Basic Biomedical Science

International Class Program

Faculty of Medicine University of Indonesia

Lecture 1
Photometry

Ade Arsianti
Department of Medical Chemisty
Faculty of Medicine University of Indonesia
2016
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 Outline
• Introduction
• UV-Vis Spectroscopy
• Infrared (IR) Spectroscopy
• Mass Spectroscopy (MS)
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Including the medical application of UV-Vis, IR,

MS and NMR spectroscopy.

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OVERVIEW
Photometry and Spectroscopy are important tools for organic and medical chemists
which are used both to gather information about the structure of a molecule
(compound), and as an analytical tool to determine the concentration of analyte in a
solution. Types of spectroscopy including: Ultra violet-Visible (UV-Vis) spectroscopy,
Infrared (IR) spectroscopy, Mass spectroscopy (MS) and Nuclear Magnetic Resonance
(NMR) spectroscopy.

Introduction
• Photometry : Measurement of the properties of light,
especially luminous intensity.
• Spectroscopy is an analytical technique which helps
determine structure.
• Spectrophotometry is the measurement of intensity
(amount) of light absorbed or transmitted by subtances
in solution. This is measured using an instrument that is
called spectrophotometer .
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 Types of Spectroscopy
• Ultraviolet (UV) spectroscopy uses electron transitions
to determine bonding patterns.
• Visible (Vis) spectroscopy measures amount of light
absorbed by colored solute in solution.
• Infrared (IR) spectroscopy measures the bond vibration
frequencies in a molecule and is used to determine the
functional group.
• Mass spectrometry (MS) fragments the molecule and
measures the masses.
• Nuclear magnetic resonance (NMR) spectroscopy
detects signals from hydrogen atoms and can be used to
distinguish isomers.
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ELECTROMAGNETIC (EM) SPECTRUM

ROY G B V 5
 V= Frequency, λ = wavelenght, E = Energy of photon

c = 299,792,458 m/s is the speed of light in vacuum

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UV-VISIBLE SPECTRUM

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 UV / VISIBLE SPECTROMETER

The main
components of
UV/Vis
Spectrometer

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 Io IT Abs = 0.51

• Io : Intensity of light before pass through the sample

• IT: Intensity of light after pass through the sample
• Transmittance (T) is the fraction of light that pass through the sample.
• Absorbance (A) is the fraction of light absorbed by sample.
For the mathematically minded:
• Transmittance (T) = IT / Io or 1/T = Io / IT
• Absorbance (A) = log (Io / IT ) or A = log (1/T)
Converting Transmittance to Absorbance
A = log (1/T), if T is expressed as %T, so that A= log (100/%T)
A = log 100 –log %T
A = 2 – log %T 8
Identifying a compound by UV 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. For
example: the nitrogen bases of nucleic acids.
NH2 NH2 O
O

N H 3C
N N N NH
NH

N N N N O N O
H N NH2 H H
H
Thymine
Guanine Adenine Cytosine
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 Nucleic Acid Absorption Properties
Base lmax Absorption Spectrum: Guanine Absorption Spectrum: Adenine

(nm) 0.5

0.4
1.0

0.8

Absorbance

Absorbance
0.3 0.6

Guanine 275 0.2 0.4

0.1 0.2

0 0.0

Adenine 260 220 240 260 280

Wavelength (nm )
300 320 220 240 260 280
Wavelength (nm )
300 320

Cytosine 265 Absorption Spectrum: Cytosine Absorption Spectrum: Thymine

0.5 0.6

0.4 0.5
Absorbance

Absorbance
Thymine 258 0.3
0.4
0.3
0.2
0.2
0.1 0.1
0.0 0.0
220 240 260 280 300 320 220 240 260 280 300 320
Wavelength (nm ) Wavelength (nm )

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 Determination of the concentration of compounds
by UV/Vis Spectrophotometry

• Quantitative UV/vis is used to determine the concentration

of an analyte usually in an aqueous solution.
• In order to be able to do this, the analyte must absorb in the
UV/Vis region. Especially for visible, the analyte should be
coloured-solution.

