SPECTROSCOPIC METHODS

seeing the unseeable things

Light interacts analytical tool

with

matter

as

an

Overview
What is light?
an electromagnetic wave that travels in small packets called photons.

Light also has particle character

Energy (E) of a single photon is given by the following formula

 Electromagnetic radiation: light and other forms of radiant energy Wavelength (): the distance between consecutive peaks on a wave >< Wave number: the number of waves per

centimeter
Frequency (): the number of full cycles of a wave passing through a fixed point in a second

a form of energy and may be considered either as a continuous wave travelling through space, or as discrete photons of the same energy

Overview
How to identify a given organic molecule ??? In organic chemistry: to determine and confirm molecular

structures, to monitor reactions and to control the purity of

compounds Spectroscopic method Most essential methods for the organic chemistry are the nuclear magnetic resonant spectroscopy (1H) and 13C NMR-spectroscopy, the mass spectrometry, the infrared

and the UV/Vis-spectroscopy.

Spectroscopy
Utilises the

Absorption and

Emission of

electromagnetic radiation by atoms

Absorption:
Low energy electrons absorb energy to move to higher energy level Using electromagnetic radiation as a probe to
obtain information about atoms and molecules that are too small to see.

Emission:

Electromagnetic radiation is propagated at the speed of light through a vacuum as an oscillating wave.

Excited electrons return to lower energy states

Absorption vs Emission
Fig. illustrates the relationships between different energy levels within a molecule. 3rd Excited 2nd States 1st Energy is absorbed as electrons jump to higher energy levels
Atoms and molecules can absorb electromagnetic radiation, but only at certain energies (wavelengths)

Energy is emitted by electrons returning to lower energy levels

Ground State

Principles of spectroscopy

Common term and Symbol Radiant Power, P, P0 Absorbance, A Transmittance, T Path Length, b Absorptivity, a Molar Absorptivity,

Alternative Name and Symbol Radiation Intensity, I, I0 Optical Density, D Transmission, T l, d Extinction Coefficient, k Molar Extinction Coefficient

Regions of the electromagnetic spectrum

We see only a very small portion of the electromagnetic spectrum

Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength.

Electromagnetic Spectrum
Different spectroscopic techniques different, limited frequency ranges (depending on the processes and magnitudes of the energy changes). Certain energies in the Visible and UV regions: cause electrons to be excited into higher energy orbitals. Very energetic photons (UV to X-ray region): cause an electron to be ejected from the molecule (ionization). Photons in the Infrared region: excite vibrations in molecules. There are many possible vibrational levels within each electronic state. Microwave radiation: cannot excite electrons in molecules, nor can it excite vibrations. It can only cause molecules to rotate.

Electron transition between energy levels result in emission or absorption lines gas atoms absorb the same wavelengths as they usually emit

no gaps in wavelength of light produced

Different elements produce different spectra due to differing atomic structure.

emission only in narrow wavelength regions produced by solids, liquids and dense gases

produced by rarefied gases

Reflection and Scattering Losses

Po Radiant Power

A (Absorbance) = lg Po/P

Reflection and Scattering Losses

When light is absorbed by a sample, the radiant power P (energy per unit time per unit area) of the beam of light decreases. The energy absorbed may stimulate rotation, vibration or electronic transition depending on the wavelength of the incident light. Reflection and scattering losses are significant and to compensate for these effects, the power of the beam transmitted by the analyte solution is ordinarily compared with the power of the beam transmitted by an identical cell containing only the solvent. An experimental absorbance that closely approximates the true absorbance is then obtained with the equation: A = lg Psolvent/Psolution ~ lg Po/P

Quantitative Spectroscopy
Light absorbed (Absorption)
is directly proportional to the

Concentration of the absorbing component.

Transmittance vs Absorbance
Measurement of the amount of radiation absorbed: Transmittance, T = Psolution/Psolvent ~ P / P0 % Transmittance, %T = 100 T Absorbance, A = lg P0 / P worth remembering because it allows A = lg 1 / T you to easily calculate absorbance from percentage transmittance data. A = lg 100 / %T A = 2 - lg %T Absorbance can be defined as the base-ten logarithm of

the reciprocal of the transmittance :

A = lg 1/T = -lgT = -lg P/Po

Transmittance vs Absorbance
The relationship between absorbance and transmittance

If all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite.

UV-Visible (UV-Vis) Spectroscopy

Lets Talk About Beer

More molecules means more light is caught

Why are carrots orange?

