Absorbance powerpoint

Absorbance
Part A
Determination of Lambda Max

Maximum Absorbance
• Learning objectives:
– Determination of the wavelength of maximum
absorbance of a given solution
– Learn how to use spectrophotometer
– Calculate absorbance

Spectrophotometry
is a technique used to measure how much
energy a substance absorbs at varying
wavelengths of light.
Wavelength (nm)
Absorbance
0.0
2.0
200 250 300 350 400 450

What is wavelength?
Forms of electromagnetic radiation like radio waves or light waves
make characteristic patterns as they travel through space. Each wave
has a certain shape and length. The distance between peaks (high
points) is called wavelength.

UV-Vis spectrophotometry
5
•

UV-Vis Spectrophotometry
6
• Color of a solution:

• Visible light is only a
small portion of the
entire electromagnetic
spectrum
• it includes the colors
commonly observed
(red, yellow, green, blue
and violet).
• The visible spectrum
consists of electro-
magnetic radiation
whose wavelengths
range from 380nm to
nearly 760nm.
Background :

Wavelength (, Greek lambda):
Distance from one wave peak
to the next.
Units: m, cm, m, nm or

A
Light waves can be characterized By:

Spectrophotometer
To measure the amount of light that a sample absorbs
Visible spectrophotometer: uses light over visible range (400 - 700 nm) of
electromagnetic radiation spectrum.

11
UV spectrum of
phenolphthalein

UV-visible spectrum
The two main properties of an
absorbance peak are:
1. Absorption wavelength
max
2. Absorption intensity
Amax
Housecroft and Sharpe, p. 466

Identifying a compound by
spectrophotometry
• If a compound absorbs light its absorption
spectrum is a unique property of that
compound.
• The molecular structure is responsible for
the absorption properties
• The most common feature of absorbing
compounds are conjugated double bonds,
often as an aromatic ring

Spectrophotometry
By measuring the Absorption Spectrum of a
substance, it is possible to identify it or at
least place it in a particular class of
compounds.
Max λ

Absorbance vs Wavelength
Different compounds absorb best at different wavelengths

Transmittance (T) is defined as the fraction of
incident light which is transmitted, ie, passes
through, a sample.
Thus, T = I/Io,
Transmittance

• Defined as the ratio of the intensity of light
emerging from the solution (I) to that of
incident light entering (Io)
Transmittance

Transmittance is usually expressed as a
percentage:
%T = (I/Io) x 100
100% transmittance means no light is absorbed
by the solution so that incident light is 100%
transmitted
Transmittance

Absorbance (A), or optical density, is a
logarithmic function of T and is expressed as:
A = 2 – log(%T)
Note that absorbance has no units.
Absorbance

So, for example, at 100% transmittance, A = log
1.0 = 0.
At 50% transmittance, A = 2 -log (50) = 0.30.
Transmittance and Absorbance

The spectrophotometer displays:
Absorbance -- a number between 0 and ∞
Transmittance -- a number between 0 and
100%.

Applications/Use
• Identify food dyes
• Protein assays of milk and protein drinks
• Test rates of photosynthesis
• Bacterial growth
• Use enzyme preparations to break down complex
sugars
• Determine unknown concentration of solution
• Determine equilibrium constant of a reaction
involving ions
• Purity of protein or nucleic acid preps (A260 /A280)

Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry
How to use Spectrophotometer

Turn on the Spec 20 to warm it up for 10
minutes (left front knob).
Set wavelength using the
dial on top
Step 1:

Prepare a BLANK cuvette by adding WATER
A BLANK is used to calibrate the Spec 20 so
that any absorbance attributable to the
solvent and/or glass cuvette can be
compensated.
By zeroing the Spec 20 to the blank, you will
measure only the absorbance due to the
substance in question.
Step 2:

With no tube in the
holder, adjust the meter
needle to read infinite
absorbance (= 0%
transmittance) using the
left front knob (= power
switch)
Step 3:

Using a Kimwipe, wipe off/polish the outside of the
cuvette.
Using a Sharpie, make a small vertical mark at the top
of each cuvette for alignment in the sample holder.
Step 4:

Step 5:
Insert the cuvette to line up with the line
on the sample holder.
Close the lid.

