Principles of Spectrophotometry- Part 1 W8: L2

The Nature of Light:

The term light is used to describe radiant energy with wavelengths visible to the human eye or with wavelengths bordering on those visible to the human eye.

PHOTONS:

discrete packets of energy traveling in the form of waves.

-Photons interact with matter (atoms, ions, molecules)

-When photons hit matter, the photon is absorbed and the energy of the photon changes the matter (EXCITATION)

-excites matters

Review of Light:

The wavelength of light is the linear distance between waves
Wavelength is measured in nm or 10-9m
Frequency: describes the number of wave peaks per given unit of time
Amplitude: the height of the wave

Electromagnetic spectrum:

The range of all possible electromagnetic radiation.

The spectrum of an object is the range of electromagnetic radiation that it emits, reflects, or transmits.

Colour and wavelengths

Solutions contain particles that absorb certain λs and transmit others.
The λ of light transmitted by a solution is recognized as color by the human eye

Analytical Wavelength:

describes the position within the electromagnetic spectrum at which we “read” a solution.

--we have been setting the spects to certain wavelengths so that the spect allows light at that wavelength to hit the solution

Photometry:

the measurement of the luminous intensity of light or the amount of luminous light falling on a surface

-measurement of intensity

Spectroscopy:

the measurement of the intensity of light falling on a surface at selected wavelengths

-Originally, the study of how visible light dispersed by a prism according to its wavelength!

-a measurement of that intensity at a specific wavelength

Spectrophotometry

The quantitative measurement of the absorption or transmission properties of a material (solution) as a function of wavelength

-Molecules absorb only those wavelengths of light that contain just the right amount of energy to move the electrons to a higher energy state

Spectrophotometry cont.

We want to use wavelengths which excite electrons and move them to a higher energy state=Requires a Spectrophotometer

Isolates a narrow wavelength (λ) of light required for measurement of a solution

Spectrophotometry (aka colorimetry):

Measurement is based upon the reaction between a substance to be measured (the analyte) and a reagent that produces a COLOR

The amount of color produced is dependent upon the amount of analyte in the sample

We have been using ABSORBANCE as a signal in the lab to detect the intensity of the color

more intense colour=more light it will absorb= higher absorbance reading

What happens when we add a volume of sample to a volume of reagent?

For most assays, a color develops.

But how do we convert a color change into a quantitative test result?
-principles of Spectrophotometry

Reagent:

any substance employed to produce a chemical reaction
-Reagents are solutions containing the chemicals required for the reaction to occur when a SAMPLE is added

In spectrophotometry, the reaction is a change in color that can be detected at a specific wavelength of light

Basics of Light:

Light can be transmitted, absorbed, or reflected

Transmittance:

Light hitting a solution can pass through the solution.
The light passing through is considered to be the transmitted light
The portion of the original (incident) light that has passed through is the % transmittance (%T)

%Transmittance (%T):

- the % of light intensity that passes through a solution

Incident light (Io): light intensity hitting the solution
Transmitted light (I): intensity of light exiting a solution
%T =ratio of transmitted light energy (intensity) to the incident light energy

%T = (I/Io) x 100
=the % of incident light reaching a detector

1. Transmittance:

Concentration of a substance is inversely proportional (decreases exponentially) to the logarithm of the transmitted light

If no light is transmitted: %T = 0 (none of the light makes it thru)
If all light is transmitted: %T = 100 (all light makes it thru)

Absorbance (A):

the amount of light absorbed by the solution
The light NOT transmitted has been absorbed by the solution

Abs = 1/logT
Hard to do math so manipulated to:

A = 2-log%T

(Translation: to calculate A, take the log of the %T, add a negative sign to it and then add 2)

1. ABSORBANCE

Concentration of a substance is directly proportional (increases linearly) to the amount of light absorbed by the substance

Reminder: -this is an indirect measurement based upon %T

Optical Density (O.D.) is an older term used for absorbance.

