separations - s ee text for chapters on each topic 1. solvent extraction 2. what is chromatography...
TRANSCRIPT
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Separations - see text for chapters on each topic
1. Solvent Extraction
2. What is Chromatography
3. Efficiency of Separation
4. Why Bands Spread
5. Electrophoretic Separations (gel and capillary)
6. Electrochromatography
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Separations – two-phase separation: partitioning
Liquid Solid Gas GLC GSC distillation sublimation vapor phase chrom. (VPC) Liquid L-L extraction liquid chrom. (e.g., HPLC) Hg electrochem deposition Solid precipitation adsorption chrom. electrodeposition zone refining (melting)
Single phase separation: electrophoresis; ultracentrifugation ; diffusion; mass spectrometry; excited state reactions
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Solvent extraction
21
1
1
1
2
11
22
1
2
:dunextractefraction where
/
/)1(
/
/
KVV
Vq
m
mq
Vqm
Vmq
Vm
Vm
S
SK
Partition coefficients and undissociated speciesPartition coefficient:
i phase of volume:
totalmoles :
i phasein moles :
i
i
V
m
m
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Multiple extractions
n
n
KVV
Vq
21
1
For multiple extraction with each extraction using same volume of V, : the fraction unextracted after n extractions
For “infinite number of extractions (i.e., the limit):
2
1
)( V
VK
eq
Dependence of multiple extractions on Particitition Coefficient
(V1=V2)
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
Number of extractionsFr
actio
n un
extra
cted
K=1
K=0.2
K=0.1
K=0.05
K=0.01
n=99%95%90%82%37%
e.g. Consider Kp=2 and single vs 5
extractions (V1 = 100 mL; V2(total) = 1 L)
Single: q = 100/[100+(K*1,000) = 0.048 (4.8% unextracted or 95.2% extracted)Multiple: q=[100/(100+(K*200)]5 = 0.00032 ( 0.032% unextr. or 99.998% ext.)
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][
1 phasein ion concentrat total
2 phasein ion concentrat totalt coefficienon Distributi
HK
KKD
KHBBH
D
a
a
a
Extraction of acid/base species
B +H+
B
BH+
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Extraction of metal chelates
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Counter current extraction (equilibrium-based partitioning)
1 2 3
1 2 3 1 2 3
1 2 31 2 3
1 2 3
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Calculations after many extractions
1 2 3
r: tube #
fraction present in the r th tube after n extractions
nr
qqrnr
nf rnr
rn
where
)1()!(!
!,
For large values of n:
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stationary
mobile
Counter current vs. “chromatography”
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Chromatography
Separating Molecules
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Science of Chromatography
Separation (relative speeds of molecules) depends on Polarity of solvent Polarity of substrate Other molecular properties (b.p., chirality, etc.)
Like moves fastest with like Polar molecules move fastest with polar solvents
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Chromatography
Two phases• Stationary phase
Solid or liquid Spatially fixed throughout experiment
• Mobile phase Liquid or gas In motion relative to the stationary phase
Chromatography is classified as to type of mobile phase and stationary phase • LC (liquid mobile) . . . . liquid or solid stationary• GC (gas mobile) . . . . usually liquid stationary
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Types of chromatography
adsorption partition ion-exchange molecular exclusion (size exclusion) affinity chiral separations
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Subtypes of Chromatography
Paper chromatography Thin layer chromatography (TLC) Gas chromatography (GC) High-performance liquid chromatography
(HPLC) And several others
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Liquid Chromatography
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Paper Chromatography
Spottingsample(pencil marknear spot)
Developingchamber
Solventfront
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Original spot(pencil line)
Final position ofsolvent front (pencil line)
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Visualizing Spots
May be visible If not, try
• UV light• I2
• Mark spots with pencil when visualized
Lichen extract
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Retardation Factor, Rf
R Ad
dfA
S
( )
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Rf is relatively imprecise Cut out spots, extract solute and run further tests
to complete identification In paper chromatography,
• The mobile phase is a• The stationary phase is a• The chromatographic classification is LSC
liquid
solid?
?
?
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Cellulose
Absorbed water in thepolar regions is actually the stationary phase
Thus, paper chromatography’s actual classificationis LLC
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Thin Layer Chromatography (TLC)
Stationary layer is a thin layer of a solid bonded to an inert plastic or glass binder• Silica (SiO2)
• Alumina (Al2O3)
• Binder is often plaster of Paris
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Silica
Surface O’s converted into O-H’sSmall particles mean very enormous surface areaAlumina works similarly
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TLC is an example of LSC TLC is exactly like paper chromatography in terms
of the actual procedure Rf values are more stable than on paper In forensics, TLC is routinely used for identifying
and/or individualizing• Inks• Dyes• Drugs
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Thin Layer Chromatography
What components
are in the unknown
from the case?