Step 6:
The spectrophotometer is now
calibrated to this BLANK.
Using the right front knob, adjust the
meter needle to read absorbance = 0.0 (=
100 % transmittance).

Remove BLANK and insert cuvette with
your sample. Close lid.
Read transmittance (upper scale) for your
sample.
Repeat steps 3-8 for each wavelength
Step 7:
Step 8:
Step 9:
Measuring Absorbance or Transmittance

NOTE: When taking several measurements at the
same wavelength over a short time period, you do
not need to re-blank for each.
If you are taking readings over an extended
period or change the wavelength, you must re-
zero the instrument.,
Re-blank the spectrophotometer

Summary
1. Turn instrument on
2. Select correct wavelength  (400-700 nm)
3. Block light, set Zero (no light = infinity
absorption = 0% T)
4. Choose and clean test-tubes
5. Open light, insert Blank (maximum light =
no absorption = 100% T)
6. Measure transmittance of samples
7. Repeat from step 2

Method:
1) Prepare solution of Basic Fuchsin Dye
a) 1 ml of Dye into 9 ml of water (total 10 ml)
2) Determine spectrum:
a) Set up spectrophotometer
b) Measure %T using lambda (400 – 700 nm)
c) Convert %T to Absorbance

Data Analysis
• Plot your data on a graph
– Wavelenght is plotted on the X-axis
– Absorbance is plotted on the Y-axis
• Put units on each axis
• Determine the wavelength of maximum
absorbance
– Now you are ready to proceed to part B

LABORATORY #4
Part B:
Verification of Beer-Lambert Law

Beer-Lambert Law
• Learning objectives:
– What is Beer-Lambert Law?
– Determine the relationship between concentration
and absorbance of the solution
– How to calculate dilutions

Transmittance (T)
Absorbance (A)
A = 2 - log10 (%T)

The Beer-Lambert law describes that
Absorbance, A, is directly proportional to
concentration (M):
Beer-Lambert Law

Absorbance increases linearly with concentration
Standard curve

What is concentration and how we
can calculate it…

Mole: is simply the amount of a substance that contains
6.02 x 1023 particles.
Avogadro's Constant: The number of objects in a mole;
6.02 x 1023
Molarity: A unit of concentration equal to the number
moles of solute in a 1 liter of solution.
VOCABULARY:

What is mole?
• measurement for the amount of substance
• a mole of any pure substance has a mass
in grams exactly equal to that substance's
atomic or molecular mass

What is mole?
• atomic mass for C = 12 g

What is mole?
• MW = 12 g/mole

The Mole (n)
• 1 dozen cookies = 12 cookies
• 1 mole of cookies = 6.02 X 1023 cookies
• 1 dozen cars = 12 cars
• 1 mole of cars = 6.02 X 1023 cars
• 1 dozen Al atoms = 12 Al atoms
• 1 mole of Al atoms = 6.02 X 1023 atoms
Note that the NUMBER is always the same,
but the MASS is very different!

Suppose we invented a new collection unit
called a rapp. One rapp contains 8 objects.
1. How many paper clips in 1 rapp?
a) 1 b) 4 c) 8
2. How many oranges in 2.0 rapp?
a) 4 b) 8 c) 16
3. How many rapps contain 40 gummy bears?
a) 5 b) 10 c) 20
Learning Check

6.02 x 1023 particles
1 mole
or
1 mole
6.02 x 1023 particles
Note that a particle could be an atom OR a molecule!
Avogadro’s Number as
Conversion Factor

1. Number of atoms in 0.500 mole of Al
a) 500 Al atoms
b) 6.02 x 1023 Al atoms
c) 3.01 x 1023 Al atoms
2.Number of moles of S in 1.8 x 1024 S atoms
a) 1.0 mole S atoms
b) 3.0 mole S atoms
c) 1.1 x 1048 mole S atoms
Learning Check

