In the Laboratory

The Mysterious Death: An HPLC Lab Experiment W

An Undergraduate Forensic Lab
Douglas J. Beussman
Department of Chemistry, St. Olaf College, Northfield, MN 55057; *beussmad@stolaf.edu

One of the greatest challenges educators face is captur-
ing the interest of the students they are attempting to in-
struct. This can be accomplished with demonstrations,
anecdotes, or by relating the material to a situation that the
student has experienced or may likely experience in the fu-
ture. These same challenges exist both in the classroom as
well as in the laboratory. Indeed, laboratory experiments of-
ten present unique teaching challenges. There is a tendency
for students to follow the laboratory procedure without ever
considering what they are doing, why they are doing it, or
fully understanding the concepts being taught. This cook-
book recipe approach to laboratory experiments allows the
student to successfully complete the assigned task, but often
does not impart any knowledge, understanding, or insight
into the phenomenon they are studying. The experiment pre-
sented here challenges the students to use their chemical
knowledge to solve a potentially real-world problem, while
at the same time forcing the students to critically think about
what they are doing, interpret the results on their own, and
make decisions to complete the assignment and arrive at an
answer.
With the above in mind, a laboratory experiment was
designed to expose students to a high-performance liquid
chromatography (HPLC) separation. Owing to student cu-
riosity about the forensic science field, the experiment was
designed using four drugs that have clinical relevance and
have lethal doses (Figure 1 and Table 1). It is applicable for
any student taking an analytical chemistry course where
HPLC is taught, typically in the third year of the curricu-
lum, and may be applicable for introductory forensic science
courses as well. Adding a bit of mystery to the outcome of
an experiment often can capture the attention of the students
and compel them to make an extra effort to both understand
the concepts being presented as well as correctly solve the
mystery.

Experimental Overview
Scenario
The HPLC experiment is presented to the students in
the context of a mysterious death. Each lab group is presented
with a patient (male, 48 years, 200 lb, 61) who has been
found dead in his bed with no signs of foul play. Next to the
bed are several prescription medications, and a drug over-
dose is suspected as the cause of death. Each group is given a
different mixture, representing a blood extract containing one
or more of the four drugs found on the nightstand, and is
challenged with determining whether the death was a drug
overdose and if so, whether it was accidental or a suicide.
Several previous lab experiments have been published describ-
ing the HPLC analysis of pharmaceutical agents, including
Figure 1. Structures of prescription drug analytes.
an asthma medication (1), antipsychotics (2), and several deal-
ing with analgesics (36), but none of these experiments have
used the drugs presented here, and none of these have built
the experiment around the context of a forensic mystery that
Table 1. Therapeutic and Toxic Drug Ranges the students must solve.
Therapeutic Level/ Toxic Level/
Drug Group Organization
(g/L) (g/L)
Disopyramide 0.0020.005 > 0.005 To more accurately model what the students will encoun-
Lidocaine 0.00150.0050 > 0.005 ter outside of the academic environment, they are randomly
divided into groups of four students at the beginning of the
Procainamide 0.0040.010 > 0.016
semester. The lab format is based on the format developed
Quinidine 0.0020.005 > 0.010 and used by John Walters, with each group member having
NOTE: The data are from ref 10. a specific role to play during the experiment (79). The roles

www.JCE.DivCHED.org Vol. 84 No. 11 November 2007 Journal of Chemical Education 1809