Carrots contain the pigment carotene which absorbs blue light strongly and reflects orange red and so the carrot appears orange.
400nm 500nm 600nm 700nm

BLUE

GREEN

Y E L LO W

O R A N G E

RED

420nm

520 nm

600nm

Beers Law quantifies the relationship between color and concentration

Uses of Beers Law:

Relates concentration to the optical measurement of absorbance combined with spectrophotometry can be used to

distinguish and compare different molecules in solution

* Conceptual Basis of Beers Law

Light of a particular wavelength enters the sample. Light scatters from particles in solution (no momentum change) reducing light transmission. Light is absorbed by molecules/particles (momentum

change) and remitted at different wavelengths, reducing

light transmission

Beers Law: more in depth

slit source detector cuvette

A = bc (no units, since A = lg P0 / P) : molar absorptivity (unique for a given compound, units of M-1 cm-1) b: path length of the cuvette or sample holder (usually 1 cm) c: concentration of the compound in solution (M)

Beam of radiation of radiant power Po

Po b

Beers Law predicts linear behavior between concentration and absorbance but not between amount of power

coming out of sample.

Typical Beers Law Plot

Calibration curve showing absorbance as a function of metal concentration
Absorbance ( no units)
1.2 1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 Concentration (mg L-1) y = 0.9982x R2 = 0.9996

Characteristics of Beers Law Plots

One wavelength
Good plots have a range of absorbances from 0.010 to 1.000 Absorbances over 1.000 are not valid and should be avoided

 Beers law is valid at low concentrations, but breaks

down at higher concentrations For linearity, A < 1

Standard Practice
Prepare standards of known concentration

Measure absorbance at lmax

Plot A vs. concentration

Obtain slope
Use slope (and intercept) to determine the unknown concentration of the analyte

 If your unknown has a higher concentration than your

highest standard, you have to ASSUME that linearity still holds (However, its NOT GOOD for quantitative analysis) Unknowns should ideally fall within the standard range

A<1
If A > 1 ???? Dilute the sample Use a narrower cuvette (usually 1 mm, 1 cm or 10 cm)

UV-Vis spectrophotometer
A UV-vis spectrophotometer measures the amount of energy absorbed by a sample.
The optics of the light
source in UV-visible spectroscopy allow either

visible [approx. 400nm

(blue end) to 750nm (red end) ] or ultraviolet

(below 400nm) to be
directed at the sample under analysis.

BASIC design components

1) LIGHT SOURCE
2) SAMPLE Chamber 3) OPTICS 4) DETECTOR 5) OUTPUT

Figure 1

Illustration of a single beam UV-vis instrument.

Figure 2

Illustration of a double beam UV-vis instrument.

Figure 3 Illustration of a simultaneous UV-vis instrument.

 Some have a filter for selecting one wavelength at a time. The single beam instrument (Figure 1) has a filter or a monochromator between the source and the sample to analyze one wavelength at a time. The double beam instrument (Figure 2) has a single source and a monochromator and then there is a splitter and a series of mirrors to get the beam to referencesample analyzed allows for more accurate readings. The reference beam intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement displayed is the ratio of the two beam intensities. In contrast, the simultaneous instrument (Figure 3) does not have a monochromator between the sample and the source; instead, it has a diode array detector that allows the instrument to simultaneously detect the absorbance at all wavelengths. The simultaneous instrument is usually much faster and more efficient.

Choice of solvent
Solvent UV absorbance cutoff (nm)

Acetone
Benzene Dimethylformamide (DMF) Ethanol Toluene Water

329
278

267
205 285 180

The solvent cutoff is the wavelength below which the solvent itself absorbs all of the light.

The solvents for these determinations are often water for water soluble compounds, or ethanol for organic-soluble compounds.

Organic solvents may have significant UV absorption;

not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths. Solvent polarity and pH can affect the absorption spectrum of an organic compound.

Tyrosine, for example, increases in absorption maxima and

molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.

Choice of container
Material Glass Plastic Fused quartz

Wavelength range
(nm) 380 - 780 380 - 780 below 380

The molecular structure of Rose Bengal (4,5,6,7-tetrachloro2',4',5',7'-tetraiodofluorescein).

UV-vis spectra of different concentrations of Rose Bengal.

What information can be obtained from UV-vis spectra?

qualitative and quantitative information of a given compound or molecule. it is important to use a reference cell to zero the instrument for the solvent which the compound is in. quantitative information: calibrating the instrument using known concentrations of the compound (in question) in a solution with the same solvent as the unknown sample. If the information needed is just proof that a compound is in the sample being analyzed, a calibration curve will not be necessary; however, if a degradation study or reaction is being performed, and concentration of the compound in solution is required, thus a calibration curve is needed.

