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ANALYTICAL CHEMISTRY CHEM 3811
CHAPTER 21
DR. AUGUSTINE OFORI AGYEMANAssistant professor of chemistryDepartment of natural sciences
Clayton state university
CHAPTER 21
CHROMATOGRAPHYAND
MASS SPECTROMETRY
CHROMATOGRAPHY
- The most powerful tool for separating mixtures
- Used for both qualitative and quantitative analysis
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
CHROMATOGRAPHY
- A column is packed with the stationary phase
- Mobile phase passes through the stationary phase
- Separation process involves the interaction of themobile phase (a mixture) with the stationary phase
CHROMATOGRAPHY
A
B
eluent
eluate
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
CHROMATOGRAPHY
Elution- The process of passing a liquid or gas through a column
Eluent- Fluid entering the column
Eluate- Fluid exiting the column
CHROMATOGRAPHY
Gas Chromatography (GC)
- Mobile phase is a gas
Liquid Chromatograpgy
- Mobile phase is a liquid
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
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
CHROMATOGRAPHY
Partition Chromatography
- Stationary phase is a thin liquid coated on the surfaceof a solid support
- Mobile phase is a liquid or a gas
- Solute equilibrates between the stationary and mobile phases
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
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
CHROMATOGRAPHY
Affinity Chromatography
- Very selective
- Based on specific interactions between a type of solute moleculeand another molecule covalently attached to the stationary phase
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
THE CHROMATOGRAM
tr
tr
h
1/2h
w1/2 = 2.35σ
w = 4σ
Det
ecto
r re
spon
se
Time
THE CHROMATOGRAM
- An ideal chromatogram has a Gaussian shape
- h = height of peak
- σ = the standard deviation of the peak
- w = base width = 4σ
- w1/2 = width at half height (w at 1/2h) = 2.35σ
- tr and w can be measured in time or volume units
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)
21/2
2r
w
5.55tN
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
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
To Test a Column for Degradation
- Inject standards periodically
- Look forPeak asymmetry
Change in number of plates
THEORETICAL PLATES
- Peak separation (Δtr) divided by the average peak width (wav)
- Better resolution implies more complete separationbetween neighboring peaks
RESOLUTION
1/2(av)
r
av
r
w
t0.589
w
ΔtResolution
- Doubling the length of a column (2L) increases resolution by √2
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 differentretention times on different stationary phases
QUANTATIVE ANALYSIS
- Chromatographic peak area is proportional to quantity of solute
- A good measure of solute concentration is obtained byusing internal standards
- Internal standards eliminate the effect of variable conditions
- Conditions mostly vary from run to run
QUANTATIVE ANALYSIS
Conditions Include
- Sample injection errors or changes
- Column changes
- Detector variations
QUANTATIVE ANALYSIS
Internal Standard Method
- Concentration of analyte (canalyte) can be determined using the concentration of internal standard (cIS) and both peak areas
analyte
IS
analyte
IS
Area
Area
c
c
SCALING UP
Analytical Chromatography
- For small-scale analysis
Preparative Chromatography
- For large-scale analysis
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
2
radiuscolumnsmall
radiuscolumnlarge
(g)loadsmall
(g)loadlarge
BAND BROADENING
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
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
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
BAND BROADENING
van Deemter Equation
- The plate height equation as a result of the three band broadening mechanisms
Cuu
BAH
Multiplepaths
Longitudinaldiffusion
Equilibrationtime
BAND BROADENING
van Deemter Equation
u = flow rate
A, B and C are constants dependent on - Column
- Stationary phase- Mobile phase- Temperature
OPEN TUBULAR COLUMN
- Hollow capillary column
- Inner wall is coated with thin layer of stationary phase
- Gives better separation than packed columnNo 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)
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 behindthe concentrated region
ASSYMETRIC PEAKS
- Tailing is when the trailing part is elongated
- Occurs when the stationary phaseis 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
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
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)
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
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
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
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
MASS SPECTROMETRY
Types of Mass Spectrometers (Analyzers)
Electrostatic
Magnetic
Time of flight
Ion trap (quadrupole ion storage)
Quadrupole mass spectrometer
THE MASS SPECTRUM
- Fragmentation patterns from the mass spectrum provideinformation 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
THE MASS SPECTRUM
Isotope Pattern- Information is obtained from relative intensities at M+1 and M+
- M+1 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