 In the Laboratory
Figure 2. HPLC of a mixture of 0.04 g/L procainamide, 0.5 g/
L lidocaine, 0.25 g/L disopyramide, and 0.05 g/L quini- in the unknown samples vary from 0.010.5 g
L for
dine, with dihydroquinidine contaminant. Injection volume of 20
L, separation conditions as listed in the text.
include a group manager, chemist, instrumentation special-
ist, and software specialist. For each new experiment the roles
rotate among the group members so that everyone has a
chance to experience, and is equally challenged by, each role
over the course of the semester. The successful completion
of the experiment requires that all group members success-
fully complete their assigned role. A discussion of group dy-
namics is beyond the scope of this manuscript, but can be
found in the Walters publications (79).
Group Roles
The group manager is the key person for each experi-
ment. He or she is in charge of the group and is directly re-
sponsible for all aspects of the lab experiment. The group
manager directs the other members of the group, ensures that
the experiment runs smoothly, and handles any problems
within the group. It is his or her responsibility that all mem-
bers of the group understand their roles, complete their tasks,
and understand what other group members are doing even
though each member has a separate task. After completion
of the experiment, the group manager also must complete a
short 1015 minute interview with the lab instructor or
teaching assistant to discuss the results of the lab.
The chemist is responsible for making all solutions for
the experiment including dilutions of the drug standards. He
or she must understand how to use any equipment needed
and how to perform any calculations required to make the
solutions. It is also the chemists responsibility to handle all
chemicals safely and dispose of any waste properly.
The instrumentation specialist is in charge of learning
how to operate the HPLC correctly and safely as well as per-
forming the analysis. During the first 15 minutes of the lab
period, the instrumentation specialists from each group are
given a short tutorial on how to operate the HPLC systems
and how to collect and process the data. He or she may also
refer to the instrument manuals provided in the lab.
The software specialist is in charge of documenting the
procedures, performing any calculations or data manipula-
tions, and helping to write the final report for the group man-
ager to present to the instructor or teaching assistant. He or
she also must determine the clinical relevance of each drug,
the normal dosages, and toxic levels.
1810 Journal of Chemical Education Vol. 84 No. 11 November 2007
Procedure
Chemicals
The four drugs used in this experiment are disopyramide,
lidocaine, procainamide, and quinidine (Figure 1). Prior to
the start of the laboratory, the instructor prepares standard
(2.5 g
L) samples of each compound in 50:50 water:HPLC
mobile phase (63:30:6.5:0.5 water:methanol:acetic
acid:triethylamine). Several unknown blood extract samples
containing one or more of the standards are also made by di-
luting the standards with water. To remain in the linear work-
ing range of the HPLC system, the concentrations of the drugs
disopyramide, 0.0252.5 g
L for lidocaine, 0.001250.04
g
L for procainamide, and 0.001250.1 g
L for quini-
dine. These concentrations have been determined to provide
good signal-to-noise ratios in the chromatogram without over-
loading the column and are within the linear UV-absorbance
region of the instruments. While the entire working concen-
tration range for disopyramide and lidocaine are above the
toxic levels, the concentration ranges for procainamide and
quinidine span both the therapeutic and toxic levels. Thus,
the instructor can choose to include drugs in the blood sample
at therapeutic or toxic levels as desired. These ranges assume
that there is no sample preconcentration incorporated into
the hypothetical extraction protocols, which are not discussed
here. Alternatively, the instructor could indicate that the blood
extracts have already been concentrated by a factor of 10 or
more, which would allow even disopyramide and lidocaine
to be observed in the linear working range of the instrument
but represent therapeutic ranges in the patient. Students would
then have to take this concentration step into account when
calculating the original blood concentrations of the drugs.
A representative chromatogram is shown in Figure 2. As
can be seen, the quinidine standard yields two peaks, with
the small, later-eluting peak due to the contaminant
dihydroquinidine. Each HPLC system is equipped with a 4.6
mm 15 cm C18 column and a 20 L injection loop. An
isocratic flow rate of 1 mL
min is used and UV absorbance
is measured at 254 nm. We currently use four isocratic HPLC
systems (Alltech 426 pump and LINBAR UVIS 200 detec-
tor), which allows a maximum of sixteen students per labora-
tory section. Each HPLC system is connected to a
data-collection computer running an in-house written data
acquisition program using National Instruments LabVIEW
software. This software collects time and absorbance values
and allows them to be imported into Microsoft Excel for fur-
ther mathematical processing if desired. Any HPLC system
capable of collecting UV data should be adequate for this ex-
periment.
Group Responsibility
During the prelab lecture, the instructor presents the
problem that each group must attempt to solve. Each group
manager chooses a simulated blood extract and the instru-
mentation specialists are called together for a short tutorial
presented by the instructor or TA on how to operate the
HPLC systems. The group manager is then in charge of mak-
ing all the decisions, hopefully in consultation with the other
www.JCE.DivCHED.org

