Chem 340 - Exam I – Fall 2015
Name
Date
SHOW ALL WORK
Points Earned
#1
(12 pts)
#2
(12 pts)
#3
(6 pts)
Possible Useful Equations
Rs =
N  1
k


4

1 k
N = 16(tr/wb)2
vep = (q/f)E
#4
(20 pts)
#5
(10 pts)
OR
#6
(10 pts)
#7
(20 pts)
OR
#8
(20 pts)
#9
(10 pts)
N = (µepV)/(2D)
IMPORTANT!!!!
There are some questions in which
you do not have to answer every
part. Please do not try to answer all
of them if the question indicates for
you to choose 2 of the 3 for
example. If you answer all 3 then I
will grade the first 2.
OR
#10
(10 pts)
#11
(10 pts)
TOTAL BONUS
Total Points
IMPORTANT!!!!
Some of the problems have
embedded bonuses. You can only
receive the bonus if you answer that
particular part of the problem.
1. (12 pts) Answer 2 of the following 3 questions related to UV-Vis – BE SPECIFIC
a.
PMTs are common detectors in spectrophotometery. Using a labeled diagram of a PMT,
explain the advantage of using these as detectors. BONUS (+2 pts) A diode array is
another type of detector. Explain how these work in terms of a reverse bias.
b. If you were to build a UV/Vis spectrophotometer for the analysis of unknowns, what
instrument parts would you buy? Be specific. BONUS (+3 pts) Why is it uncommon for
HPLC manufacturers to sell HPLC-IR instruments?
c. Would 2-methylbenzene blue shift or red shift when it is analyzed as an aqueous solution
after being analyzed as a non-aqueous solution. Why does this occur? Explain IN DETAIL
and make sure to use energy level diagrams.
2. (12 pts TOTAL) Answer 2 of the following 4 questions in relation to chromatography– BE
SPECIFIC
a.
If you were doing a research project where you had to separate acetaminophen from aspirin
with reversed phase chromatography and your data had terrible selectivity, what
specifically about the chromatography system would you adjust and why? BONUS (+2
pts) Which molecule is acetaminophen and which one is aspirin?
b. Explain in detail how you would use IEC to separate the following molecules. BONUS (+4
pts) Draw the structure of each amino acid that you will have in your experiment.
Amino Acid
pI
Arginine
10.8
Lysine
9.8
c. Explain the difference between electrophoretic mobility and electroosmotic flow. BONUS
(+2 pts) The mobile phase in HPLC moves due to high pressure. Explain, in context to
what the mobile phase is, the mechanism behind how the mobile phase in CZE moves?
d. Define equilibrium in terms of liquid chromatography. Explain one way in which you can
achieve perfect equilibrium when performing liquid chromatography and one way in which
your experiment may divert from equilibrium.
3. (6 pts) What does each acronym stand for? Skip 2 of them
a. GPC
e. HPLC
b. GFC
f. CZE
c. GC
g. SEC
d. IEC
h. EOF
4.
(20 pts) ONE or TWO WORD Answers
a. What is the term that is used to refer to the mobile phase in HPLC?
b. What is the term that describes the type of elution method in which you don’t change the
composition of the mobile phase during a HPLC experiment?
c. What is the term that describes the column that is used in liquid chromatography to protect
your actual column?
d. What does the “A” term in the Van Deempter equation stand for?
e. Cuvettes that transmit UV light would be made out of this material.
f. What is a typical dispersion element used in UV/Vis spectrometers?
g. What is the term that describes the peak shape that results when you overload the HPLC
column with sample?
h. What is the “shift” in UV/Vis spectra that results from hydrogen bonding between the
nonbonding electrons of the analyte and the solvent?
i. What is the term that describes the molecular orbital that forms when two p atomic orbitals
combine along the bonding axis?
j. What is the specific type of chromatography that uses a amine resin as the stationary phase
and a buffer as the mobile phase?
5. (10 pts TOTAL) Calculate the retention factor for each solute and the number of plates of
the following TOTAL Chromatogram of a sample that contains 2 different solutes.
a. (2 pts) retention factors
A
30
B
Arbitray Intensity Units
25
b. (4 pts) number of plates
20
15
10
5
0
0
5
10
15
20
25
30
35
retention time (minutes)
c. (4 pts) If this was an ion exchange system that was run in anion mode, comment on the
characteristics of the analytes based on their retention times.
6. (10 pts TOTAL) Is it HPLC, GC, or CZE? Explain why if you are concerned about your answer
a. Technique that contains a PDMS column which can volatilize at high temperatures.
b. Technique in which H = B/µ
c. Technique in which you have to purge the pumps.
d. Technique that separates molecules according to their volatility.
e. Technique that is useful for separating charged biomolecules.
STOP – DO #7 or #8
7. (20 pts TOTAL) The Van Deemter plot below is describing the efficiency of HPLC data upon
varying the stationary phase particle diamter.
a. (15 pts) Explain the plot below in terms of efficiency.
b. (5 pts)Write the Van Deemter Equation taking into account the change in particle size (dP)
Pharmtech.com
8. (20 pts TOTAL) The Van Deemter plot below is describing the efficiency of the analysis of C440 (top molecule) and C-450 with a C18 column and a mobile phase of water with varying
amounts of acetonitrile.
a. (15 pts) Discuss the efficiency differences that result from varying the amounts of acetonitrile
in the mobile phase.
b. (5 pts) Discuss the differences in Van Deemter plots for the two molecules within the same
mobile phase composition.
Lab Chip, 2011, 11, 3081-3088
STOP – DO #9 or #10
9. (10 pts TOTAL) You are currently working as an Analytical Chemist for Sigma-Aldrich and
your first assignment is to develop a protocol for a total vitamin assay with liquid
chromatography. This protocol should be precise and if followed will result in reproducible
data. Your boss tells you that the “former” employee was unable to separate and detect the
water-soluble vitamins from the fat-soluble vitamins on one column. Explain how to do this
with liquid chromatography and BE SPECIFIC about the experimental setup including the
detection system that you will use and give the predicted results.
10. (10 pts TOTAL) Design a HPLC experiment to analyze the relative amounts of the molecules
below in the liver. The molecule 6β-hydroxytestosterone is a metabolite of testosterone.
Specify the parameters of the experiment including the phases and parameters of the detection
method along with your predicted results.
11. (10 pts TOTAL) High-efficiency microbial separation (HEMS) through the theory of CZE
uses a dissolved polymer (PEO) in the buffer at a pH between 3 and 9 to separate different
bacterial strains (Figure a below). One can also use what is called capillary isoelectric focusing
(CIEF) to obtain HEMS (Figure b below in which three bacterial strains of the same shape are
separated).
a. (8 pts) Explain how you think each method works in terms of separation by utilizing the data
from the chromatograms below
A
B
Armstrong, D. W.; Schneiderheinze, J. M. Anal. Chem. 2000, 72, 4474–4476.
Armstrong, D. W., et al. Anal. Chem. 1999, 71, 5465–5469.
PEO
b. (2 pts) Using your theory of CZE, explain the data in the chromatogram below. What would
you need to do to prevent this problem from affecting your data?
BONUS (2 extra points for every one done correctly. No partial credit will be given)
O
O
OH
Cl