Spectrophotometry

What is light?

A form of energy that travels in waves.

What is a wavelength?

The distance between wave peaks of light.

What unit is wavelength measured in?

Nanometers (nm).

What happens when light hits an opaque surface?

Some wavelengths are reflected (scattered/redirected) and the remainder are absorbed.

What determines the color we see when light hits an opaque surface?

The wavelengths that are reflected (scattered back) — those are the colors we perceive.

What happens to the wavelengths that are NOT reflected when light hits an opaque surface?

They are absorbed by the surface.

What color is seen at wavelengths less than 380 nm?

UV (ultraviolet) — invisible to the human eye.

What color is seen at wavelengths of 380–440 nm?

Violet.

What color is seen at wavelengths of 440–500 nm?

Blue.

What color is seen at wavelengths of 500–580 nm?

Green.

What color is seen at wavelengths of 580–600 nm?

Yellow.

What color is seen at wavelengths of 600–620 nm?

Orange.

What color is seen at wavelengths of 620–750 nm?

Red.

What is seen at wavelengths greater than 750 nm?

IR (infrared) — invisible to the human eye.

What are the two ends of the visible light spectrum that are invisible to the human eye?

UV (< 380 nm) and IR (> 750 nm).  

What are the three things that can happen when light hits a transparent surface?

Reflection, absorption, and transmission.

What does "transmitted" light mean?

Light that passes through to the other side of the surface.

How does a transparent surface differ from an opaque surface in terms of what happens to light?

A transparent surface transmits light (lets it pass through); an opaque surface does not transmit light — it only reflects and absorbs.  

What is photometry?

The measurement of light intensity.

What is spectrophotometry?

The measurement of light at selected (specific) wavelengths.

Name the three types of light measurements used in laboratory assays.

Reflectance, absorbance, and transmittance.  

Describe the general principle of how spectrophotometry is used to measure albumin.

A reagent is added to the sample; if albumin is present, a color change occurs. Light is then passed through the sample at a specific wavelength, and the amount of light absorbed is measured — more albumin = more color change = more light absorbed.

What is the relationship between analyte concentration and color change in a spectrophotometry test?

More analyte (e.g., more albumin) = more color change.

What is the relationship between analyte concentration and light absorbed in spectrophotometry?

More analyte = more color change = more light absorbed.

Why is a reagent added to the sample in a spectrophotometry test?

To produce a color change (colorimetric reaction) that is proportional to the amount of analyte present.

What triggers the color change in a spectrophotometry test?

The reaction between the reagent and the analyte in the sample.  

Name the five components of a spectrophotometer in order.

Incident light, monochromator, cuvette, photodetector, and readout display.  

What is incident light?

The total, starting light that enters the spectrophotometer.

What can happen to incident light as it travels through the system?

Some is absorbed, some is transmitted, and some is reflected.

Name three types of incident light sources used in spectrophotometers.

Incandescent, UV, and laser.  

What is the purpose of a monochromator?

To select a specific wavelength of light.

Name the three types of monochromators.

Filter, prism, and diffraction grating.  

What material are filters typically made of?

Colored glass.

What is spectral bandwidth?

The range of wavelengths emitted by a filter.

Do filters have wide or narrow spectral bandwidth?

Wide spectral bandwidth.

Do filters have high or low resolution?

Low resolution.

Can more than one filter be used in sequence?

Yes — multiple filters can be used sequentially.

What is a filter photometer?

A photometer that uses filters as its monochromator.  

What do prisms and diffraction gratings do to white light?

They separate white light into its component wavelengths.

What advantage do prisms and diffraction gratings have over filters?

They allow a specific wavelength to be targeted with higher precision.

What is the most common type of monochromator?

Diffraction grating.

What type of instrument uses a prism or diffraction grating as its monochromator?

A spectrophotometer.

How does a spectrophotometer differ from a filter photometer in terms of monochromator?

A spectrophotometer uses a prism or diffraction grating (higher resolution); a filter photometer uses colored glass filters (lower resolution).

What is a cuvette?

The container that holds the sample to be tested in a spectrophotometer.

Why must a cuvette be transparent?

Light must pass through it to reach the photodetector.

Name three materials cuvettes can be made of.

Plastic, glass, and quartz.

What is the standard width of a cuvette?

1 cm wide.

Why is the standard cuvette size important?

The path length (b = 1 cm) is a constant used in Beer's Law calculations.  

What does the photodetector do?

It detects the transmitted light and converts it into an electrical signal.

What does the photodetector detect?

Transmitted light (light that passed through the sample).

What does the photodetector convert light into?

An electrical signal.  

What does the readout display show?

What was measured by the photodetector — transmittance and/or absorbance.

Which measurement is directly measured by the spectrophotometer — transmittance or absorbance?

Transmittance is directly measured.

Which measurement is calculated by the instrument — transmittance or absorbance?

Absorbance is calculated by the instrument.  

List the five components of a spectrophotometer in the correct order from light source to output.

Incident light → Monochromator → Cuvette → Photodetector → Readout display.  

What is transmittance?

The amount of incident light that is transmitted (passed) through the sample, expressed as a percentage.

What is absorbance?

The amount of incident light absorbed by the sample.

What are the units of transmittance?