group members, required to complete the laboratory experi-
ment and solve the mystery. The chemist must prepare the
standard solutions, diluted to be in the working linear range
of the instrument, of each of the four drugs to be injected
into the HPLC prior to analyzing the mixture. While the
chemist prepares the first drug standard dilution, the HPLC
column can be equilibrated for 10 to 15 minutes until a stable
baseline is obtained. The software specialist can be sent to
the library to obtain information on each of the four drugs,
or may use the Internet to attempt to find this information,
since there are no data to collect or analyze during the first
portion of the lab. Unorganized or unprepared group man-
agers will find that they have people standing around with
nothing to do during the first half of the experiment and
scrambling to get everything done at the end of the lab pe-
riod. This could be a potential topic for discussion during
the management interview after completion of the lab. In
extreme cases, the instructor may need to prompt activity in
the group and gently direct the group manager to take a more
forceful approach.
The lab instructor and TA (representing upper manage-
ment) continually circulate among the groups watching the
progress of each group. If needed, the group manager may
be asked to explain what their group is doing to force the
group manager to think about how they are accomplishing
their tasks and how to possibly make things better. For ex-
ample, if a stable baseline is observed after ten minutes, but
the group manager does not decide to inject the first stan-
dard, the instructor may ask the group manager what they
are doing or what they are looking for before making the first
injection. After the column has been equilibrated, each drug
standard that the chemist has prepared is individually injected
onto the C18 column. Again, the instructor and TA circu-
late among the groups and pose thought-provoking questions
to each group such as how can the end of the separation of a
pure compound be determined, what is the predicted elu-
tion order of the four drugs based on their chemical struc-
tures, or what accounts for the negative peak seen early in
the separation? This ensures that the group members are
thinking about the separation process and not just waiting
to make the next injection.
HPLC
The retention time of the most retained compound is
approximately 6 minutes, allowing each chromatographic run
to be completed in approximately 10 minutes, depending on
how long the group manager decides to wait to ensure a
steady baseline after each separation. Once all four standards
have been analyzed, the groups unknown simulated blood
extract is injected. Each group then must use the data to de-
cide which, if any, of the standard compounds are present in
the patients blood. This is usually accomplished by observ-
ing the number of peaks in the chromatogram and compar-
ing the retention times of the standards with the peaks in
the unknown sample.
After identifying which drugs are present in the patients
system, each group must determine the quantity of these
drugs in the patient to make an informed decision about
www.JCE.DivCHED.org Vol. 84 No. 11 November 2007
In the Laboratory
whether the patient likely died of a drug overdose, and if
so, whether it was likely accidental or intentional. While
the students are told during the prelab lecture that the elu-
ent from the HPLC column will be analyzed with a UV-
absorbance detector and that the BeerLambert law applies
to this experiment, they are not given explicit instructions
as to how to convert the data obtained from the chromato-
gram into drug levels in the blood. The BeerLambert law
and the use of spectroscopic data for quantitative analysis
is covered earlier in the semester, and the students are ex-
pected to be able to apply this previous knowledge to the
HPLC data. Several weeks prior to this HPLC experiment,
students also completed other lab experiments where they
performed absorbance-based quantitative analysis by first
generating a standard curve. Questions invariably arise dur-
ing the lab as to whether peak height or peak area should
be used. This presents another opportunity for upper man-
agement to encourage the students to consider what is hap-
pening during the HPLC analysis. What does the peak
height represent? Is it a good measure of the total quantity
of drug introduced onto the HPLC column? The students
are forced to think about what they have measured and how
they might use the information to solve the problem. Dur-
ing the prelab lecture, the students are given the linear work-
ing ranges for each drug, but the exact concentrations used
to make their calibration curves, and the number of points
to use for the curves, are left to each individual group. Once
the calibration curve for known concentrations of a drug
has been created, the quantity of drug present in the patients
blood can be calculated.
For this experiment, all four drugs can be quantitated
because the blood extracts are made by the instructor from
pure drug samples and solvent. In an actual blood sample,
the blood matrix would be very complex, even after extrac-
tion protocols. Many components of the matrix may be un-
retained and thus would elute at or near the dead time (tm),
making the quantitation of procainamide impractical using
the current separation. Students generally do not recognize
this subtle point, making for a good discussion topic during
the lab.
After determining the identity and concentration of each
drug in the patients blood, each group must reach a deci-
sion as to the cause of death. Each group can determine an
approximate level of each drug in the patients blood based
on the concentration of each drug found in the sample, an
estimation of the total quantity of blood in an average-sized
person, and the weight of the patient. By comparing these
values to the normal or toxic levels of each drug as obtained
by the software specialist, the cause of death can be specu-
lated. Note that for each unknown, the concentration of at
least one of the drugs present in the samples far exceeds the
normal and toxic doses, making the determination relatively
simple if all calculations are done correctly. Nonlethal con-
centrations could be used if desired, although this would pre-
clude the students from arriving at a cause of death. With
good preparation and time management by the group man-
ager, the entire experiment can easily be accomplished within
a four-hour lab period.
Journal of Chemical Education 1811
 In the Laboratory

