ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 21

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ANALYTICAL CHEMISTRY CHEM 3811CHAPTER 21
DR. AUGUSTINE OFORI AGYEMAN Assistant professor
of chemistry Department of natural
sciences Clayton state university
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CHAPTER 21 CHROMATOGRAPHY AND MASS SPECTROMETRY
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CHROMATOGRAPHY
- The most powerful tool for separating
mixtures - Used for both qualitative and
quantitative analysis
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CHROMATOGRAPHY
Comprises of Two Phases Stationary Phase - A
solid or liquid packed in a column (does not
move) Mobile Phase - A gas or liquid that
passes through the column
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CHROMATOGRAPHY
- A column is packed with the stationary
phase - Mobile phase passes through the
stationary phase - Separation process involves
the interaction of the mobile phase (a mixture)
with the stationary phase
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CHROMATOGRAPHY
eluent
A
B
eluate
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CHROMATOGRAPHY
Adsorption - Occurs when a solute sticks to the
surface of another species - Consider a mixture
containing solutes A and B - A is more strongly
adsorbed to the stationary phase than B - A
moves down the column more slowly than B - B
comes out of column before A
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CHROMATOGRAPHY
Elution - The process of passing a liquid or gas
through a column Eluent - Fluid entering the
column Eluate - Fluid exiting the column
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CHROMATOGRAPHY
Gas Chromatography (GC) - Mobile phase is a
gas Liquid Chromatograpgy - Mobile phase is a
liquid
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CHROMATOGRAPHY
Solutes may be retarded by the stationary phase
based on various interactions - Surface
adsorption - Relative solubility -
Charge Chromatography is classified based on
the type of interactions
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CHROMATOGRAPHY
Adsorption Chromatography - Stationary phase is
a solid - Mobile phase is a liquid or a gas -
Solute adsorbs to the surface of the solid
particles
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CHROMATOGRAPHY
Partition Chromatography - Stationary phase is a
thin liquid coated on the surface of a solid
support - Mobile phase is a liquid or a gas -
Solute equilibrates between the stationary and
mobile phases
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CHROMATOGRAPHY
Ion-exchange Chromatography - Allows separation
of ions and polar molecules - Ionic groups are
covalently attached to a stationary solid phase
- Mobile phase is a liquid - Ionic solutes are
electrostatically attracted to the stationary
phase
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CHROMATOGRAPHY
Size Exclusion Chromatography (Gel Filtration,
Gel Permeation) - Solutes are separated based on
size - Stationary phase has small pores that
exclude large molecules - Small molecules enter
the pores so spend more time in column - Large
molecules come out of column before small
molecules
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CHROMATOGRAPHY
Affinity Chromatography - Very selective -
Based on specific interactions between a type of
solute molecule and another molecule covalently
attached to the stationary phase
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THE CHROMATOGRAM
- Detector response as a function of time or
elution volume - Different peaks correspond to
different eluates Retention Time (tr) - Time
taken by a solute to reach detector after
injection
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THE CHROMATOGRAM
w1/2 2.35s
tr
tr
h
Detector response
w 4s
Time
1/2h
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THE CHROMATOGRAM
- An ideal chromatogram has a Gaussian shape - h
height of peak - s the standard deviation of
the peak - w base width 4s - w1/2 width
at half height (w at 1/2h) 2.35s - tr and w
can be measured in time or volume units
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THEORETICAL PLATES
- Imaginary way to picture the separation
process - Imaginary discrete sections of the
chromatography column - Though the process is
continuous - Retention of solutes can be
described by the number of equilibrium steps
(theoretical plates)
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THEORETICAL PLATES
The number of theoretical plates on a column (N)
The Plate Height (H) - The length of one
plate H L/N L the length of column
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THEORETICAL PLATES
- The higher the N the narrower the bandwidth -
The higher the N better the separation - The
smaller the H the narrower the peaks - The
smaller the H the better the separation
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THEORETICAL PLATES
To Test a Column for Degradation - Inject
standards periodically - Look for Peak
asymmetry Change in number of plates
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RESOLUTION
- Peak separation (?tr) divided by the average
peak width (wav) - Better resolution implies
more complete separation between neighboring peaks
- Doubling the length of a column (2L) increases
resolution by v2
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QUALITATIVE ANALYSIS
- Identify peaks by comparing retention times to
those of authentic samples - Unknown sample is
spiked (authentic sample is added) - The
relative size of a peak will increase if the
authentic sample is identical to one of the
components - Different compounds may have the
same retention time - It is more likely for
different compounds to have different retention
times on different stationary phases
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QUANTATIVE ANALYSIS
- Chromatographic peak area is proportional to
quantity of solute - A good measure of solute
concentration is obtained by using internal
