Light and Color Principles

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Questions and Answers

Which material should be used for UV light spectrophotometry measurements?

Ceramic

Plastic

Quartz (correct)

Glass

What should be done if scratches or marks are found on the optical windows of a cuvette?

Mark it for future use

Clean them with a soft cloth

Replace it immediately (correct)

Continue using it unless performance drops

What is the typical path length of cuvettes used in spectrophotometry?

1 cm (correct)

5 cm

2 cm

0.5 cm

What component in a spectrophotometer converts light into an electrical signal?

Detector (photocell) Signup and view all the answers

Which wavelength setting should be used first when measuring green dye absorbance in the provided procedure?

400 nm Signup and view all the answers

What is the primary purpose of using calibration standards in absorbance spectrophotometry?

To determine known concentrations for instrument calibration Signup and view all the answers

What component of white light allows it to be separated into a visible spectrum?

Wavelengths are refracted Signup and view all the answers

How is the concentration of an unknown solution determined in absorbance spectrophotometry?

By comparing its absorbance to that of a known standard at the same wavelength Signup and view all the answers

Which range of wavelengths is considered the visible light spectrum?

400 – 750 nm Signup and view all the answers

What does transmittance measure in relation to light?

The fraction of light that is transmitted through a sample Signup and view all the answers

How is absorbance calculated from transmittance?

A = -log(T) Signup and view all the answers

What is the relationship between the intensity of color and the concentration of a solute in a solution?

Intensity of color is directly proportional to concentration Signup and view all the answers

According to Beer-Lambert Law, what relationship exists between absorbance and concentration?

Absorbance is directly proportional to concentration Signup and view all the answers

What type of energy is utilized in absorbance spectrophotometry to measure concentrations?

Light energy Signup and view all the answers

Which of the following best describes the role of a spectrophotometer in clinical laboratories?

It quantifies the amount of light absorbed by a sample Signup and view all the answers

When white light passes through a colored solution, which wavelengths are primarily affected?

Some wavelengths are transmitted while others are absorbed Signup and view all the answers

Which statement is true regarding transmittance and absorbance?

Transmittance decreases as absorbance increases. Signup and view all the answers

In the context of absorbance spectrophotometry, which statement about Beer’s Law is correct?

It indicates that absorbance is directly proportional to concentration Signup and view all the answers

What does Beer’s Law describe in relation to absorbance spectrophotometry?

The relationship between absorbance and the concentration of a solute. Signup and view all the answers

What is the role of the monochromator in a spectrophotometer?

To split white light into separate colors or wavelengths. Signup and view all the answers

If the intensity of incident light (Io) is 0.49 W/m² and the transmitted light (It) is 0.30 W/m², what is the transmittance?

0.6122 Signup and view all the answers

Which analytical technique is NOT part of the methodologies used in clinical laboratories mentioned?

Transpiration Signup and view all the answers

Which component of a spectrophotometer is used to hold the sample solution?

Sample Cell Signup and view all the answers

What is the implication of a high absorbance value?

The solution absorbs a large amount of light Signup and view all the answers

Which of the following factors does NOT influence the quantitative measurement of absorbance?

The specific color of the solvent. Signup and view all the answers

What is a limitation of absorbance spectrophotometry?

The solution must be colored to determine concentration. Signup and view all the answers

Which lamp is commonly used for visible light analysis in a spectrophotometer?

Tungsten-Halogen lamp Signup and view all the answers

In absorbance spectrophotometry, which variable is NOT represented in Beer’s Law equation A = ε b C?

Temperature (T) Signup and view all the answers

What does the absorbance (A) in Beer’s Law indicate when it is higher?

Higher concentration of the solute. Signup and view all the answers

What is the maximum absorbance wavelength for the green dye?

630 nm Signup and view all the answers

At what wavelength does the green dye have the highest absorbance?

630 nm Signup and view all the answers

Which wavelength corresponds to the max transmittance for the green dye?

520 nm Signup and view all the answers

What color does the green dye most likely transmit at its maximum transmittance wavelength?

Green Signup and view all the answers

What color of the visible spectrum corresponds to a wavelength of 630 nm?

Red Signup and view all the answers

Which of the following wavelengths yields the lowest absorbance for the green dye?

500 nm Signup and view all the answers

Which wavelength has a higher absorbance: 580 nm or 590 nm?

590 nm Signup and view all the answers

What is the approximate absorbance value at 450 nm for the green dye?