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Thin Layer Chromatography
Identifying
gasoline
by separation
of dye
additives in
the fuel
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High Performance Liquid Chromatography (HPLC)
Directly analogous to GC• Column is relatively short (10-30 cm), with inside
diameter of 4-10 mm• Column is very tightly packed• LSC
Packed with microparticles (3-10 m) of silica or alumina
• LLC Packed with microparticles coated with a liquid
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Liquid solvents (up to four) replacecarrier gas in GC experiment
Column is at roomtemperature
Liquid pressure isenormous (in excessof 6000 psi = 45 bar)
Even at enormous pressurethe flow rate is 0.1-10 mL/min
Detector is double-beam UV-Visspectrometer, constantlyscanning a selected range of ’s
Can also hook-up the columndirectly to a MS to give LC-MS
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HPLC Data
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HPLC – use of solvent gradients
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Ion Chromatography
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Ion Chromatography
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Size Exclusion Chromatography
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Gas Chromatography
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Gas Chromatography (GC)
Column • Contains stationary phase• Long coil (2-3 m) of tubing• Relatively narrow internal diameter (2-4 mm) • Glass or metal
Stationary phase• May be solid (GSC)
Silica, alumina• May be liquid at operating temperatures (GLC)
Squalane, C30H62
Dimethyl silicone oil, (CH3)3-Si-O-[Si(CH3)2-O]n-Si(CH3)3
Coated onto inert beads or granules
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Equilibration between Phases
Molecules divide up between those in mobile phase and those in the stationary phase
Rapid equilibrium established Avoid column overloading
The ratio in each phase depends on Temperature Polarities of column material, mobile phase, and
molecules “other” factors
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Gas Chromatograms
Complex mixtures can have many peaks
Broad peaks (later) High backgrounds can
come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)
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Identification of species: retention times
Capacity factor: k’= (tr
- tm) / tm
t solute is in stationary phase = ---------------------------------------- t solute is in mobile phase
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Qualitative Analysis
Retention times, tR
• Difficult to develop data base because of the variety of factors which alter tR
Nature of stationary and mobile phases Length and diameter of column Flow rate of carrier gas Temperature, etc.
Compare original chromatogram to a known chromatogram Use GC to separate components, then identify them in some
other fashion (IR, MS, etc.)• Detector must be nondestructive
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Band broadening and resolution
van Deempter equation
'
'2
2
2
1Resolution
16
avg
r
k
kN
H
L
w
tN
• plates ( ‘theoretical plates’)• plate height, H• Height equivalent of a theoretical plate, HETP• number of plates• resolution
Improved resolution with larger N, longer retention on column (i.e., larger k’)
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Band Broadening Description –assumes Gaussian peaks
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Pressure reducers
Flow rate valve
Mobile phase iscalled “carrier gas”(He, Ar, N2)
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Column housed in oven
User selectstemperature
Must be sufficient to volatilize allcomponents
Lower temperatures lead to better resolution but longer retention times
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GC Improvements
Capillary Columns• Fused silica• 10-60 m long• 0.1-0.3 mm internal
diameter• Coated with a liquid
(GLC) Temperature
programming
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Types of columns
Packed high capacity common in many types
of chromatography adsorption, LC (e.g., HPLC), affinity, frontal
Open tubular less capacity better resolution (less
band broadening) preferred approach for
most GC applications
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Flame Ionization Detector (FID)
2 H2 + O2 → 2 H2O
Proceeds withoutionic intermediates
Cathode-
Amp
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Flame Ionization Detector (FID)
CH4 + 2 O2 → CO2 + 2 H2O
Proceeds by forming a variety of ionic intermediates
Cathode-
Amp
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Gas Chromatograms
Complex mixtures can have many peaks
Broad peaks (later) High backgrounds can
come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)
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Temperature Isothermal runs can suffer from:
Very broad peaks after long tR at low temps.
Poorly Resolved peaks at short retention times at high temps.
Use programmed temperature ramp Start at low temp
Allows resolution of early eluting cmpds.
Ramp to high temp Prevents band broadening of
late eluting cmpds.
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Quantitative Analysis
The area under each peak is proportional to the percent (by mass) of the analyte in the original sample
A1 : A2 : A3 : … = %1 : %2 : %3 : …• Get areas by
Numerical or electronic integrator Does not account for different detector response to different
molecules
Alternatively, prepare a calibration curve for each analyte • Area versus concentration
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GC-MS
GC is excellent for separating mixtures MS is excellent for qualitative analysis Make the MS the detector for the GC
• Always use a capillary column• No “regular” detector at the end• The column is plumbed directly into the MS ion source
subsystem
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Experiment starts whensample is injected into GC
At that instant, start the MSrecording spectra sequentially
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Experiment starts whensample is injected into GC
It takes about 0.6 s to record a MS.Thus, about 100 spectra are recorded (and stored) every minute
It may take 15-45 min forall the components to elutefrom the GC column
Thus the MS may record andstore 1500 – 4500 spectra duringthe lifetime of the experiment
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Most of these are blanks (only carrier gas coming through)
It may take 15-45 min forall the components to elutefrom the GC column
When an analyte componentappears, its band may take 10 sto completely elute
During that time, perhaps 17 orso spectra are recorded. The peakswill get more and more intense,and then will wane
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Quantitative Analysis For each chromatographic peak
• Calculate the Total Ion Count (TIC)• Plot TIC versus time
Has the appearance
of a regular GC Perform the same
sorts of quantitative
analysis as you
would do on any
other chromatogram
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Qualitative Analysis w/ MS
Sort through the 1500 – 4500 spectra and pick out the ones that aren’t blank
Of the sets of non-blanks, pick out the most intense from each set
Identify the component based on the MS fragmentation patterns (MS is covered in a separate section of the course)
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Problem Samples
Substances with enormous molecular masses (e.g.: polymers)• Not volatile• Not soluble
Much forensic evidence falls in this category• Paint• Grease• Tars• Fibers• Plastics
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Pyrolysis GC (PGC)
The analyte is pyrolyzed (heated to 500 – 1,000 oC in the absence of oxygen)• Giant molecules fragment (often into radicals)
Take GC of the fragments (pyrogram) Will not identify the analyte, but can be used to
individualize it• Compare complex pyrograms of known and questioned
samples
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Pyrolysis GC Data (with FIDetector)