Pharmacology Week 8
Spectroscopy and
Analyzing Mixtures with UV
Absorbance of light refresher
• Light energy travels through the sample and radiant Energy out
I
power decreases (since the sample absorbs some
energy)
• The transmittance of the solution is the fraction of Sample
incident radiation (I0) transmitted by the solution
𝐼
T=
𝐼0
• Usually, we multiply the answer by 100 and express it
as a percent Energy in
I0
Absorbance of light Contin.
• Absorbance is the inverse log of the transmittance Energy out
I
• Why is it inverse? Does it make sense that one would
increase as the other decreases and vice versa?
𝐼0 Sample
A = 𝑙𝑜𝑔
𝐼
• Why is the log used?
****Due to % T = T x 100 % sometimes A is calculated from
A = 2-log %T because log 100 is equal to 2
Energy in
I0
Beer-Lambert Law
• Gives us a quantitative relationship between attenuation and
concentration of absorbing molecules and the pathlength that the
absorption occurs over
• Light travels over a medium with the analyte
• The analyte becomes excited by absorbing energy
• Intensity of the light decreases with increasing absorbance
• The more concentrated the analyte is in the medium the more light is
absorbed
• The longer the pathlength the light passes through the more analyte is in
the lights path and the more light is absorbed
• Therefore, attenuation is equal to the A = c * l * a
“a” or the proportionality constant, absorptivity
sometimes referred to as ε (molar absorptivity)
• Absorptivity allows us to cancel out the units in l and c
• Thus we want to ensure whatever absorptivity we use has the
inverse of the unit our pathlength, l (generally cm) has and the
inverse of the c
• When c is in mol/L we use ε which is where the “molar” absorptivity
comes from
• Don’t be confused about the term “constant” when referring to a or ε
• It is a “flexible” number that depends on the circumstance used and allows our
equation to directly relate to absorbance; it is equal to A/lc
• In fact, the molar absorptivity of a species is sometimes a useful calculation
• But keep in mind absorptivity's are solvent, composition and temperature
dependent so we don’t generally refer to them as standard solutions but instead
calculate them for each analysis
Quantitative Measurements
• Most common use of absorbance measurements is to determine
concentration of test substance (s) in a sample (mixture)
• Involve indirect measurements of intensity of radiation
• Standardization Required
• Blank Sample set to zero Absorbance
• Maximum Sensitivity of measurements obtained at absorbance
maximum (𝐴𝑚𝑎𝑥 )
• Requires extinction coefficient value () at specified wavelength
() and validity of Beer-Lambert Law.
Absolute Methods
• Determine Concentration from KNOWN value of molar absorption
coefficient but always better to confirm under your set of
experimental conditions and with your equipment.
• Determination of Molar Absorption Coefficient from Plot of
Absorbance (at a specified wavelength) versus concentration
a or ε (molar absorptivity) can be calculated in
the lab
• If you make a standard curve and solve for the slope, the slope is
equal to the molar absorptivity AT the measured wavelength
****assuming the cell you are using is 1 cm****
• This means if you have two or more solutions that are different
colours mixed you can find the concentration of all the solutions
assuming you have a standard for each solution
• And a computer is nice if you have more than two standards since it is
difficult to calculate
Calculating the Unknown Concentration
from the Calibration Curve
The slope and intercept values are entered in the spreadsheet formula in Cell B12 (from the equation in A9) to
calculate the unknown concentration.