Which type of measurement is preferable?

Absorbance readings when determining concentration are preferred because its a directional proportional relationship

Analytical Wavelength:

The absorbance of a solution peaks at a particular λ of the spectrum
Peak = λmax

Assays are designed to measure an analyte at the λ that the absorbance peaks at.= analytical λ

λmax

The λmax is the wavelength at which the sample reaction mixture absorbs the most light.

At this wavelength, most light is absorbed so the exiting light is at its weakest intensity

Sensitivity:

The lowest level of a substance that can be detected by an analytic method
The Abs per unit concentration of an analyte is greatest at its λ max

-this provides maximum sensitivity.

-that is to say, the higher the absorbance, the lower the concentration that the analysis is able to reliably measure

At higher wavelengths, we can detect concentration in patients' sample even if its really really low

Bougher-Lambert Law

The amount of monochromatic light transmitted through a substance diminishes exponentially with the thickness of the substance

Therefore, the length of the light path through the cuvette affects the amount of light transmitted.

Ie: The longer the cuvette path in cm (the LONGER the cuvette) the less light transmitted.

The thicker the substance, the more distance that light has to travel (less light that can be transmitted- more is absorbed)

Bougher-Lambert Law*

Longer path means more absorbance of light!

Think of a Solution as existing in “layers”
–each layer getting hit by light:

Each layer absorbs 70% of light (in this graphic 49 represents 70% of 70)

Beer’s Law (or Beer-Lambert Law)*

Describes the relationship between absorption of light by a solution and the concentration of that solution

States that the concentration of a substance is directly proportional to the amount of light (radiant energy) absorbed or inversely proportional to the logarithm of the transmitted light (radiant energy)

Beer-Lambert Law *

Two laws combined to express:
A = abc
where A = absorbance
a = absorptivity constant
b = light path in cm
c = concentration of analyte

a: units of moles/cm2 Remains constant for a given analyte at a specified wavelength

b: remains constant as long as you use the same cuvettes!!

Therefore, if the light path and absorptivity are constant:
Absorbance is DIRECTLY proportional to Concentration A = c

a = absorptivity constant

The physical property of a solution that determines the fraction of light it can absorb at a given λ

Absorptivity constant is unique for a solution and reproducible at the given λ

Beer’s Law is only applicable under the following conditions:

-Incident radiation on the substance of interest is monochromatic (light is of a single wavelength)

-The absorption of the solvent alone is insignificant

-The solute concentration is within the given limits (not too high)

-An optical interferent is NOT present (something else in the solution isn’t mucking up the true absorbance!!)

-A secondary chemical reaction does not occur between the analyte of interest and the solvent

What is a “water” blank?

Used to “zero” or “blank” the spectrophotometer.
Incident light can be reflected or absorbed by the surface of the cell wall (cuvette walls)
Eliminates effects of any potential absorbance from the cuvette
The walls of the cuvette reflect and absorb light!

What is a Reagent blank (aka “reference” blank)?

Incident light (light source) can be reflected or absorbed by the solvent (reagent). This can be eliminated with a reagent blank!

REAGENT Blanks:

Identical conditions to sample cell except WITHOUT the sample added!

Add water in place of the sample, allow water/reagent solution to run through entire test along with sample solutions.

Measure the reagent blank and subtract its readings from the sample measurements or “zero” with the reagent blank

REAGENT Blanks allow for:*

Allows us to isolate %T or Abs from ONLY the specific compound by “blanking out” any additional absorbance from the color of the reagent

What is a Sample Blank?*

Allows us to isolate %T or Abs from a specific compound (analyte) in the sample by “blanking out” the color of the sample

SAMPLE Blanks:

The color or turbidity of a sample may cause an interference with measurements

Identical conditions to sample cell except WITHOUT the reagent added

Add water in place of the reagent, allow sample/water solution to run through entire test along with sample solutions.

Measure each sample blank and subtract its readings from its sample measurement