Chapter 2c — Spectrophotometry

What is spectrophotometry?

A method using light absorption to determine the concentration of a substance.

What does a spectrophotometer measure?

How much light is absorbed or transmitted by a sample.

What are the main parts of a spectrophotometer?

Light source, filter/monochromator, cuvette and detector.

What is monochromatic light?

Light with one defined wavelength.

Why is monochromatic light used?

Different substances absorb best at specific wavelengths.

What is a cuvette?

A transparent container holding the sample solution.

What is the usual path length of a cuvette?

1 cm.

What is incoming light called?

I₀.

What is transmitted light called?

Iₜ.

What is transmittance?

The fraction of light passing through the sample.

What is absorbance?

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

How does absorbance change with concentration?

Higher concentration usually gives higher absorbance.

What is the Beer-Lambert law?

A = ε × b × c.

What does A mean in Beer-Lambert law?

Absorbance.

What does ε mean in Beer-Lambert law?

Molar absorptivity.

What does b mean in Beer-Lambert law?

Path length.

What does c mean in Beer-Lambert law?

Concentration.

What is a blank?

A solution used to correct background absorbance.

Why is a reagent blank used?

To subtract absorbance caused by reagent and cuvette.

How is spectrophotometry connected to GOD-PAP?

It measures the colored quinoneimine dye.

How is spectrophotometry connected to hexokinase?

It measures NADH absorbance in the UV range.

How is spectrophotometry connected to cholesterol and triglyceride protocols?

It measures colored dye formed from H₂O₂ reactions.

How is spectrophotometry connected to total protein and albumin protocols?

It measures the colored complexes formed by Biuret and BCG reactions.