standards - Internal standards eliminate the
effect of variable conditions - Conditions
mostly vary from run to run
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QUANTATIVE ANALYSIS
Conditions Include - Sample injection errors or
changes - Column changes - Detector variations
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QUANTATIVE ANALYSIS
Internal Standard Method - Concentration of
analyte (canalyte) can be determined using the
concentration of internal standard (cIS) and
both peak areas
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SCALING UP
Analytical Chromatography - For small-scale
analysis Preparative Chromatography - For
large-scale analysis
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SCALING UP
- A developed procedure for analytical
chromatography can be scaled up and used for
preparative chromatography - Maintain column
length and increase cross-sectional area -
Volume flow rate should also be increased by the
same factor
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BAND BROADENING
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BAND BROADENING
May be due to Diffusion - Diffusion of solute
molecules away from the center of the band in
both directions - Longitudinal diffusion - The
faster the flow rate the sharper the peaks -
Broadening is inversely proportion to flow rate
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BAND BROADENING
May be due to Solute Equilibration - If solute
equilibrates slowly between mobile and
stationary phases - Solute in stationary phase
tends to lag behind solute in mobile phase -
Broadening is directly proportional to flow rate
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BAND BROADENING
May be due to Irregular Flow Paths - Occurs
since column is packed with solid particles -
There are random multiple paths for solute
particles - These multiple paths are unequal -
Independent of flow rate
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BAND BROADENING
van Deemter Equation - The plate height equation
as a result of the three band broadening
mechanisms
Multiple paths
Longitudinal diffusion
Equilibration time
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BAND BROADENING
van Deemter Equation u flow rate A, B and C
are constants dependent on - Column - Stationary
phase - Mobile phase - Temperature
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OPEN TUBULAR COLUMN
- Hollow capillary column - Inner wall is
coated with thin layer of stationary phase -
Gives better separation than packed column No
multiple paths (A 0) Can be much longer (gives
less resistance to gas flow) Smaller plate
height - Only useful for analytical
chromatography (can only handle small samples due
to less stationary phase)
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ASSYMETRIC PEAKS
- When a band is overloaded by too much
solute - Band emerges gradually in front - An
abrupt cut off is observed behind the
concentration region - Overloading leaves very
little trails of solute behind the concentrated
region
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ASSYMETRIC PEAKS
- Tailing is when the trailing part is
elongated - Occurs when the stationary phase is
strongly polar has highly adsorptive sites (-OH
groups) Salinization - Chemical treatment to
reduce tailing - Converts -OH groups to nonpolar
-OSi(CH3)3 groups - Column should be replaced
when tailing increases
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MASS SPECTROMETRY
- Measures the masses and abundances of ions in
the gas phase - Detector is sensitive to low
analyte concentrations - Distinguishes
different substances with the same retention
time - Used for both qualitative and
quantitative analysis
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MASS SPECTROMETRY
- Molecules are converted to ions prior to
separation - Molecules entering the ionization
chamber of a mass spectrometer are converted
into ions - Ions are separated based on
mass-to-charge ratio (m/z)
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MASS SPECTROMETRY
Two Common Methods of Ionization Electron
Ionization (EI) - Electrons emitted from a hot
filament are accelerated by 70 V - Molecules are
ionized by striking electrons as they absorb
energy M e- ? M e- e- M is
called the molecular ion M breaks into
fragments after ionization
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MASS SPECTROMETRY
Two Common Methods of Ionization Electron
Ionization (EI) - The most intense peak from
fragments is called the base peak - Other peaks
are expressed as percentages of the base peak
intensity
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MASS SPECTROMETRY
Two Common Methods of Ionization Chemical
Ionization (CI) - Ionization chamber contains a
reagent gas (CH4) - Pressure is maintained at
about 1 mbar - Energetic electrons convert gas
into a variety of products
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MASS SPECTROMETRY
Two Common Methods of Ionization Chemical
Ionization (CI) CH4 e- ? CH4 2e- CH4
CH4 ? CH5 CH3 CH5 then protonates
the analyte CH5 M ? CH4 MH -
Fragmentation is less than EI
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MASS SPECTROMETRY
Types of Mass Spectrometers (Analyzers) Electrost
atic Magnetic Time of flight Ion trap
(quadrupole ion storage) Quadrupole mass
spectrometer
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THE MASS SPECTRUM
- Fragmentation patterns from the mass spectrum
provide information about the structure of
analyte molecule Nominal Mass - Integer mass of
the species with the most abundant isotope of
each element For benzene (C6H6) - The most
abundant isotopes are 12C and 1H Norminal mass
(6 x 12) ( 6 x 1) 78
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THE MASS SPECTRUM
Isotope Pattern - Information is obtained from
relative intensities at M1 and M - M1 is one
mass unit above the molecular ion Nitrogen
Rule - Used to propose composition of molecular
ions - Odd nominal mass implies compound has odd
number of N atoms - Even nominal mass implies
compound has even number of N atoms