MDP10108 (20 September 2016)

Spectrophotometry

The Electromagnetic Spectrum

Relationship between wavelength (

) and types of radiation
White light = continuous distribution of wavelengths

Beers Law
The amount of light passing through

Concentration

Lamberts Law
The amount of light passing through (pure water)

Distance travelled

Beer-Lambert/Beers Law

= extinction coefficient/absorbtivity constant

l = length/path of light traveled

Amount of light absorbed is proportional to concentration & optical path length

Absorbance can be used to calculate concentration of a substance in solution

MDP10108 (20 September 2016)

Spectrophotometry

Limitation of the Beers Law

1. Linear (except at high conc.)

2. Passes through the origin.

Prefer in absorbance instead of percent transmission

When is Beers law inapplicable?

Chemical factors
Presence of particulates
Fluorescence or phosphorescence
Refractive index (i.e., insufficient dilution)
Instrumentation
Stray light
Non-monochromatic radiation

What if cannot use Beers Law?

Use standard/calibration curve

What if the solution is colourless?

Use of suitable reagents for indirect colour development.

MDP10108 (20 September 2016)

Spectrophotometry

When light interacts with matter

What is spectrometry?
(qualitative/quantitative) Study of
atoms/molecules/physical processes.
Interaction between the electromagnetic
radiation with molecules.

Components:
Light Source
Wavelength selector

1
Sample Holder
Detector

or
(glass, Quartz or UV-transparent plastic cuvette)

Output/Display
(pedestal)

MDP10108 (20 September 2016)

Spectrophotometry

Specialised
sample holder

Specialised cuvette

Colorimeter vs. Spectrophotometer

2
1

Detector

Colorimeter

Spectrophotometer

MDP10108 (20 September 2016)

Spectrophotometry

Monochromator = Wavelength selector

1. Select prism/grating
2. Focus/Direct exit slit

Applications of spectrophotometry?
To quantify (ie, amount)
Plasma cholesterol
Serum ALT (liver function test)

To identify a substance
Serum ferritin (iron status)
Blood urea (renal function test)

To monitor changes (ie, activity)

Cardiac troponin (MI diagnosis)
Oxygen saturation

Nuclear Magnetic Resonance (NMR)

Pulse oximeter for

pO2 saturation

Magnetic Resonance Imaging (MRI)

Bone Densitometry
Dual-energy x-ray absorptiometry
(DXA or DEXA)
Low dose x-ray
Central or peripheral (wrist,
finger, leg, heel)

MDP10108 (20 September 2016)

Spectrophotometry

Important terms

Absorbance
Absorbance spectrum
Absorbance maximum (
max)
Reference or Blank cell
Serial dilution
Standard curve/Calibration curve
Chromophore
Beers law
Wavelength (
)
Transmitted light
Percent transmission (%T)

UCLA, USA
www.chem.ucla.edu/~gchemlab/colorimetric_web.htm
Sheffield Hallam University, UK
teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm
How to prepare a serial dilution?
www.wikihow.com/Do-Serial-Dilutions
How to draw & use a standard curve?
www.ruf.rice.edu/~bioslabs/methods/protein/protcurve.html
How to present data in graph form?
www.ruf.rice.edu/~bioslabs/Graphing_tutorial/index.htm