Download 4/3 Lecture Slides

Chem. 31 – 4/3 Lecture
Announcements I
• Lab Resubmissions
– Cl lab – due today
– Water Hardness – due Wednesday
• Quiz 4 this Wednesday
– On shape of precipitation curve
– Spectroscopy
– Chromatography: Extractions plus sections
covered today
Announcements II
• Upcoming Assignments
– Atomic Absorption Lab Report (4/12)
– Additional Problem 3 (4/17)
– Formal Lab Report (Part A – will post
materials soon – due 4/17)
– Exam 2 (4/19; about 1 lecture behind
expectations)
Announcements III
• Today’s Lecture
– Chapter 23 – Chromatography
• Partitioning
– liquid – liquid example
– in chromatography
• Chromatography Equipment
• Transport and Retention in Chromatography
Chromatography
Partitioning
• Example of Effect of
Aqueous Reactions on
Compound Distribution
• Compound A is nearly as
polar as B
• However, acidity affects
distribution between
water and organic layer
• Compound B will undergo
dissociation in water:
HA ↔ H+ + A-
• Distribution of B given by:
D = [HA]org/{[HA] + [A-]}aq
O
H3C
H3C
O
Compound A
K = 7.59
Not very
acidic
OH
Compound B
K = 6.17
pKa = 4.62
If aqueous phase is buffered at pH >
pKa (e.g. pH = 6), most of B will be in
anion form and very little of B will be
in organic phase
With a low pH buffer, D ~ K
Chromatography
Partitioning - Questions
1.
2.
3.
A compound with an octanol water partition coefficient of 52 is
placed in a separatory funnel with water and octanol and shaken.
The concentration of it in octanol is found to be 0.150 M. What is
its concentration in water?
It is desired to separate the following two compounds:
CH3(CH2)3OH and CH3(CH2)3NH2.
The two compounds have similar KOW values (around 11) but the
second compound is basic. What can be done to separate the
two?
It is desired to transfer butanol (left compound in #2) from water
to an organic phase. Would it be transferred most efficiently
using 1-octanol, a less polar solvent (e.g. octane), or a more
polar solvent (e.g. 1-hexanol) as the organic solvent?
Chromatography
Partitioning in Chromatography
• Separation Occurs in Column
• Partitioning Requires Two Phases:
– Mobile phase
•
•
•
•
fluid flowing through the column
type of fluid determines type of chromatography
fluid = gas means gas chromatography (GC)
liquid chromatography (high performance liquid
chromatography or HPLC)
• supercritical fluid (SFC) [supercritical fluid = fluid at high
temperature and pressure with properties intermediate
between liquid and gas]
– Stationary phase (solid or liquid within column)
• most commonly liquid-like substance on solid support
Chromatography
More on Stationary Phases
Open Tubular – in GC
(end on, cross section view)
Packed column (side view) (e.g.
Silica in normal phase HPLC)
Column Wall
Mobile phase
Stationary phase
(wall coating)
Expanded View
Stationary Phase
Chemically bonded to
packing material
Packing Material
Packing Material (solid)
Stationary phase is surface (larger
area than shown because its
porous)
Bonded phase (liquid-like)
Chromatography
Overview – The Good, the Bad, and the Ugly
• The Good: Differential
partitioning of solute
between a mobile and
a stationary phases
• The Bad: Band
broadening
• The Ugly: Non-ideal
peak shapes (we will
see this in the GC
lab)
More realistic picture
Concentration profile
Chromatography
Equipment
• Chromatograph = instrument
• Chromatogram = detection vs. time (vol.) plot
Chromatograph Components
Sample In
Chromatographic
Column
Flow/Pressure
Control
Mobile Phase
Reservoir
Injector
Signal to data
recorder
Detector
Waste or fraction
collection
Chromatogram
Chromatography
Flow – Volume Relation
• Relationship between volume (used with gravity
columns) and time (most common with more
advanced instruments):
V = t·uV
V = volume passing through column part in time t at
flow rate uV
Also, VR = tR·uV where R refers to retention
time/volume (time it takes component to go through
column or volume of solvent needed to elute
compound)
• Can also use linear velocity (ux)
ux = L/tR where L = column length
Chromatography
More on Volume
• Hold-up volume = VM = volume occupied
by mobile phase in column
• Stationary phase volume = VS
• Calculation of VM:
VM = tM·uV, where tM = time needed for
unretained compounds to elute from column
Unretained compound = compound 100% in
mobile phase
Chromatography
Reading Chromatograms
• Determination of parameters from reading chromatogram (HPLC
example)
• tM = 2.374 min. (normally determined by finding 1st peak for
unretained compounds – contaminant below)
• VM = uV·tM = (1.0 mL/min)(2.37 min) = 2.37 mL
Chromatography
Some Questions
1. What are the required two phases in
chromatography called?
2. List two ways in which a stationary phase is
“attached” to a column?
3. List 3 main components of chromatographs.
4. A GC has the flow rate set to 2.0 mL/min.
When methane is injected (it is not retained at
all on the stationary phase), it takes 2.85 min
to appear. What is the mobile phase volume?
5. What phase is the mobile phase in HPLC?
Chromatography
Partition and Retention
•
•
•
•
•
Partition Coefficient = K = [X]S/[X]M
Higher K means slower elution
K is constant if T and/or solvent remain constant
K is not used that frequently in chromatography
Retention Factor = k = main measure of
partioning/retention in column
• k = (moles X)S/(moles X)M = K(VS/VM)
• Retention Factor is more commonly used
because of ease in measuring, and since VM/VS
= constant, k = constant·K (for a given column)
• Note: kColumn1 ≠ k Column2 (if VM/VS changes)
Chromatography
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
• Note: t’R = tR – tM = adjusted retention
time
Chromatography
Reading Chromatograms
• 1st peak, tR = 4.958 min.; k = (4.958 - 2.374)/2.374 = 1.088
Chromatography
What do all these Parameters Mean?
• Large k value (or K value) means analyte
prefers stationary phase
• “Adjusting k (or K)” - in GC:
– k value will depend on volatility and polarity
(analyte vs stationary phase)
– k value adjusted by changing T (most
common)
– higher T means less retention (lower k and K)
Chromatography
What do all these Parameters Mean? II
• Adjusting k – in HPLC
– k value will depend on analyte vs. mobile phase and
stationary phase polarity
– Oldest type (= Normal phase) stationary phase is
polar and mobile phase is non-polar
– Most common type (= Reversed phase) has nonpolar stationary phase and polar mobile phase
– k value adjusted by changing mobile phase polarity
– k value is decreased by making mobile phase more
like stationary phase (= using “stronger” eluent)
– In reversed phase HPLC, less polar mobile phase
means less retention
analyte
more
polar
water
methanol
stationary
phase
less
polar
Chromatography
What do all these Parameters Mean? III
• Retention Factor is a more useful measure of partitioning
because value is related to elution time
• Compounds with larger K, will have larger k, and will
elute later
• Practical k values
– ~0.5 to ~10
– Small k values → overlapping peaks likely
– Large k values → must wait long time
Chromatography
More Questions
2.
A chemist is analyzing
samples by normal phase
HPLC using a mobile
phase containing 90%
hexane and 10% 2propanol (2-propanol is
the more polar solvent).
The analysis is taking too
long. How can she
decrease k values?
From the chromatogram
to the right, calculate kX.
Chromatogram 1
3.1
2.9
2.7
response
1.
2.5
2.3
2.1
1.9
1.7
1.5
0.0
0.5
1.0
1.5
2.0
2.5
time (min.)
unretained
peak
X
Y