Calibration and reference

A blank reference will be needed
at the very beginning of the

analysis of the solvent to be used. If concentration analysis needs to be performed, calibration solutions need to be made accurately. If the solutions are not made accurately enough, the actual

concentration of the sample in question will not be accurately determined. Calibration curve of Rose Bengal. Equation of line: y = 0.0977x 0.1492 (R2 = 0.996)

 quantitative determination of different analytes

transition metal ions ( color ) highly conjugated organic compounds (water/water soluble compounds, or ethanol/ organic-soluble compounds )

biological macromolecules.

Determination is usually carried out in solutions. Certain parts of an Organic Molecule will ABSORB light energy to create PEAKS on a SPECTRUM for Quantitative (primarily) and Qualitative Analysis

Limitations of UV-Vis spectra

UV-Vis spectroscopy works well on liquids and solutions, if the sample is more of a suspension: the sample will scatter the light more than absorb the light and the data will be very skewed.

Infrared (IR) Spectroscopy

No two molecules of different structure will have exactly the same natural frequency of vibration, each will have a unique infrared absorption pattern or spectrum. Two Common Uses: to distinguish one compound from another. Absorption of IR energy by organic compounds will occur in a manner characteristic of the types of bonds and atoms in the functional groups present in the compound; thus, infrared spectrum gives structural information about a molecule Easy to use Unique, used as fingerprint Gas, liquid or solid

Infrared Spectrometer Design

Interaction of light with matter

All organic compounds absorb electromagnetic radiation, of only certain wavelengths and transmit the rest.

When organic molecules absorb energy in the UV-Vis

region, they undergo electronic transitions (electrons in the ground state go to higher states known as excited state) (Absorption spectroscopy) Photon energies associated with infrared are not large

enough to excite electrons, but may induce vibrational

excitation of covalently bonded atoms and groups (Infrared spectroscopy)

 IR involves absorption phenomenon: the absorption of radiation depends on increasing energy of vibration or rotation associated with covalent bond in molecule provide that such an increasing in energy causes a change in the dipole moment of molecule Hence in order to absorb IR radiation, a molecule must go

a net change in dipole moment due to its vibration or

rotation motion means that nearly all molecules containing covalent bond will show some degree of selective IR absorption

Molecular vibrations
The covalent bonds in molecules are not rigid sticks or rods, but are more like stiff springs that can be stretched and bent.

A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. In short, the stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding.

The atoms in a CH2 group, commonly found in organic compounds, can vibrate in six different ways:

Infrared spectra are usually plotted as % transmittance (%T)

Make absorption band appear as dips in the curve than as maxima as in the case of UV-Vis. Each dip in the spectrum is called a band or peak and represented absorption of IR radiation at that frequency by

the sample
The transmittance is 0% if all the radiation is absorbed and the transmittance is 100% for no absorption

Infrared (IR) Spectroscopy

Frequency of the incident radiation is varied and the quantity of radiation absorbed or transmitted by the sample is obtained.

wavenumber: 4000 - 400 cm-1

Note: IR spectra are typically presented in units called wave number, or more correctly, reciprocal centimetres (cm-1). Increasing wave number corresponds to increasing frequency.

Infrared Spectra

in cm-1

 Stretching: a change in the length of a bond, such as C-H or C-C Bending: a change in the angle between two bonds, such as the HCH angle in a methylene group Rocking: a change in angle between a group of atoms, such as a methylene group and the rest of the molecule. Wagging: a change in angle between the plane of a group of atoms, such as a methylene group and a plane through the rest of the molecule, Twisting: a change in the angle between the planes of two groups of atoms, such as a change in the angle between the two methylene groups.