• Lambert-Beer's Law is a linear relationship

between absorbance and concentration.
• A = a * b * c, where c is concentration, A is
absorbance, b is path length (usually 1 cm) and a is
the molar absorbtivity.
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 Determination of the concentration of protein albumin
by Visible Spectrophotometry
• Many compounds are not themselves colored but can be made to absorb light in the
visible region by reacting them with suitable reagent.
Protein + Biuret reagent  Violet complex of copper-protein (lmax = 550 nm)
• Prepare : variety concentration of standard solutions, blank solution, sample solution
Solution Volume of 20 Volume of Volume of Concentration
mg/ml standard water Biuret reagent of standard
stock Protein (mL) (mL) (mL) Protein (mg/mL)
Blank - 1,00 9.00 0
Standard 1 0.50 0.50 9.00 1.0
Standard 2 0.75 0.25 9.00 ?
Standard 3 1.00 - 9.00 ?

V1 x C1 = V2 X C2, so that C2 = V1 x C1/V2

For standar 1, C2 = (0.5 mL x 20 mg/mL)/(0.5 + 0.5 + 9 mL) = 10/10 = 1.0 mg/mL
Could you please calculate the concentration of standar 2 and standard 3 ?
• Blank solution contains the solvent and the chemical reagents , but not contain
the substance being assayed. Before measuring the absorbance value of
standar and sample solutions. The blank solution is set to zero absorbance.
• Sample solution : 1 mL of unknown sample + 9.00 mL of biuret reagent 13
 • Make a calibration curve between A vs C of standard solution at paper graph ,
and determine the line equation A=mC + b with :
A = Absorbance value measured
C = Concentration of protein standard (mg/mL)
m = Gradient of dy/dx
b = intersection value of line at ordinate axis

• Data (example)

Solution Concentration of Absorbance

A

.
standard protein value
(mg/ml)(C) (A)
Blank 0 0 0.35
Standard 1
Standard 2
Standard 3
Sample
1.0
1.5
2.0
Cs = ?
0.10
0.22
0.35
0.25
0.25
0.22

. .
0.10
Cs
• The concentration of the unknown sample (Cs) 0
is then extrapolated from this calibration graph, 1.0 1.5 2.0
or by entering A value of sample at line Cons. of protein (mg/mL)
equation , A = mC + b, then C can be calculated.
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 INFRARED SPECTRUM

• Wavelengths usually 2.5-25 mm.

• More common units are wavenumbers, or cm-1, the reciprocal of the wavelength in
centimeters. Wavenumbers are proportional to frequency and energy.
• Infrared (IR) is used to determine functional groups in a molecule
• IR radiation is absorbed by molecules and converted into energy of molecular vibration
(streching and bending vibration). The fingerprint region is the complex vibrations in
infrared spectrum ranging of 600 to 1400 cm-1 which is specific for each molecule. 15
 Carbon-Carbon Bond streching Carbon-Hydrogen streching
• Stronger bonds absorb at higher frequencies:
– C-C 1200 cm-1 Bonds with more s character absorb at a
higher frequency.
– C=C 1660 cm-1
– CC 2200 cm-1 (weak or absent ) – sp3 C-H, just below 3000 cm-1
• Conjugation lowers the frequency: – sp2 C-H, just above 3000 cm-1
– isolated C=C 1640-1680 cm-1 – sp C-H, at 3300 cm-1
– conjugated C=C 1620-1640 cm-1
– aromatic C=C approx. 1600 cm-1

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 O-H and N-H Stretching
• Both of these occur around 3300 cm-1, but they look different.
– Alcohol O-H, broad with rounded tip. Secondary amine (R2NH), broad with one sharp spike.
– Primary amine (RNH2), broad with two sharp spikes. No signal for a tertiary amine (R3N)

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Specific Medical Applications of Vibrational IR spectroscopy
1. Near Infrared Spectroscopy (NIRS) uses near infrared light between 650
and 950 nm to non-invasive measurement of the amount and oxygen
content of blood hemoglobin in brain, muscle and other tissues and is
used e.g. to detect changes induced by brain activity, injury, or disease.

This is the absorption

spectra of oxygenated and
deoxygenated hemoglobin
and water for typical
concentrations found in the
brain of healthy patient.

Any change in this NIRS

spectra indicates the injury
or disease (sickle cell
anemia or Thalasemia.

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Specific Medical Applications of Vibrational IR spectroscopy
2. Urine Analysis in Diabetic Patient
Near Infra Red Spectroscopy region at wavelenght 700 -2000 nm is useful for urine
analysis in diabetic patient.