Calculating Standard Deviation of the
Sample Concentration
The sample standard
deviation (Sc) is determined
from the calibration curve,
based on:
number of measurements
(N); slope (m); average
absorbance (yave); sum of
squares of deviations (Sxx);
standard deviation about
regression (Sr)
Let excel do the work…
Analysis of Mixtures
• Remember *the total absorbance of a solution at any given
wavelength is equal to the sum of the absorbances of the
individual components*
• The A+B spectrum is always a
combination of both
• We find the max of A and the
max of B and determine the
molar absorptivity's at those
points (both A and Bs max)
• We then use the max
wavelengths to determine two
different absorption equations
𝐴1 = 𝜀𝐴𝑚𝑎𝑥𝐴 𝑏𝑐𝐴 + 𝜀𝐵𝑚𝑎𝑥𝐴 𝑏𝑐𝐵
𝐴2 = 𝜀𝐴𝑚𝑎𝑥𝐵 𝑏𝑐𝐴 + 𝜀𝐵𝑚𝑎𝑥𝐵 𝑏𝑐𝐵
Example: Analysis of Mixtures
A steel sample has been analyzed using atomic absorption spectrometry and
determined to contain titanium and vanadium oxides. A standard solution of titanium
is run as well as a standard solution of vanadium. The following information is
obtained. What is the concentration of the titanium and vanadium oxides in the steel?
Species Max Absorbance Calculated ε at 400 Calculated ε at 460 Absorbance at 400 Absorbance at 460
nm nm nm nm nm
Titanium 400 644 L/mol cm 321 L/mol cm NA NA
Vanadium 460 145 L/mol cm 232 L/mol cm NA NA
Steel 400 + 460 NA NA 0.172 0.116
How does a Mass Spectrometer
work?
• 3 fundamental parts: the ionization source, the analyser,
the detector
• Samples easier to manipulate if ionized
• Separation in analyser according to mass-to-charge
ratios (m/z)
• Detection of separated ions and their relative abundance
• Signals sent to data system and formatted in a m/z
spectrum
How does a Mass Spectrometer work?
Using a Mass Spectrum (needed for
assignment 5)
• Find the molar mass (use the mass spectrum)
• Mass Spec: A method for measuring the mass of atoms/molecules/fragments** of
molecules
• Species are put into the gas state, ionized, accelerated, then separated by their
mass to charge ratio (m/z)
• For ions with a charge of +1, m/z will equal the mass of the ion
• For ions with a charge of +2, m/z will equal the ½ of the mass of the ion
• Generally we are looking for the peak with the largest m/z value, that is
signficant (NOT THE LARGEST PEAK!)
• If you have the correct peak it is equal to the ionized molar mass (which is
the same as the regular molar mass)
Fun Facts about Mass Spectra
• Generally, it is easy to identify with a quick glance whether a
compound does or does not have:
• Nitrogen
• Chlorine
• Bromine
• Nitrogen: usually the molecular weight is odd
• Chlorine and Bromine has very unique patterns because the
percent abundance of their isotopes are different than most
atoms
Most Atoms…
• Have one isotope that is a lot more plentiful than the others
• Ex: Oxygen – 99.63 % 16, 0.0375 % 17 and 0.1995 % 18
• Chlorine – 75.8 % 35; 24.23 % Cl
• Bromine – 50.7 % 79; 49.3 % 81
[Link]
Electrospray Ionization
• Sample dissolved in polar, volatile buffer (no salts) and
pumped through a stainless steel capillary (70 - 150 m)
at a rate of 10-100 L/min
• Strong voltage (3-4 kV) applied at tip along with flow of
nebulizing gas causes the sample to “nebulize” or
aerosolize
• Aerosol is directed through regions of higher vacuum until
droplets evaporate to near atomic size (still carrying
charges)
Multiply Charged Ions
ESI spectrum of
HEW Lysozyme
MW = 14,305.14
Determination of Mass of Protein from
ESI
Determination of Mass of Protein from
ESI
Each peak is given by:
m/z = (MW + nH+)
n
m/z = mass-to-charge ratio of each peak on spectrum
MW = MW of parent molecule
n = number of charges (integer)
H+ = mass of hydrogen ion (1.008 Da)
Determination of Mass of Protein from
ESI Charge (n) is unknown, Key is to determine MW
Choose any two peaks separated by 1 charge
1431.6 = (MW + nH+) 1301.4 = (MW + [n+1]H+)
n [n+1]
2 equations with 2 unknowns - solve for n first
n = 1300.4/130.2 = 10
Substitute 10 into first equation - solve for MW
MW = 14316 - (10x1.008) = 14305.9 14,305.14
Electrospray Ionization Mass Spectrum of
Cytochrome C