Instruments & Application

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