Percentage (%).

What are the units of absorbance?

No units — it is a dimensionless logarithmic value.

What is the relationship between transmittance and absorbance?

They have an inverse relationship — as transmittance increases, absorbance decreases, and vice versa.

What does 100% transmittance equal in absorbance?

0.000 absorbance.

What does 1.00 T (100% T) correspond to in absorbance?

0.000 absorbance.

Why does absorbance have no units?

Because it is a logarithmic conversion of transmittance, not a direct measurement.  

Write all four equations for converting between transmittance and absorbance.

A = –log(T) / A = log(1/T) / A = 2 – log(%T) / T = 10^(–A)

What is the formula: A = 2 – log(%T) used for?

To convert percent transmittance (%T) to absorbance (A).

What is the formula A = –log(T) used for?

To convert transmittance (as a decimal) to absorbance.

What is the formula A = log(1/T) used for?

To convert transmittance (as a decimal) to absorbance.

What is the formula T = 10^(–A) used for?

To convert absorbance to transmittance (as a decimal).   --- SLIDE 19: Conversion Practice — 50% T to Absorbance ---

What is the absorbance of a sample with 50% transmittance?

A = 0.301.

Using A = 2 – log(%T), show the steps to convert 50%T to absorbance.

A = 2 – log(50) = 2 – 1.699 = 0.301.

Using A = –log(T), show the steps to convert 50%T (0.50 T) to absorbance.

A = –log(0.50) = –(–0.301) = 0.301.

Using A = log(1/T), show the steps to convert 0.50 T to absorbance.

A = log(1/0.50) = log(2) = 0.301.

Using T = 10^(–A), verify that 50%T equals 0.301 absorbance.

0.50 = 10^(–A) → –A = log(0.50) = –0.301 → A = 0.301. ✓

To how many decimal places is absorbance typically reported?

Three decimal places (e.g., 0.301).   --- SLIDE 20: Conversion Practice — 0.468 A to Transmittance ---

What is the %T of a sample with an absorbance of 0.468?

%T = 34%.

Using A = 2 – log(%T), show the steps to convert 0.468 A to %T.

0.468 = 2 – log(%T) → –1.532 = –log(%T) → log(%T) = 1.532 → %T = 10^1.532 = 34%.

Using T = 10^(–A), show the steps to convert 0.468 A to transmittance.

T = 10^(–0.468) = 0.34, so %T = 34%.   --- SLIDE 21: Practice Problems (Set 1) ---

Convert 64%T to absorbance.

A = 0.194.

Convert an absorbance of 0.537 to %T.

%T = 29%.   --- SLIDE 22: Practice Problems (Set 2) ---

Convert 0.48 T (as a decimal) to absorbance.

A = 0.319.

Convert an absorbance of 0.738 to %T.

%T = 18%.   --- SLIDE 23: Using Light Measurements to Find Concentration ---

Once you know transmittance or absorbance, what can you calculate?

The concentration of the analyte in the sample.

Why is absorbance preferred over transmittance for calculating concentration?

Because absorbance has a linear relationship with concentration, making calculation easier.   --- SLIDE 24: Transmittance vs. Concentration ---

What type of relationship exists between transmittance and concentration?

A logarithmic (non-linear) relationship.

Why is transmittance not directly used to calculate concentration?

Because the relationship between transmittance and concentration is logarithmic, not linear.   --- SLIDE 25: Absorbance vs. Concentration ---

What type of relationship exists between absorbance and concentration?

A linear relationship.

Why is absorbance used to calculate concentration rather than transmittance?

Because absorbance has a linear relationship with concentration, making it directly proportional and easier to calculate.   --- SLIDE 26: Calculating Concentration — Beer's Law Overview ---

Name the three methods used to calculate analyte concentration from absorbance.

Beer's Law (A = abc), the colorimeter formula, and a standard curve.

What does Beer's Law state?

The concentration of a substance is directly proportional to the amount of light absorbed, or inversely proportional to the logarithm of the transmitted light.

What is the colorimeter formula?

A variation of Beer's Law that uses a standard with a known concentration to calculate an unknown concentration.

What is a standard curve?

A graph of absorbance values for multiple known standard concentrations used to determine the concentration of unknowns.  

Write the Beer's Law equation.

A = abc

In Beer's Law (A = abc), what does A stand for?

Absorbance.

In Beer's Law (A = abc), what does "a" stand for?

Absorptivity — the absorbance per unit of concentration for a substance at a particular wavelength.

In Beer's Law, is "a" a constant or variable?

It is a constant for a specific substance at a specific wavelength, as long as testing conditions (temperature, pH, etc.) remain constant.

In Beer's Law (A = abc), what does "b" stand for?

The distance light travels through the solution (path length), measured in cm.

What is the standard value of "b" (path length) used in spectrophotometry?

1 cm (the standard cuvette width).

In Beer's Law (A = abc), what does "c" stand for?

The concentration of the substance being measured.

What conditions must remain constant for "a" (absorptivity) to remain constant?

Temperature, pH, and other testing conditions.   --- SLIDE 28: Beer's Law — Limitations ---

Name three conditions required for Beer's Law to apply.

The solution must be homogeneous, the solution must be transparent, and the concentration must not be too high.