GenChem
Week 11
Patient Project: Glucose
Determination
Today’s Agenda:
 Review patient project.
 Review understanding of absorption
spectroscopy.
 Develop the day’s problem.
 Perform the challenge: determine
[glucose] in sample.
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Looking Ahead
Gen. Chem. Exam 3 This FRIDAY!
14
15
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Last Makeup
Lab
Ion-selective electrodes
(12)
21
18
22
23
24
25
29
30
1
2
Building a Poster
(13)
28
Lab
LabFinals
Final ExamLab Finals
(14)
Lab Finals
Poster Day: Mon, May 5
Lab Finals
Gen. Chem.
Exam 4
Final Exam: ???
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But First: What do you think?
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The Patient Project
GenChem
 Patient case studies
 Patient interview data
 Urine analysis (test strips)
 CK and LD tests
 Glucose test
Ion-selective electrodes
 Diagnosis and presentation as a poster
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Testing for glucose
The question posed to you is “how much
glucose is in your samples?” You will use a
common clinical test to get an answer.
What do you know about these tests:
 The test strip
 The enzymatic test.
What information do they provide?
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Glucose
Pre-lab
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Let′s think!
Using Spectroscopy to measure concentrations
A   bC
In your groups:
 Discuss how you can use Beer’s Law to
determine the concentration of something in
solution.
Given values for  and b, A can be
measured and C calculated.
Do we really need
?
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If we construct a calibration plot from values
measured with standards, we can read
unknown values directly from the graph.
Consider a sample
having an
absorbance of 0.37
Let′s think!
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Creating a Calibration Plot
In your groups review your responses to the following
pre-lab question and come up with a consensus answer.
 Indicate how many standard glucose solutions you expect to make and
why you think that will be a sufficient number.
 List the glucose concentrations of the standards you expect to make.
 Explain why one of the standard solutions needs to contain no glucose.
How many total solutions will you need to be
measuring the absorbance of?
HK GLUCOSE Determination Flowchart (per 4 students)
Solutions on the reagent bench:
DO THIS FIRST
Dilute 25 mL HK reagent with 25
mL DI water (for 50 mL total)
Solutions on your bench
Solutions in the fume hood
HK Reagent
Dilute HK Reagent in a small beaker
50 mL in a small beaker
Glucose Standard in a small beaker
GenChem
Glucose Standard
Patient Sample XX
10 mL in a 6” test tube
Patient Sample XX in a 6” test tube
HK GLUCOSE Determination Flowchart (per 4 students)
Solutions on the reagent bench:
Dilute 25 mL HK reagent with 25
mL DI water (for 50 mL total)
DO THIS FIRST
Solutions on your bench
Solutions in the fume hood
HK Reagent
GenChem
Glucose Standard
10 mL in a 6” test tube
50 mL in a small beaker
Dilute HK Reagent in a small beaker
Patient Sample XX in a 6” test tube
Glucose Standard in a small beaker
DO THIS SECOND
Parallel dilutions
2.00 mL
“Glucose
Standard”
+
8.00 mL DI H2O
Glucose 2:10 dilution in tube “1”
4.00 mL
“Glucose
Standard”
+
6.00 mL DI H2O
Glucose 4:10 dilution in tube “2”
6.00 mL
“Glucose
Standard”
+
4.00 mL DI H2O
Glucose 6:10 dilution in tube “3”
Patient Sample XX
8.00 mL
“Glucose
Standard”
+
2.00 mL DI H2O
Glucose 8:10 dilution in tube “4”
HK GLUCOSE Determination Flowchart (per 4 students)
Solutions on the reagent bench:
Dilute 25 mL HK reagent with 25
mL DI water (for 50 mL total)
DO THIS FIRST
Solutions on your bench
GenChem
Solutions in the fume hood
HK Reagent
Glucose Standard
10 mL in a 6” test tube
50 mL in a small beaker
