1. What Is a Spectrophotometer?
A spectrophotometer is an instrument used to measure absorbance values using the principle of light absorption, specifically using ultraviolet light. It is a combination of two devices: a spectrometer and a photometer./ lemari asam
2. What Are a Spectrometer and a Photometer?
A spectrometer produces light from a spectrum at specific wavelengths, while a photometer measures the intensity of light that is either transmitted or absorbed.
3. Who Invented the First Spectrophotometer?
The spectrophotometer’s origins date back to the 19th century, specifically in 1802, when scientist William Hyde Wollaston discovered black lines in the solar spectrum, contradicting the previous belief that solar spectra were composed of continuous color gradients. This discovery was later confirmed by Josef von Fraunhofer in 1814. Their findings showed that the black lines were due to the absorption of certain chemical elements in the sun.
In 1905, an instrument resembling a tube, known as a simple spectrometer, was invented, paving the way for the modern spectrophotometer.
4. What Is the Function of a Spectrophotometer?
The primary function of a spectrophotometer is to identify the absorbance values of a sample using ultraviolet light. It can measure absorbance values both qualitatively and quantitatively by comparing them to a positive control. Qualitative testing is based on shape and color, while quantitative testing provides numerical values, such as absorbance levels.
5. How Does a Spectrophotometer Work?
The working principle of a spectrophotometer is based on light dispersion, which involves splitting light into different color spectra using a prism mirror. It also follows Lambert-Beer’s Law, which relates the absorption of light to the concentration of the sample.
6. What Are the Types of Spectrophotometers?
There are several types of spectrophotometers, including:
UV-Vis Spectrophotometer: Uses both UV and visible light sources to measure absorbance.
Infrared Spectrophotometer (FTIR): Observes electromagnetic radiation in the wavelength range of 0.75 – 1,000 µm.
Atomic Absorption Spectrophotometer (AAS): Determines metal elements and was first developed in 1955.
Nuclear Magnetic Resonance (NMR) Spectrophotometer: Analyzes molecular structures and was developed by Edward Purcell and Felix Bloch.
Molecular Fluorescence/Phosphorescence Spectrophotometer: Measures radiation absorption with higher wavelength values.
7. How to Use a Spectrophotometer?
- Ensure the light source functions properly.
- Align the spectrophotometer with the light source.
- Calibrate the spectrometer.
- Place the prism mirror to measure the refractive index.
- Adjust the telescope to view spectral lines at each wavelength.
- Record the dispersion angles of the spectrum.
- Calculate the refractive index and wavelength.
8. What Samples Can Be Tested with a Spectrophotometer?
Samples such as plant extracts, chemical solutions, and various medications can be tested.
9. What Are the Terms Used in Spectrophotometry?
Absorbance: The amount of light absorbed by the solution.
Transmittance: The amount of light passing through the solution.
Cuvette: A container for holding the sample solution.
Drive Cell: The holder for the cuvette.
Blank: A reference sample to correct for light reflected by the cuvette and absorbed by other sample components.
10. How to Maintain a Spectrophotometer?
- Warm up the instrument for 15-20 minutes.
- Avoid direct sunlight exposure.
- Store in a stable temperature environment on a permanent table.
- Keep cuvettes clean and dry.
- Regularly calibrate wavelength and absorbance.
11. What Is the Difference Between Single Beam and Double Beam Spectrophotometers?
Single Beam: Used for quantitative measurements with a simpler process and lower cost. It has a wavelength range of 190 to 1,000 nm.
Double Beam: Has two beams; one passes through the blank, the other through the sample, covering a wavelength range from 190 to 750 nm.
12. How to Calibrate a Spectrophotometer?
Wavelength Calibration: Use holmium oxide glass filters and compare the obtained spectrum with reference data.
Absorbance Calibration: Prepare potassium dichromate solutions in sulfuric acid and compare results with reference samples.
13. What Is the Price Range of a Spectrophotometer?
As of this year, the price range for spectrophotometers is between 30 million and 300 million IDR, depending on the brand.