Gas Chromatographic-Mass Spectrometric and High-Performance Liquid Chromatographic
Chem. 230 – 9/23 Lecture. Announcements Exam 1 today – first 40 min. Second Homework Set will be...
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Transcript of Chem. 230 – 9/23 Lecture. Announcements Exam 1 today – first 40 min. Second Homework Set will be...
![Page 1: Chem. 230 – 9/23 Lecture. Announcements Exam 1 today – first 40 min. Second Homework Set will be online soon Today’s Topics – Chromatographic Theory –Basic.](https://reader030.fdocuments.in/reader030/viewer/2022033103/56649eba5503460f94bc2733/html5/thumbnails/1.jpg)
Chem. 230 – 9/23 Lecture
![Page 2: Chem. 230 – 9/23 Lecture. Announcements Exam 1 today – first 40 min. Second Homework Set will be online soon Today’s Topics – Chromatographic Theory –Basic.](https://reader030.fdocuments.in/reader030/viewer/2022033103/56649eba5503460f94bc2733/html5/thumbnails/2.jpg)
Announcements
• Exam 1 today – first 40 min.• Second Homework Set will be online soon• Today’s Topics – Chromatographic Theory
– Basic definitions (flow – time relationship, distribution constant, retention factor, velocities, plate number, plate height, asymmetry factor, resolution, separation factor)
– How to read chromatograms– Meaning of parameters (more when we cover
optimization)
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Chromatographic TheoryQuestions on Definitions
1. When is chromatographic separation needed vs. only simple separations?
2. An analyte interacts with a stationary phase via adsorption. The stationary phase is most likely:
a) Liquid b) Liquid-like c) Solid3. What are the required two phases in
chromatography called?4. What are advantages and disadvantages
with the three common stationary phases (liquid, liquid-like, and solid)?
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Chromatographic Theory Definition Section – Flow – Volume Relation
• Relationship between volume (used with gravity columns) and time (most common with more modern instruments):V = t·F V = volume passing through column part in
time t at flow rate FAlso, VR = tR·F where R refers to retention
time/volume (time it takes component to go through column or volume of solvent needed to elute compound)
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Chromatographic Theory Definition Section – More on Volume
• Hold-up volume = VM = volume occupied by mobile phase in column
• Stationary phase volume = VS
• Calculation of VM:
VM = Vcolumn – Vpacking material – VS
VM = tM·F, where tM = time needed for unretained compounds to elute from column
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Chromatographic Theory Definition Section – Partition and Retention
• Distribution Constant (= Partition Coefficient from LLE) = KC = [X]S/[X]M
• KC is constant if T and/or solvent remain constant
• Retention Factor (= Capacity Factor = Partition Ratio) = k = (moles X)S/(moles X)M = KC/(VM/VS)
• k = KC/β where β = VM/VS
• Retention Factor is more commonly used because of ease in measuring, and since β = constant, k = constant·KC (for a given column)
• Note: kColumn1 ≠ kColumn2 (because β changes)
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Chromatographic Theory Definition Section – Partition and Retention
• Since the fraction of time a solute molecule spends in a given phase is proportional to the fraction of moles in that phase,k = (time in stationary phase)/(time in mobile phase)
• Experimentally, k = (tR – tM)/tM
• The same equations can be made with volumes instead: k = (VR – VM)/VM
• Note: t’R = tR – tM = adjusted retention time
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Chromatographic Theory Reading Chromatograms
• Determination of parameters from reading chromatogram (HPLC example)
• tM = 2.37 min. (normally determined by finding 1st peak for unretained compounds – contaminant below)
• VM = F·tM = (1.0 mL/min)(2.37 min) = 2.37 mL (Note: 4.6 x 250 mm column, so total vol. = (π/4)(0.46 cm)2(25 cm)(1 mL/cm3) = 4.15 mL
• Vol. of packing material + stationary phase = 4.15 mL – 2.37 mL = 1.78 mL (note only VS is useful)
• 1st peak, tR = 5.93 min.; k = (5.93 -2.37)/2.37 = 1.50
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Chromatographic TheoryWhat do all these Parameters Mean?
