Spectroscopic Structure Determination
Chromatography (GC, LC, TLC, HPLC, electrophoresis):
Separation of different molecules by their solubility, size, or charge;
identification by comparison with known. Sometimes destructive (depending on detectors).
Mobile Phase - Gas or Liquid
Stationary Phase - Liquid or Solid
Solid Support (for stationary phase) - Paper, Diatoms, Capillary column wall
Detectors - Flame Ionization; Radioactivity; Mass Spectrometer; Refractive Index; Ultraviolet Spectrophotometer; Colored Indicators (iodine
and others)
Sample - milligram quantities; solid (in solution), liquid, or gas
Column Spectrum - sharp randomly spaced peaks rising from the baseline
Measurements - Distance or Time; Amount
Distance - how far the sample travels in a given time
Time - how long it takes to travel through a column of known length
Mass Spectrometry (MS):
Identification of an unknown molecule by analysis of spectrum or by matching the spectrum of a known
compound. The insrument ionizes the molecule and determines the mass of the cation radicals (molecular ion and the fragments
formed as some ions break apart) formed. Destructive.
Sample - a milligram or so; may be gas, liquid, or solid; must be pure and have a low enough boiling point to vaporize at low pressure
(~1x10-7torr) without decomposing.
Spectrum - very sharp peaks rising from baseline.
Measurements - peak intensity; mass/charge ratio (m/z)
Intensity: Base Peak (100%) - tallest peak, all other peaks measured relative to this
m/z ratio: because z is usually 1 this ratio represents the mass of the radical cations
Molecular Ion (Parent Ion; M+.) - indicates the molecular weight (MW)
Fragment Masses - indicates atom groupings formed as some of the radical cations break apart
Our Use - To determine the molecular formula from the MW by: a) calculating formulas consistant with the MW and distinguish based on
either a very accurate MW or the isotopic ratio (M+./M+1+.); or b) using MW with elemental analysis to determine the molecular
formula (see Molecular Formula below); or
Molecular Formula:
Using the percent composition (from elemental analysis) and the MW to determine the molecular formula.
Determine Mass of Element:
Determine # Atoms of Element:
percent of element
mass of element
mass of element =
x molecular weight
# atoms =
100
atomic weight of element
Unsaturation Number:
Unsat. # =
Indicates the total number of pi bonds plus rings.
(2x#C+2)+#N-(#H+#X)
2
Other than the elements in the formula, this is our first information about the functional groups present in our unknown.
Infrared Spectrophotometry (IR):
Identification by analysis of spectrum or by matching the fingerprint region with a known spectrum.
Light is absorbed when the stretching or bending of bonds in the molecule requires an amount of energy equivalent to the energy in
the photons of that particular frequency of light. Not destructive.
Sample - 10mg without solvent (neat) or with solvent; may be gas, liquid, or solid (in solution, nujol mull, or KBr pellet) held in or between
salt windows (NaCl, KBr, AgI).
Spectrum - sharp or broad peaks descending from the baseline; position and strength of peak indicate the type of bond absorbing the light.
Measurements
Percent Transmittance, %T, (or Absorption) - size
Interpreting IR Spectra
of peak
Peaks descend from "baseline" at the top of the spectrum.
Wavenumbers, 1/cm, (or wavelength, microns) The IR spectrum is especially handy when trying to get information about atoms
position of peak; wavenumbers are linear for
that have no hydrogens attached because these will not show up in the NMR.
The peaks we will concentrate on stand out either because of their location or their
grating, wavelengths are linear for prism.
size so there is little ambiguity.
Our Use - to determine the functional groups.
O-H
C=C
C=N
Fingerprint
Region
-C-H
4000
3500
3000
C=O
2500
2000
1800
1600
1/cm
1
1400
1200
1000
800
600
400
The N-H stretch indicative of amines appears in the same region as the O-H
stretch
but is somewhat smaller. Two peaks at the tip means a primary amine (2 H's)
while a weaker single peak shows the presence of a secondary amine (1 H).
The O-H of a carboxylic acid is a very strong and broad peak which covers the
same
region as the C-H but extends out on each side.
The sharp peaks at the other end of the spectrum allow us to distinguish between
ortho, meta, and para disubstituted benzenes: ortho 770-730(s);
meta 810-750(s), 725-680(m); para 860-800(s).
4000
2°
1°
m
N-H
C=C-H
p m
o
C=C
3500
3000
2500
2000
1800
1600
1400
1200
1000
800
600
400
1/cm
Nuclear Magnetic Resonance (NMR):
Identification by analysis of unknown spectrum. In proton magnet resonance (PMR) the signals
represent the nuclei of hydrogen atoms in the molecule. Energy is absorbed by the sample when the radio frequency equals the
precession frequency of the hydrogen nucleus in a magnet field. These frequencies range from 60MHz to 400MHz depending on
the strength of the magnetic field. Each signal indicates a) the environment of the nucleus, b) how many nuclei share that
environment, c) how many similar nuclei are nearby, and d) which signal corresponds to those nearby nuclei. Not destructive.
Sample - 50mg of solid or liquid in a solvent; sample held in a quartz tube; tetramethylsilane (TMS) is added to the sample to provide a
reference point in the spectrum.
Spectrum - usually sharp peaks rising from the baseline; each signal may have one or more peaks grouped in a pattern; TMS signal represents
zero on the delta (∂) scale.
Measurements
Number of Signals - each chemically different set of hydrogens has a signal.
Integration - area under the signal’s peaks; proportional to the number of equivalent hydrogens the signal represents.
Coupling - the splitting of a signal into two or more peaks caused by hydrogens on an adjacent atom; points to remember:
1. Hydrogens of the same signal do not couple.
2. # peaks = number of equivalent hydrogens on adjacent atoms + 1
or
# peaks = (# hydrogensset a + 1) x (# hydrogensset b + 1) x ...
3. Peaks of coupled signals have the same spacing - coupling constant
Chemical Shifts - measured in PPM (parts per million) relative to TMS (zero); indicates the shielding (upfield, toward TMS) or
deshielding (downfield, away from TMS) of the nucleus by nearby atoms or bonds.
2