2c. Spectrophotometry

What is spectrophotometry?

A method that uses color intensity to determine the concentration of a solution.

What does a spectrophotometer measure?

It measures how much light passes through or is absorbed by a colored solution.

Why is spectrophotometry useful in clinical biochemistry?

It allows quantitative determination of analytes such as glucose by measuring absorbance.

What is a cuvette?

A small transparent container that holds the sample solution in the spectrophotometer.

What are the main parts of a spectrophotometer?

Light source, filter or monochromator, sample cuvette, and detector.

Why is white light not used directly in spectrophotometry?

Because measurements need light of a specific wavelength.

What is monochromatic light?

Light with one precisely selected wavelength.

How is monochromatic light obtained in simple spectrophotometers?

By passing white light through a colored filter.

What is the visible light range?

Approximately 400–700 nm.

What happens when monochromatic light passes through a colored solution?

Part of the light is absorbed and part is transmitted.

What is I0 in spectrophotometry?

The intensity of incoming light before it passes through the sample.

What is It in spectrophotometry?

The intensity of transmitted light after it passes through the sample.

Why is It lower than I0?

Because some light is absorbed by the colored solution.

What is transmittance?

The fraction of light that passes through the sample.

What is the formula for transmittance?

T = It / I0.

What is absorbance?

A measure of how much light is absorbed by the sample.

What is the formula for absorbance?

A = log(I0 / It).

How are absorbance and transmittance related?

A = log(1 / T).

What happens to transmittance when concentration increases?

Transmittance decreases.

What happens to absorbance when concentration increases?

Absorbance increases.

What does stronger color usually mean in spectrophotometry?

Higher absorbance and usually higher concentration.

What is the Beer-Lambert law?

A = ε × b × c.

What does A mean in the Beer-Lambert law?

Absorbance, the value read by the instrument.

What does ε mean in the Beer-Lambert law?

Molar absorptivity, a constant showing how strongly a substance absorbs light.

What does b mean in the Beer-Lambert law?

Path length, usually the width of the cuvette.

What is the usual path length of a cuvette?

Usually 1 cm.

What does c mean in the Beer-Lambert law?

Concentration, usually in mol/L.

What is the practical meaning of the Beer-Lambert law?

Absorbance is proportional to concentration within the linear range.

What is a blank solution?

A reference solution used to set absorbance to zero.

Why is a blank prepared?

To remove background absorbance from solvent and reagents.

What should absorbance be at 0 M concentration?

Zero.

What is a calibration curve?

A graph made from known concentrations and their absorbance values.

How is an unknown concentration found?

Measure its absorbance and read the concentration from the calibration curve.

What is a best-fit line used for?

To estimate the relationship between absorbance and concentration.