• KC is just like KP in liquid – liquid extractions for HPLC or KH (Henry’s law constant) for GC
• Large KC value means analyte prefers stationary phase• In GC:
– KC value will depend on volatility and polarity (analyte vs stationary phase)
– KC value adjusted by changing T (most common)– The mobile phase or carrier gas (e.g. He vs. N2) has no effect
on KC
• In HPLC– KC value will depend on analyte vs. mobile phase and
stationary phase polarity– KC value adjusted by changing mobile phase polarity
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Chromatographic TheoryWhat do all these Parameters Mean? II
• Retention Factor is a more useful measure of partitioning because value is related to elution time
• Compounds with larger KC, will have larger k, and will elute later
• Practical k values– ~0.5 to ~10– Small k values → usually poor selectivity– Large k values → must wait long time – Higher k values are more practical for
complicated samples while low k values are desired for simpler samples to save time
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Chromatographic Theory Definition Section – Velocity
• Mobile phase velocity (u) and analyte average speed (v) can be useful quantities
• u = L/tM (L = column length)
• v = L/tR
• R = retardation factor = v/u (similar to RL used in TLC based on distance migrated)
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Chromatographic Theory Reading Chromatogram – cont.
• u = L/tM = 250 mm/2.37 min = 105 mm/min• v(1st peak) = L/tR = 250 mm/5.93 min = 42.2
mm/min• R = 42.2/105 = 0.40
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Chromatographic TheoryShape of Chromatographic Peak
• Gaussian Distribution
• Normal Distribution Area = 1
• Widths– σ (std deviation)– wb (baseline width) = 4σ– wh (peak width at half height)
= 2.35σ– w’ = Area/ymax = 2.51σ (often
given by integrators)
Gaussian Shape (Supposedly)
2
21
exp2
1xx
y
2σ
Inflection lines
wb
Height
Half Height
wh
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Chromatographic TheoryMeasures of Chromatographic Efficiency
• Plate Number = N (originally number of theoretical plates – similar to number of liquid-liquid extractions or distillations)
• N = (tR/σ)2 (= 16(tR/wb)2 )• N is an absolute measure of column
efficiency but depends on length• Plate Height = H = length of column
needed to get N of 1• H = L/N, but H is constant under specific
conditions, while N is proportional to L
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Chromatographic TheoryMeasures of Chromatographic Efficiency
• Measuring N and H is valid under isocratic conditions• Later eluting peaks normally used to avoid effects from
extra-column broadening• Example: N = 16(14.6/0.9)2 = 4200 (vs. ~3000 for pk 3)• H = L/N = 250 mm/4200 = 0.06 mm
Wb ~ 0.9 min
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Chromatographic TheoryNon-ideal Peak Shapes
40
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13.00 13.20 13.40 13.60 13.80 14.00
time
Res
po
nse
Tailing Peak (actually slow detector)
Tailing Factor = TF = b/a > 1 (tailing peak)
a b
Fronting Peak (TF < 1)
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Chromatographic TheoryDefinitions - More on Peak Shapes
• A Gaussian peak shape is assumed for many of the calculations given previously (e.g. peak width and N)
• For non-Gaussian peaks, the equations relating specific widths to σ are no longer valid.
• New equations are required for equations that have width in them
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Chromatographic Theory Definitions - Resolution
• Resolution is a measure of the ability to separate two peaks from each other
• Resolution = RS
where d = (tR)B – (tR)A
and ave w = [(wb)A + (wb)B]/2
bS w
dR
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Chromatographic Theory Definitions - Resolution
• Resolution indicates the amount of overlap between peaks
• RS < 1, means significant overlap
• RS = 1.5, means about minimum for “baseline resolution” (at least for two peaks of equal height)
• RS > 2 often needed if it is important to integrate a small peak near a large peak
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Chromatographic Theory Definitions - Resolution
• RS calculation examples:– 1st two peaks:
• tR(1st pk) = 4.956 min., w (integrator) = w’ = 0.238 min, so wb = 0.238·(4/2.5) = 0.38 min.
