1. Chem. 31 – 10/30 Lecture

2. Announcements I AA Lab – Due today Quiz 4 Today’s Lecture On Wednesday
On Acids part of Ch. 6/Chapter 7 materials/Chapter 18 materials (extractions)
Today’s Lecture
Chapter 18 – Spectroscopy
Beer’s Law
Spectrometers

3. Announcements II Today’s Lecture – cont. Chapter 23 – Chromatography
Overview
Partitioning (in extractions and chromatography)
Chromatographs
Transport (if time)

4. Spectroscopy Beer’s Law
Transmittance = T = P/Po
Absorbance = A = -logT
sample in cuvette
Light source
Absorbance used because it is proportional to concentration
A = εbC
Where ε = molar absorptivity and b = path length (usually in cm) and C = concentration (M)
Light intensity in = Po
Light intensity out = P b
ε = constant for given compound at specific λ value

5. Spectroscopy Beer’s Law Question
Half of the 284 nm light is absorbed when benzoic acid at a concentration of M is in a cuvette with a path length of 0.50 cm. What is the molar absorptivity of benzoic acid at this wavelength?

6. Spectroscopy More on Beer’s Law
Useful for determination of analyte concentrations
Some limitations
Law not valid for high concentrations
Deviations to law appear to occur when multiple wavelengths of light used or when multiple species exist but absorb light differently
Uncertainties are lowest when 0.1 < A < 1
Example of deviations to Beer’s Law:
Unbuffered Indicator with ε(In-) = 300 M-1 cm-1, ε(HIn) = 20 M-1 cm-1; pKa = 4.0
HIn ↔ H+ + In-

7. Spectroscopy Spectrometers
light detector – measures light intensity by converting it to an electrical signal
sample in cuvette
Data processor
light source
light discriminator: monochromator (passes only a small range of wavelengths)
Components can look very different in different types of spectrometers, but spectrometers will have all of the major components (except other methods of wavelength discrimination may replace monochromators)

8. Spectroscopy Example Measurement: Ozone
Ozone (O3) is a pollutant (lower atmosphere) and in stratosphere provides UV protection
Instrument is used for measurement at station or in airplane
compares absorbance through sample cell vs.
absorbance through reference cell
Can also make measurements remotely (e.g. absorbance between two skyscrapers)
light source (l = 254 nm)
chopper
air in
reference cell
sample cell
O3 scrubber
light detector

9. Chromatography – Ch. 23 Introduction
Purpose of Chromatography
To separate and detect components of a mixture
Analytical chemists are more interested in the detection part
Advantages of Chromatography
Can handle more complex samples than typical spectroscopic methods
Also results in purification of mixtures (if desired)
Disadvantage of Chromatography
Separation takes time (so generally not as fast as pure spectroscopic methods)
Basis for Separation:
differential partitioning between a stationary and a mobile phase

10. Chromatography Partitioning – Ch. 23 Sect. 1
Covering to understand partitioning in chromatography
Partitioning can occur between any two phases (as long as one phase is a fluid)
Liquid-liquid is chosen as an example
Partitioning governed by equilibrium equation
X(org)
X(aq)
K = partition coefficient (a constant)
note: technically, upper conc. is for “raffinate” phase while lower is for “extractant” phase

11. Chromatography Partitioning
Partitioning coefficient depends on stability in solvents (related to solubility in solvents)
Most common rule is likes dissolve likes
Example of water – hexane partitioning
Other Effects on Partitioning
K gives distribution if compound does not react further in either phase
However, compounds may react further (e.g. acid HA → H+ + A- in aqueous phase)
Ions (e.g. A-) will be found almost exclusively in aqueous phase
Distribution coefficient (D) gives ratio of total species concentration (only covering qualitatively)
larger K
Although both have K > 1, the OH group makes right molecule more polar (favors water more vs left)

12. 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
Compound A
Compound B
K = 7.59
K = 6.17
pKa = 4.62
Not very acidic
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

13. Chromatography Partitioning - Questions
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 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?
3. 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?

14. 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

15. 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
Packing Material (solid)
Stationary phase is surface (larger area than shown because its porous)
Stationary phase (wall coating)
Expanded View
Bonded phase (liquid-like)
Stationary Phase
Chemically bonded to packing material
Packing Material

16. 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

17. Chromatography Equipment
Chromatograph = instrument
Chromatogram = detection vs. time (vol.) plot
Chromatograph Components
Sample In
Chromatographic Column
Detector
Flow/Pressure Control
Mobile Phase Reservoir
Waste or fraction collection
Injector
Chromatogram
Signal to data recorder

18. 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

19. 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