Post on 16-Jul-2015
UV-visible spectroscopy
How They Work
What is Spectroscopy?
• The study of molecular structure and
dynamics through the absorption,
emission and scattering of light.
Spectroscopy
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)
Spectrophotometer (Spec)
An instrument that measures the
amount of light that passes through
(is transmitted through) a sample.
Transmission and Color
The human eye sees the complementary color to that which is
absorbed
Absorbance and
Complementary Colors
Molecules are whatever color of
light that they do not absorb.
Green molecules appear green
because they absorb most
wavelengths of visible light,
except the green wavelengths.
Ultraviolet (UV) Spectrophotometers.Uses ultraviolet light of wave lengths from 200 nm to 350 nm.
Visible (VIS) Light Spectrum Spectrophotometers.Uses visible light (white light) of wave lengths from 350 nm to 700 nm.
Conventional
Spectrophotometer
Schematic of a conventional single-beam spectrophotometer
Conventional
Spectrophotometer
Optical system of a double-beam spectrophotometer
Cells
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
Open-topped rectangular standard cell (a)
and apertured cell (b) for limited sample volume
Cell Types I
Cell Types II
Micro cell (a) for very small volumes and flow-through cell (b)
for automated applications
Light Sources
UV Spectrophotometer
Hydrogen Gas Lamp
Visible Spectrophotometer
Tungsten Lamp
The concentration of an unknown sample can be
determined by comparing the absorbance data to
standards of known concentration.
The data generated with the set of known
standards is called a standard curve.
Transmittance and Path
Length: Beer’s Law
Concentration
The Beer-Bouguer-
Lambert Law
cbIIIITA /log/loglog 00
R- Transmittance
R = I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
Log is proportional to C (concentration of solution)
also proportional to L (length of light path
through the solution).
I
I0
I
I0
1
T
I
I0
STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD
1. Run the sample for
spectrum
2. Obtain a monochromatic
wavelength for the
maximum absorption
wavelength.
3. Calculate the concentration
of your sample using Beer
Lambert Equation: A = KCL
Wavelength (nm)
Absorbance
0.0
2.0
200 250 300 350 400 450
Slope of Standard Curve = A
C
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
Absorbance at 280 nm
There is some A vs. C where graph is linear.
NEVER extrapolate beyond point known where
becomes non-linear.
SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
1 2 3 4
0.4
0.8
1.2
Absorbance at 540 nm
Conc entration (g/l) glucose
Avoid very high or low absorbencies when drawing a
standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
straight line
• Every instrument has a useful range for a
particular analyte.
• Often, you must determine that range
experimentally.
• This is done by making a dilution series of
the known solution.
• These dilutions are used to make a
working curve.
What concentration do you think the
unknown sample is?
In this graph, values above A=1.0 are not linear. If we
use readings above A=1.0, graph isn’t accurate.
Spectrophotometry
1. Turn instrument on
2. Select correct wavelength
3. Choose and clean cuvette
4. Open light, insert Blank (maximum light = no absorption = 100% T)
5. Measure absorption of Standards, Controls and Patient samples to 3rd decimal place
Standards
• Precisely prepared = known concentration
• Usually pure solution of single compound
• Plot absorbance vs concentration: standard
curve
How to Ensure Accuracy?
• Repeat tests many times and take average
• Run another sample that was tested before
along with patient samples and make sure
its result is close to what it should be
Control Samples
• Similar in composition to patient sample
• Usually pooled from many donors
• Tested at least 30 times to calculate the
average (target value) and allowable range
of variation