ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 21 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of...
-
Upload
myles-mcgee -
Category
Documents
-
view
229 -
download
2
Transcript of ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 21 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of...
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