Healthy human urine: high concentration of urea but no glucose was observed
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NIRS analysis of Human Urine : Normal vs Diabetic Patient

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 Mass Spectrometry
• Molecular weight can be obtained from a very small sample.
• It does not involve the absorption or emission of light.
• A beam of high-energy electrons breaks the molecule apart.
• The masses of the fragments (m/z) and their relative abundance
reveal information about the structure of the molecule.

• High Resolution MS (HRMS) can measure more exact mass of

molecule. For example : a molecule with mass of 44 could be
C3H8, C2H4O, CO2, or CN2H4.
If a more exact mass is 44.028, pick the correct structure
from the list below:

C3H8 C2H4O CO2 CN2H4

44.06260 44.02620 43.98983 44.03740
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 The GC-MS
A mixture of compounds is separated
by gas chromatography, then identified
by mass spectrometry.

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Mass Spectrum with Bromine

Mass Spectrum with Chlorine

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Medical Aplication of Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) as a tool for the
analysis of volatile organic compounds (VOCs). Many VOCs are produced within the human
body in metabolic processes. The abundance of acetonitrile and acrylonitrile in exhaled
breath provides good markers for active and passive Smoking. Nearly all the VOCs contained in
tobacco smoke are removed from the body quite rapidly via enzymatic reactions and excretion.

Figure beside shows how the

concentration of acetonitrile
and acrylonitrile increase
rapidly in the breath of a test
person smoking three
cigarettes. Following cigarette
smoking, breath
concentrations of acrylonitrile
decline rapidly to initial level,
but acetonitrile is removed
very slowly from the body, so
it will accumulate in proportion
to the amount of this
compound inhaled by a test
person . For this reason
acetonitrile can be used for
quantification of passive and
active smooking.
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 Nuclear Magnetic Resonance (nmr)
•The nuclei of many atoms do not spin: 2H, 12C, 16O, …but, some atoms have
nuclear spin: 1H, 13C, 19F, …
•If we place these nuclei in a powerful magnetic field (EM radiation of about
300 MHz (radio waves), they can line up with or against the field by spinning
clockwise or counter clockwise.

N N

N - spin state, S - spin state,

favorable, unfavorable,
lower energy higher energy
S N
S S

A spinning nucleus with it's magnetic field A spinning nucleus with it's magnetic field
aligned with the magnetic field of a magnet aligned against the magnetic field of a magnet

• So, if we bombard the molecule with 300 MHz radio waves, the protons will absorb that
energy and we can measure its absorbance which appear as a number called “the chemical
shift” (also called d). What makes it useful is that different protons usually appear at
different chemical shifts (d). So, we can distinguish one kind of proton from another.

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30
 Integral and line splitting pattern
Consider ethyl acetate. There are three kinds of protons in this molecule, the CH3 next to the
carbonyl, the CH2 next to the O and the CH3 next to the CH2. The ratio of the signals arising
from each of these kinds of protons should be 3 to 2 to 3, respectively. So, if we look at the
height of the integrals they should be 3 to 2 to 3. With this information, we can know which is
the CH2 signal (it’s the smallest one, quartet line with intensity 1:3:3:1, because it has 3
neighboring protons). To distinguish the other two methyl group, we have to be able to predict
their chemical shifts. The chart on the previous page and H-H coupling splitting allow us to
make that assignment (the CH3 next to the C=O should appear at ~ 2 ppm with singlet line
because no neighboring proton, while the other CH3 next to CH2 should be at ~ 1 ppm, triplet
line (intensity 1:2:1, because has 2 neighboring protons of CH2).

3H'S
O

O CH3 O
O
H H 3C O
O H
3H'S

2 H'S

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Magnetic resonance imaging (MRI) is a medical imaging technique used to visualize
internal structures of the body in detail. MRI uses the property and principle of nuclear
magnet resonance (NMR) to image nuclei of atoms inside the body.
MRI scans require strong magnetic fields and non-ionizing radiation electromagnetic
fields in the radio frequency range. It provides good contrast between the different
soft tissue of the body, which makes it especially useful in imaging the brain, muscle,
the heart, and cancers. MRI is primarily used to demonstrate pathological alterations
of living tissue and distinguish pathologic tissue from normal tissue.

Magnetic resonance imaging (MRI) of brain

abscess shows a hyperintense necrotic core
with a hypointense capsule, surrounded by
hyperintense edema. 34