Carbon-Carbon Bond Stretching

Stronger bonds frequencies:
C-C C=C CC

absorb

at

higher

1200 cm-1 1660 cm-1 2200 cm-1 (weak or absent if internal)

Conjugation lowers the frequency:

isolated C=C conjugated C=C aromatic C=C 1640-1680 cm-1 1620-1640 cm-1 approx. 1600 cm-1

Carbon-Hydrogen Stretching
Alkanes C-H sp3 stretch < 3000 cm-1 Alkenes C-H sp2 stretch > 3000 cm-1 Alkynes C-H sp stretch ~ 3300 cm-1

Carbon-Hydrogen Bending
CH2 bending ~ 1460 cm-1 CH3 bending (asym) appears near the same value CH3 bending (sym) ~ 1380 cm-1

Alkane

*
CH3 bend CH2 bend

CH3 CH2 CH2 CH2 CH2 CH3

C-H stretch

Alkene

sp2 C-H

C=C stretch

sp3 C-H stretch

CH2 CH CH2 CH2 CH2 CH3

Alkyne

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. Acid O-H, very broad Secondary amine (R2NH), broad with one sharp spike. Primary amine (RNH2), broad with two sharp spikes. No signal for a tertiary amine (R3N)

Alcohol

Acid

O
O-H stretch C=O stretch CH3 CH2 CH2 C

OH

Amine

N-H stretch doublet

sp3 C-H stretch

N-H bend CH2 CH3 bend

CH3 CH2 CH2 CH2 NH2

Carbonyl Stretching
The C=O bond of simple ketones, aldehydes, and carboxylic acids absorb around 1710 cm-1. Usually, its the strongest IR signal.

Carboxylic acids will have O-H also.

Aldehydes have two C-H signals around 2700

and 2800 cm-1.

Ketone

sp3 C-H stretch C=O stretch

O CH3 C CH2 CH3

Aldehyde

Variations in C=O Absorption

Conjugation of C=O with C=C lowers the stretching frequency to ~1680 cm-1. The C=O group of an amide absorbs at an even lower frequency, 1640-1680 cm-1. The C=O of an ester absorbs at a higher frequency, ~1730-1740 cm-1. Carbonyl groups in small rings (5 Cs or less) absorb at an even higher frequency.

Amide

sp3 C-H
O

N-H stretch (pair)

C=O and

N-H bend

C CH3 CH2 NH2

Ester

sp3 C-H C=O stretch

C-O stretch
O C CH3 CH2 CH2 O CH2 CH3

Carbon - Nitrogen Stretching

C - N absorbs around 1200 cm-1. C = N absorbs around 1660 cm-1 and is much stronger than the C = C absorption in the same region. C N absorbs strongly just above 2200 cm-1. The alkyne C C signal is much weaker and is just below 2200 cm-1 .
=>

Nitrile

sp3 C-H

C N

CH3 CH2 C

Example of an approach for analyzing IR Spectra

Step 1. Check for the presence of the Carbonyl group (C=O) at 1715 cm-1. If molecule is conjugated, the strong (C=O) absorption will be shifted to the right by ~30 cm-1,i.e., ~1685 cm-1 If the Carbonyl absorption is present, check for: Carboxylic Acids - Check for OH group (broad absorption near 3300-2500 cm-1) Amides - Check for NH group (1 or 2 absorptions near 3500 cm-1) Esters - Check for 2 C-O groups (medium absorptions near 1300-1000 cm-1) Anhydrides - Check for 2 C=O absorptions near 1810 and 1760 cm-1 Aldehydes - Check for Aldehyde CH group (2 weak absorptions near 2850 and 2750 cm-1) Ketones - Ketones (The above groups have been eliminated)

Step 2. - If the Carbonyl Group is Absent Check for Alcohols, Amines, or Ethers. Alcohols & Phenols - Check for OH group (Broad absorption near 3600-3300 cm-1. Confirm present of C-O near 1300-1000 cm-1 Amines - Check for NH stretch (Medium absorptions) near 3500 cm-1 Primary Amine - 2 Peaks Secondary Amine - 1 Peak Tertiary Amine - No peaks N-H Scissoring at 1560 - 1640 cm-1 N-H Bend at 800 cm-1 Ethers - Check for C-O group near 1300-1000 cm-1 and absence of OH

Step 3. Refine the Structure Possibilities by Looking for Double Bonds, Triple Bonds and Nitro Groups Double Bonds - Unsaturated C=C (and CC) stretch show absorptions on the left side of 3000 cm-1 Alkene C=C 2 weak absorptions near 1650 cm-1 Aromatic C=C (4 absorptions 1450-1650 cm-1) (Verify Aromatic at 1667 2000 cm-1) Triple Bonds - R-C N Nitrile - medium, sharp absorption (stretch) near 2250 cm-1 R C C R Alkyne - weak, sharp absorption (stretch near 2150 cm-1) R C C H Terminal Acetylene (stretch near 3300 cm-1) Nitro Groups - Two strong absorptions 1600 1500cm-1 and 1390 - 1300 cm-1