0.418 Signup and view all the answers

Study Notes

White Light

White light is a combination of different wavelengths of light known as polychromatic light
When white light passes through a prism, filter or diffraction grating it can be broken down into a visible spectrum
Infrared wavelengths are not visible to the human eye. These are below the visible light spectrum 750 nm
Visible light ranges between 400 - 750 nm

Transmitted or Absorbed

When light strikes an object, some wavelengths are transmitted and some are absorbed
The transmitted or reflected light determines the color we see

Colored Solutions & Light

When white light is shone at a colored solution, certain energies of light are transmitted, and other energies are absorbed.
Transmittance is the fraction of light that passes through a sample
Absorbance is the fraction of light absorbed by the sample
Absorbance is calculated by -log (T). Where T = It/Io.
Io = Intensity of incident light
It = Intensity of transmitted light
T = Transmittance
A = Absorbance

Transmittance & Absorbance

Transmittance decreases with increasing absorbance
Absorbance decreases with increasing transmittance

Beer-Lambert Law

States that there is a linear relationship between absorbance and the concentration of a solution
The concentration of a substance in a solution is directly proportional to the amount of light absorbed, or inversely proportional to the logarithm of the transmitted light.
Beer Lambert law equation: A = ε b C
A = Absorbance
ε = Molar absorptivity coefficient
b = Path length
C = Concentration of the solute

Clinical Methodologies

Quantitative techniques are used to measure the concentration of unknown solutions
The methodologies used in analytical instruments are based on traditional technologies like photometry, spectrophotometry, ion-selective electrodes, electrophoresis, nephelometry, immunoassays, mass spectrometry, fluorescence, and molecular diagnostic techniques.

Absorbance Spectrophotometry

The process of measuring the concentration of an unknown solution using the properties of light energy (electromagnetic spectrum)
The concentration of an unknown solution can be determined by comparing its absorbance to a known standard
The intensity of the color is directly proportional to the concentration of the substance.

Calibration Standards

Standards are chemicals that contain a known concentration of an analyte.
They provide a reference to determine unknown concentrations or calibrate analytical instruments.

Wavelength

Every chemical compound absorbs, transmits, or reflects light of a specific wavelength or range of wavelengths
Light visible to the human eye is referred to as the visible light spectrum
The visible light spectrum ranges from 400 (violet) – 750 (red) nm

Beer's Law

Absorbance is directly proportional to the concentration of a solution, when the path length and the molar absorptivity coefficient are constant (A ~ C).

Absorbance Spectrophotometry Uses

Determine the quantity of various solutes in solution
Can be found in many analytical instruments
Range from visible light to UV.

Limitations

The solution needs to be colored to use this technique

Spectrophotometer

A device that measures the light transmitted by a solution to determine the concentration of an absorbing substance.
A quantitative measurement is dependent on two main factors:
- The color itself
- The intensity of the color

Spectrophotometer Components

Light Source
- Different lamps are used for different spectral ranges: - Tungsten-Halogen lamp is used for visible light - Xenon & Mercury lamp is used for UV & visible light
  - Deuterium lamp is used for UV light
Collimator
- A lens that concentrates the light
- May not be used in every spectrophotometer
Monochromator
- Produced by a prism or diffraction grating to split white light into separate colors of the visible spectrum (wavelengths)
- When we change the wavelength on the instrument, this component is changed
Wavelength Selector (Slit)
Selects a specific color (wavelength) - Remains stationary
Sample Cell/ Cuvette
- A vessel that holds the solution
Detector (Photocell)
Converts light energy into an electrical signal
Amplifier
- Amplifies the electrical signal from the detector
- Photomultiplier & photodiodes amplify low intensity light
Meter
- Displays the data from the detector
- The results are printed out and displayed on the screen.

How the Spectrophotometer Works

The light source illuminates the sample cell
The light transmits through the solution
The transmitted light is measured by the detector
The detector converts the light energy into an electrical signal and displays the result on the meter

Green Dye Determination

Absorption Curve:
- Determines the wavelength of maximum absorbance of a known standard
  - Greatest sensitivity for the method
  - Minimizes interferences
- Procedure:
  - Set the spectrophotometer to the starting wavelength (ex. 400nm)
  - Insert a blank solution into the cuvette holder and press zero absorbance. The display should read 0.000.
  - Take an aliquot of the known standard (green dye solution) and place in the cuvette holder. - Note the absorbance reading (ex: 0.581). Record the absorbance value.
  - Repeat at the next wavelength increment.
An absorption curve is plotted using the absorbance readings from different wavelengths

Relationship Between Colors

Max Absorbance represents the complementary color wavelength of the colored solution being tested - If the maximum absorbance is 630nm Red, the solution will appear Green. - If the maximum absorbance is 520nm Green, the solution will appear Red.

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Description

Explore the fascinating properties of light with this quiz on white light and color perception. Understand how white light can be separated into a spectrum and how different materials interact with light. Test your knowledge on transmittance, absorbance, and the visible spectrum.