masking agents to form more stable compounds with interfering ions, preventing interference.

• Use reference solutions to eliminate the effects of certain colored interfering ions.

• Select appropriate working wavelengths to avoid interference.

• Employ suitable separation methods.

Selection of Absorbance Measurement Conditions

1. Selection of Incident Light Wavelength

Based on the absorption spectrum curve, the wavelength at which the solution has maximum absorption should be selected as the incident light wavelength. For example, the cobalt complex with the color reagent has maximum absorption at 420 nm. Measuring at this wavelength could result in interference from the unreacted color reagent, reducing accuracy. Therefore, measurements should be made at 500 nm where the color reagent does not absorb, but the cobalt complex has an absorption plateau.

2. Selection of Reference Solutions

• If only the reaction product of the target substance and the color reagent absorbs light, pure solvent can be used as the reference solution.

• If the color reagent or other reagents have slight absorption, a blank solution (without the sample) should be used as the reference solution.

• If other components in the sample absorb light but do not react with the color reagent, and the color reagent does not absorb light, the sample solution can be used as the reference. If the color reagent slightly absorbs, an appropriate masking agent can be added to the sample solution to mask the target component, then the color reagent is added to create the reference solution.

3. Selection of Absorbance Reading Range

Experimental evidence shows that absorbance measurements within the 0.2-0.5 range yield the least relative error.

Two methods can adjust the absorbance of the measured solution:

• Control the concentration of the measured solution by adjusting the sample volume or changing the dilution or concentration factor.

• Choose different cuvettes; longer path length cuvettes for solutions with low absorbance, and shorter path length cuvettes for solutions with high absorbance.