Spectrophotometry

CLINICAL CHEM

what does spectrophotometry measure?

the intensity of light at selected wavelengths

what is the relationship between frequency and energy?

direct

what is the relationship between frequency and wavelength?

inverse

what is the relationship between wavelength and energy?

inverse

does spectrophotometers measure transmitted, absorbed, or scattered light?

transmitted

What is A = ε b c?

A = absorbance

ε = molar absorptivity (L/(mole)(cm))

b = light path (cm)

c = concentration (mole/L)

What’s the second equation of Beer’s law you should memorize?

A = 2.00 - [log(%T)]

What’s the third equation of Beer’s Law you should memorize?

C_unknown = A_unknown (C_standard/A_standard)

List the spectrophotometer light sources

Tungsten lamp, quarts-halogen, vapor lamp, LED, laser

What does a monochromator do in a spectrophotometer?

isolates single, specific wavelengths of light from a light source

define constructive interference

when 2+ wavelengths are added up, creating a bigger combined wave

define destructive interference

when 2+ wavelengths are added, they cancel each other out

what is interference filter?

a filter that reflects specific wavelengths of light while transmitting others through constructive/deconstructive interference

what is the monochromator bandpass?

the specific range of wavelengths that reaches the cuvette with at least 50% peak transmittance

is it better to have a smaller or bigger monochromatic bandpass?

smaller (a greater bandpass can lead to a false absorbance reading)

how does a double beam in space spectrophotometer work?

it splits a single beam of light into 2 beams: one for the sample and one for the reference, which then both hit the detectors

what is an automated spectrophotometer?

similar to a standard spectrometer without requiring a person to change every cuvette by hand (what we have in the student lab)

what is a nanodrop used for?

a type of spectrophotometer only used for reading DNA/RNA

how do we achieve wavelength accuracy in spectrophotometer performance?

by using certified reference standards (running tests of materials with well-defined peaks