Chem. 133 – 5/7 Lecture

Announcements I

• Exam 3• on Tuesday (will give summary of material

to know later)• Format will be similar to other exams• I will allow use of a 3” x 5” notecard• Covers Ch. 20 (Emission

Instrumentation and interferences), NMR, Ch. 21, Ch. 22 (just through retention and relative retention)

• Last Homework (+bonus) and Quiz (replacement) today

Announcements II

• Final Exam:• Thurs. May 21 (12:45 – 2:45)• Will be ~ 50% multiple choice• You will be allowed 1 8.5 x 11” sheet

with notes on it• Besides Exam 1 to 3 material, will also

most recent chromatography topics• Term Project

• Should finish up by next Tuesday with check out on next Thursday

Announcements III

• Term Project – cont.• I will give you your poster reviewing

assignments on Thursday• Poster Presentation on Friday of Finals

Week (5/22)• Today’s Lecture

•Review for Exam 3•Chromatography

•Band Broadening (last topic on Exam 3)•Next Thursday, we will complete Ch. 22 (resolution and optimization•I expect Ch. 22 will be last chapter covered for final exam

Exam 3 Topics (cont.)

A. Chapter 20 (Atomic Spectroscopy)1. Methods for Elemental Analysis (solid + liquid

samples)2. Basic theory of atomic transitions3. Types of interferences and means of dealing

with them4. How to use Standard Addition to calculate

concentrations*5. Block diagrams of AAS, AES, and ICP-MS

instruments including specific components used

Exam 3 Topics

B. NMR Topics (Rubinson and Rubinson Ch. 11)1. How to relate resonance frequency to magnetic field strength*2. Types of nuclei that are NMR active and number of and spins of states based on I value of nucleus*3. Origin of sensitivity problem and factors that affect senstivity in NMR*4. Causes and effects of relaxation (spin-lattice and spin-spin)5. Causes of deshielding6. Relative position of protons in spectra due to amount of deshielding7. Magnetic anisotropy (know electron circulation in aromatics)

Exam 3 Topics

B. NMR Topics (cont.)8. Relationship between ppm and Hz scales*

9. Source of splitting and predicting splitting patterns from 1 set of neighboring equivalent nuclei

10. General Proton Interpretation (# equivalent nuclei, relative # of each, relative position, # neighboring nuclei)11. Understand magnetic field requirements12. Understand main spectrometer components in NMR instruments (e.g. light source)

Exam 3 Topics

C. Mass Spectrometry Topics1. Main Instrument Components2. Methods of ionization in mass spectrometry:

phase used for, ions produced, and amounts of fragmentation

3. Methods to separate ions and practical uses4. Resolution* and Resolution Needs (nominal

vs. high resolution)5. Isotope effect calculations*6. Multiple charging calculations to determine M

and z*7. MS-MS use

Exam 3 Topics

D. Chromatography1. Liquid – liquid partitioning equation*2. How reactions in water (e.g. acid/base) affect

distribution between two phases3. Partitioning Equation in Chromatography*4. Qualitative understanding of effect of partition

coefficient on retention on a chromatographic column

5. Determination* and meaning of retention factor

6. Determination* and meaning of relative retention

7. Practical ways to change retention in GC and HPLC

8. What factors affect relative retention

Exam 3 Topics

D. Chromatography – Cont.9. Relate partition coefficient and capacity factor

(including calculation of volumes)*10.Be able to calculate N and H from

chromatograms*11.Main components of chromatographs12.Understand meaning of N and H

ChromatographyColumn Efficiency – The Bad

• Original theory developed from number of simple separation steps (e.g. from fractional distillation columns)

• N = number of theoretical plates (or now plate number) = best absolute measure

• N = 16(tr/w)2 or = 5.55(tr/w1/2)2

– w = peak width at baseline– w1/2 = peak width at half height

ChromatographyShape of Chromatographic Peak

• Gaussian Distribution

• Normal Distribution Area = 1• Widths

– σ (std deviation)– w = 4σ– w1/2 = 2.35σ

– w’ = Area/ymax = 2.51σ (commonly given by integrators)

Gaussian Shape (Supposedly)

2

21

exp2

1xx

y

2σ

Inflection lines

w

Height

Half Height

w1/2

ChromatographyColumn Efficiency

• Good efficiency means:– Large N value– Late eluting peaks still have narrow peak widths

• Relative measure of efficiency = H = Plate height = L/N where L = column length

• H = length of column needed to get a plate number of 1

• Smaller H means greater efficiency• Note: H is independent of L, N depends on L

large N Valuelow N value

ChromatographyMeasurement of 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

W ~ 0.9 min

ChromatographyCauses of Band Broadening

• There are three major causes of band broadening (according to theory)

• These depend on the linear velocity (u = L/tm)

• Given by van Deemter Equation:

– where H = Plate Height, and A, B, and C are “constants”

Cuu

BAH

ChromatographyBand Broadening

u

H

Most efficient velocity

A term

B termC term

ChromatographyBand Broadening

• “Constant” Terms– A term: This is due

to “eddy diffusion” or multiple paths

– Independent of u– Smaller A term for:

a) small particles, or b) no particles (best)

XXX

dispersion

ChromatographyBand Broadening

• B Term – Molecular Diffusion– Molecular diffusion is caused by random

motions of molecules– Larger for smaller molecules– Much larger for gases– Dispersion increases with time spent in mobile

phase– Slower flow means more time in mobile phase

XXX

Band broadening

ChromatographyBand Broadening

• C term – Mass transfer to and within the stationary phase– Analyte molecules in stationary phase are not

moving and get left behind– The greater u, the more dispersion occurs– Less dispersion for smaller particles and

thinner films of stationary phase

XX

dispersion

Column particle

ChromatographySome Questions

1. 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)?

2. Which van Deemter term is negligible in open tubular GC?

3. How can columns in HPLC be designed to decrease H? In open tubular GC?

4. Why is it usually more difficult to improve the relative retention (a) when there are a larger number of analytes/contaminants?

5. Both using a longer column or using a column of smaller H will improve resolutions? Which method will generally lead to a better chromatogram? Why?