Molarity (M)
A concentration that expresses the
moles of solute in 1 L of solution
Molarity (M) = moles of solute (n)
1 liter solution (V)

Molarity (M)
Molarity (M) = moles of solute
1 liter solution
The solvent is the liquid in which something
is dissolved in, usually water
Solute is a dissolved substance
Solution is a type of homogenous mixture in
which the solute is distributed uniformly
throughout another substance, the solvent

Units of Molarity
2.0 M HCl = 2.0 moles HCl
1 L HCl solution
6.0 M HCl = 6.0 moles HCl
1 L HCl solution

Molarity Conversion Factors
A solution is a 3.0 M NaOH.. Write the
molarity in the form of conversion factors.
3.0 moles NaOH and 1 L NaOH soln
1 L NaOH soln 3.0 moles NaOH

How to calculate molarity
Example 1:
What is the molarity of a 5.00 liter solution
that was made with 10.0 moles of KBr ?
http://www.fordhamprep.org/gcurran/sho/sho/lessons/lesson64.htm

What is the molarity of a 5.00 liter solution that was made
with 10.0 moles of KBr ?
Solution:
# of moles of solute
Molarity = --------------------------
Liters of solution
Given: # of moles of solute = 10.0 moles
Liters of solution = 5.00 liters
10.0 moles of KBr
Molarity = -------------------------- = 2.00 M
5.00 Liters of solution
Answer = 2.00 M
http://www.fordhamprep.org/gcurran/sho/sho/lessons/lesson64.htm

GOLDEN RULE:
ALWAYS TO CHECK UNITS!!!

Learning Check M1
A KOH solution with a volume of 400
mL contains 2 mole KOH. What is the
molarity of the solution?
1) 0.8 M
2) 5 M
3) 0.005 M
Molarity (M) = moles of solute
1 liter solution

Solution M1
A KOH solution with a volume of 400 mL
contains 2 moles of KOH. What is the
molarity of the solution?
2) 5 M
M = 2 mole KOH = 5 M
0.4 L

Learning Check M3
Stomach acid is a 0.10 M HCl solution. How
many moles of HCl are in 1500 mL of stomach
acid solution?
1) 15 moles HCl
2) 1.5 moles HCl
3) 0.15 moles HCl

Solution M3
3) 1500 mL x 1 L = 1.5 L
1000 mL
1.5 L x 0.10 mole HCl = 0.15 mole HCl
1 L
(Molarity factor)

What is dilution?
The act of diluting a solution is to
simply add more water (the
solvent) thus leaving the amount
of solute unchanged

What is dilution?
the amount of solute is unchanged
= 13
= 13

Amount or moles of solute before dilution (nb)
and the moles of solute after the dilution (na)
are the same:
nb = na
MOLARITY & DILUTION
= 13
= 13

MOLARITY & DILUTION
MbVb = MaVa
Remember that M =
So, the moles for any solution can be calculated by
n=MV
Moles (n)
1 Liter (V)

MOLARITY & DILUTION
MbVb = MaVa
Since… n=MV
A relationship can be established such that
MbVb = nb = na = MaVa
Or simply : MbVb = MaVa

MOLARITY & DILUTION
MbVb = MaVa

How to calculate serial dilutions
• Dilution Equation: MbVb = MaVa
• Mb = Molarity of initial solution
• Vb = Volume of initial solution to be used
• Ma = Molarity of final (diluted) solution
• Va = Desired volume of final dilute
solution.

• Va = Desired volume of final dilute solution.
2 ml
2 ml of water
1 M

2 ml
2 ml of water
1 MMb
Vb
Va = 2ml +2 ml = 4 ml
Ma
= ???
MbVb = MaVa
Ma
=
MbVb
Va
=
(1M)x(2ml)
4 ml
= 0.5 M

• Va = Desired volume of final dilute solution.
2 ml
2 ml of water
1 MMb
Vb
Va = 2ml +2 ml = 4 ml
Ma
= ???