Lab Report antiarrhythmics and thus have physiological effects. While

only a small quantity of each drug is used in this experiment,
The final portion of the experiment entails the group
reasonable care must be taken to prevent unnecessary expo-
preparing a short (12 pages) summary of the results of the
sure. Methanol is an irritant and may cause central nervous
experiment. This report is given to the group manager who
system depression and respiratory and digestive tract irrita-
must then discuss the results with the lab instructor during
tion. It may be fatal or cause blindness if swallowed. Triethyl-
an out-of-lab interview. The group manager must defend the
amine is corrosive and causes burns. It is harmful by ingestion,
cause of death determination and explain how the group ar-
inhalation, and if absorbed through the skin and may cause
rived at the conclusion. The group manager may be asked to
liver damage.
explain any of the thought-provoking questions that the in-
structor has asked the groups during the lab period. Finally
the group manager is asked for input on how to make the Conclusions
lab better for the next year.
The HPLC experiment presented here not only helps
As described above, the instructor or TA can initiate stu-
teach principles of liquid chromatography, but also helps fos-
dent learning throughout the lab by asking a variety of ques-
ter group interactions and teaches the principles of division
tions that the student groups can reason through during the
of responsibilities. Student interest in the experiment is main-
waiting time of each HPLC analysis. For example, students
tained and in fact heightened by directly relating the objec-
may be asked to explain why there is a negative peak early in
tives of the experiment to a potentially real forensic situation.
the chromatogram (the tm peak, due to water from the sample
As many of our students are considering careers in the health
not being retained and having less absorbance than the mo-
profession, they also generally enjoy learning about actual
bile phase as well as mobile phase displacement due to the
pharmaceutical agents. Recognition of the applicability of the
injection solvent), why procainamide could not be quanti-
concepts taught in the lab to what they might encounter af-
tated using this separation in a real blood extract, why 254
ter graduating, along with the desire to solve the mystery,
nm is used as the detection wavelength (all four drugs are
provides incentive to the students to put extra effort into the
aromatic), what role the acetic acid plays in the mobile phase
lab and really understand what is being presented. Both the
(sets the pH and thus the quantity of stationary phase silanol
role-playing style and the concept of using HPLC techniques
deprotonation), or what role triethylamine plays in the mo-
to solve a mysterious death have been well received by the
bile phase (interacts with any deprotonated silanol groups
students.
present on the stationary phase, limiting the degree of silanol
interaction with the analytes). Students can also be asked to
predict before the lab, or rationalize afterwards, the order of
W
Supplemental Material
elution of the four compounds. Depending on the instruc-
Instructions for the students and notes for the instruc-
tor and focus of the course, more or less emphasis can be
tor are available in this issue of JCE Online.
placed on the actual workings of the HPLC and injection
valve, the pharmacology of the drugs, or the forensic appli-
cations of HPLC. Many of these discussion points could be Literature Cited
incorporated into the prelab lecture or lab manual if less in-
1. Mueller, Brian L.; Potts, Lawrence W. J. Chem. Educ. 1988,
structor interaction with the students during the lab is de-
65, 905906.
sired.
2. Ferguson, Glenda K. J. Chem. Educ. 1998, 75, 16151618.
While we currently do not operate the lab with an in-
3. Kagel, R. A.; Farwell, S. O. J. Chem. Educ. 1983, 60, 163
ternal standard, lidocaine could be used as an internal stan-
166.
dard since it does not co-elute with any of the other drugs.
4. Haddad, Paul; Hutchins, Stephen; Tuffy, Michael. J. Chem.
This would still leave three drugs to be used as unknowns.
Educ. 1983, 60, 166168.
As we currently only put two drugs into each simulated blood
5. Beaver, Rodney W.; Bunch, John E.; Jones, Louis A. J. Chem.
extract, there could still be different unknowns given to dif-
Educ. 1983, 60, 10001001.
ferent lab groups. Alternatively, N-acetylprocainamide could
6. Ferguson, Glenda K. J. Chem. Educ. 1998, 75, 467469.
be used as an internal standard.
7. Walters, John P. Anal. Chem. 1991, 63, 977A985A.
8. Walters, John P. Anal. Chem. 1991, 63, 1077A1087A.
Hazards
9. Walters, John P. Anal. Chem. 1991, 63, 1179A1191A.
Gloves are provided to the students, and safety goggles 10. Data obtained from MedlinePlus Web site. http://
are required for all personnel in the lab. Disopyramide, www.nlm.nih.gov/medlineplus/ency/article/003430.htm (accessed
lidocaine, procainamide, and quinidine are all class 1 Jul 2007).

1812 Journal of Chemical Education Vol. 84 No. 11 November 2007 www.JCE.DivCHED.org