• tR(2nd pk) = 5.757 min., wb = 0.44 minRS = 0.801/0.410 = 1.95 (neglecting non-Gaussian peak shape)
– Last two peaks, RS = 3.0
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Chromatographic Theory Definitions - Resolution
• Higher resolution values are needed to quantify small peaks next to large peaks
• RS = 1.61 (assuming wb 1st peak equals 2nd peak)
• RS is not sufficient for accurate integration of 1st peak (but o.k. for integration of 2nd peak)
Expansion of above box
Large integration error on 1st pk
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Chromatographic Theory Definitions - Peak Capacity
• Peak Capacity is the theoretical maximum number of peaks that can be separated with RS = 1.0 within a given time period.
• We won’t cover calculation, but for example, about 2X more peaks could be possible between 5 and 13 min.
• Peak capacity 2.3 to 20 min. would be ~27 peaks.• Greater peak capacity is typical with temperature/gradient
programs (like in example).
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Chromatographic Theory Definitions - Separation Factor
• Separation Factor = = ratio of distribution constants = KB/KA = kB/kA = (t’R)B/(t’R)A
Where (tR)B > (tR)A so that > 1• Smaller (closer to 1) means more difficult separation• In example chromatogram, (1st 2 peaks)
= (5.77 – 2.37)/(4.96 – 2.37) = 1.31
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Chromatographic Theory Definitions - Overview
• The “good” part of chromatography is separation, which results from differences in KC values giving rise to > 1
• The “bad” part of chromatography is band broadening or dispersion, leading to decreased efficiency (and also reducing sensitivity)
• The “ugly” part of chromatography is non-Gaussian peak shapes (leads to additional band broadening plus need for new equations)
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Chromatographic TheoryQuestions on Definitions
1. List two ways in which a stationary phase is “attached” to a column?
2. What column component is present in packed columns but not open-tubular columns?
3. In HPLC, typical packing material consist of μm diameter spherical particles. Even though tightly packing the spheres should lead to > 50% of the column being sphere volume, the ratio of VM/Column Volume > 0.5. Explain this.
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Chromatographic TheoryQuestions on Definitions
4. List 3 main components of chromatographs.5. A chemist perform trial runs on a 4.6 mm
diameter column with a flow rate of 1.4 mL/min. She then wants to scale up to a 15 mm diameter column (to isolate large quantities of compounds) of same length. What should be the flow rate to keep u (mobile phase velocity) constant?
6. A chemist purchases a new open tubular GC column that is identical to the old GC column except for having a greater film thickness of stationary phase. Which parameters will be affected: KC, k, tM, tR(component X), β, .
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Chromatographic TheoryQuestions on Definitions
7. What “easy” change can be made to increase KC in GC? In HPLC?
8. A GC is operated close to the maximum column temperature and for a desired analyte, k = 10. Is this good?
9. If a new column for problem 8 could be purchased, what would be changed?
10. In reversed-phase HPLC, the mobile phase is 90% H2O, 10% ACN and k = 10, is this good?
11.Column A is 100 mm long with H = 0.024 mm. Column B is 250 mm long with H = 0.090 mm. Which column will give more efficient separations (under conditions for determining H)?
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Chromatographic TheoryQuestions on Definitions
• Given the two chromatograms to the right:– Which column shows a
larger N value?– Which shows better
resolution (1st 2 peaks top chromatogram)?
– Which shows better selectivity (larger ; 1st 2 peaks on top)?
– Should be able to calculate k, N, RS, and α
min0 2.5 5 7.5 10 12.5 15 17.5
mV
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200
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1000
ADC1 A, ADC1 CHANNEL A (LILLIAN\102507000009.D)
0.9
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7.1
73
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15.
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VWD1 A, Wavelength=210 nm (LILLIAN\102507000009.D)
0.8
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.201
1.4
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.613
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ADC1 A, ADC1 CHANNEL A (LILLIAN\102507000006.D)
0.9
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.042
12.
754
VWD1 A, Wavelength=210 nm (LILLIAN\102507000006.D)
0.8
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.204
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.616
2.6
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Unretained pk
min0 1 2 3 4 5 6 7 8
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ADC1 A, ADC1 CHANNEL A (MONIQUE\062608000004.D)
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ADC1 B, ADC1 CHANNEL B (MONIQUE\062608000004.D) VWD1 A, Wavelength=205 nm (MONIQUE\062608000004.D)
2.8
42