MOLARITY & DILUTION
Calculate the molarity of a solution prepared by
diluting 25.0 mL of 0.05 M potassium iodide with 50.0
mL of water.
Mb = 0.05 mol/L Ma = ?
Vb = 25.0 mL Va = 50.0 + 25.0 = 75.0 mL

MOLARITY & DILUTION
Calculate the molarity of a solution prepared by diluting 25.0 mL
of 0.05 M potassium iodide with 50.0 mL of water.
Mb = 0.05 mol/L Ma = ?
Vb = 25.0 mL Va = 50.0 + 25.0 = 75.0 mL
MbVb = MaVa
Mb Vb = Ma = (0.05 mol/L) (25.0 mL) = 0.0167 M of Ka
Va 75.0 mL

Example:
• What volume of 0.01 M solution do we
need to make 20 mL of a 0.001 M
solution?

Example:
need to make 20 mL of a 0.001 M solution?
• Use the dilution equation, MbVb = MaVa
• Mb=0.01 M
• Vb = unknown
• Ma= 0.001 M
• Va = 20 mL
Solve for Vb

Example:
• Use the dilution equation, MbVb = MaVa
• Mb=0.01 M
• Vb = unknown
• Ma= 0.001 M
• Va = 20 mL
Vb=(0.001M)x(20ml) = ???
0.01M

Example:
• Got 2 mL. This is the amount of the 0.01 M
solution that we need. The total volume is
20 mL, so we need to add 18 mL of water.
• So, measure 2 mL of your 0.01 M solution
and add it to 18 mL of water to make a
0.001 M solution.

Methods:
1) Prepare serial dilutions using Basic Fuschin Dye
a) Stock (1 ml Dye + 9 ml of water)
b) Set up different concentrations of dye
2) Determine %T:
a) Set up spectrophotometer
b) Measure %T using maximum lambda from Part A
c) Convert %T to Absorbance
d) Plot Absorbance vs. Concentration

Plot Absorbance vs. Concentration

How to make dilutions (flow chart)
9 ml Water (mL)4.5 4.0 3.5 3.0
Stock solution:
1 ml dye + 9 ml water
0.5 1.0 1.5 2.0 Stock (mL)
2.5 2.5 ml H2O
1
6
5432
Mb=0.0024M
MbVb = MaVa

• Show your calculations, proper graphs and
results
• Be neat
• Part A and Part B are due before the class
next week (week 5)

Just for fun
More info for chemistry nerds!!!

87
Concentration
Absorbance,A
0
0.5
1
Concentration
Transmittance,T
A=bc
certain 
constant b
One analyte
T=10-A =10- bc
Beer’s law is a relation between
absorbance and concentration
which is a straight line passes by
origin at constant pathlength, b,
and at certain wavelength, .
Transmittance decreases
exponentially as concentration
increases
Beer’s law is obeyed for
monochromatic light
50%
25%
12.5%
P0 = 100%
Solution with T = 50%
not zero
Slope = b

UV/visible: Applications
UV/visible is still used in current research,
especially for heme-containing proteins, which have
absorbance in the Soret region that is sensitive to
the state of the protein
3d shell of Fe2+ has 6 electrons
High Spin Low Spin

UV/Visible Applications
This paper looks at iron-sulfur clusters in a native and a
mutant protein
C196S Mutant lacks broad
absorption band between 400-600
nm which is diagnostic of an 2Fe-
2S cluster
JBC (1998)Vol. 273, No. 35;28 pp. 22311–22316

Applications of Spectrophotometer
• Spectroscopy
• Chemical Analysis: concentration ,trace
analysis, pH and remote monitoring
• Geology.
• Astronomy.
• Particle size.
• Thin film characterization
• Color matching
• Optics

• For the mathematically minded:
• Transmittance = IT/I0*100
• Absorbance = log10(I0/IT)
Converting Transmittance to Absorbance
• %T/100 = IT/I0  100/%T = I0/IT
Taking logs on both sides
• Log 100 – log %T = log I0/IT
• 2 – log%T = Absorbance

Absorbance powerpoint

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Absorbance powerpoint