Dilute HK Reagent in a small beaker
Patient Sample XX
Patient Sample XX in a 6” test tube
Glucose Standard in a small beaker
DO THIS SECOND
Parallel dilutions
2.00 mL
“Glucose
Standard”
+
8.00 mL DI H2O
4.00 mL
“Glucose
Standard”
+
6.00 mL DI H2O
Glucose 2:10 dilution in tube “1”
DO THIS THIRD
4.00 mL Dilute HK Reagent
+
1.00 mL DI H2O
Dilutions
with HK
added
Test Tube “0 ”
6.00 mL
“Glucose
Standard”
+
4.00 mL DI H2O
Glucose 4:10 dilution in tube “2”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “1”
Test Tube “1 ”
Glucose 6:10 dilution in tube “3”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “2”
Test Tube “2 ”
8.00 mL
“Glucose
Standard”
+
2.00 mL DI H2O
4.00 mL
Dilute
HK Reagent
+
1.00 mL “3”
Test Tube “3 ”
Glucose 8:10 dilution in tube “4”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “4”
Test Tube “4 ”
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Glucose
Standard”
Test Tube “5 “
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Patient
Sample XX”
Test Tube “XX”
HK GLUCOSE Determination Flowchart (per 4 students)
Solutions on the reagent bench:
Dilute 25 mL HK reagent with 25
mL DI water (for 50 mL total)
DO THIS FIRST
Solutions on your bench
GenChem
Solutions in the fume hood
HK Reagent
Glucose Standard
10 mL in a 6” test tube
50 mL in a small beaker
Dilute HK Reagent in a small beaker
Patient Sample XX
Patient Sample XX in a 6” test tube
Glucose Standard in a small beaker
DO THIS SECOND
Parallel dilutions
2.00 mL
“Glucose
Standard”
+
8.00 mL DI H2O
4.00 mL
“Glucose
Standard”
+
6.00 mL DI H2O
Glucose 2:10 dilution in tube “1”
DO THIS THIRD
4.00 mL Dilute HK Reagent
+
1.00 mL DI H2O
Dilutions
with HK
added
Test Tube “0 ”
6.00 mL
“Glucose
Standard”
+
4.00 mL DI H2O
Glucose 4:10 dilution in tube “2”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “1”
Test Tube “1 ”
8.00 mL
“Glucose
Standard”
+
2.00 mL DI H2O
Glucose 6:10 dilution in tube “3”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “2”
Test Tube “2 ”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “3”
Test Tube “3 ”
Glucose 8:10 dilution in tube “4”
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Glucose
Standard”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “4”
Test Tube “4 ”
Test Tube “5 “
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Patient
Sample XX”
Test Tube “XX”
DO THIS FORTH
IMPORTANT!
Configure (set-up)
spectrometer to
read at 340 nm
before taking any
readings!
Absorbance
readings
with Logger
Pro
Use as the
“blank” in
calibrating the
spectrometer
Read the
absorbance of
dilution in tube
“1”
Read the
absorbance of
dilution in tube
“2”
Read the
absorbance of
dilution in tube
“3”
Read the
absorbance of
dilution in tube
“4”
Read the
absorbance
of
“5”
Read the
absorbance of
“XX”
IMPORTANT! Patient Sample
“XX” must be read at the same
time as the calibration dilutions!
HK GLUCOSE Determination Flowchart (per 4 students)
Solutions on the reagent bench:
Dilute 25 mL HK reagent with 25
mL DI water (for 50 mL total)
DO THIS FIRST
Solutions on your bench
GenChem
Solutions in the fume hood
HK Reagent
Glucose Standard
10 mL in a 6” test tube
50 mL in a small beaker
Dilute HK Reagent in a small beaker
Patient Sample XX
Patient Sample XX in a 6” test tube
Glucose Standard in a small beaker
DO THIS SECOND
Parallel dilutions
2.00 mL
“Glucose
Standard”
+
8.00 mL DI H2O
4.00 mL
“Glucose
Standard”
+
6.00 mL DI H2O
Glucose 2:10 dilution in tube “1”
DO THIS THIRD
Test Tube “0 ”
Glucose 4:10 dilution in tube “2”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “1”
4.00 mL Dilute HK Reagent
+
1.00 mL DI H2O
Dilutions
with HK
added
6.00 mL
“Glucose
Standard”
+
4.00 mL DI H2O
Test Tube “1 ”
8.00 mL
“Glucose
Standard”
+
2.00 mL DI H2O
Glucose 6:10 dilution in tube “3”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “2”
Test Tube “2 ”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “3”
Test Tube “3 ”
Glucose 8:10 dilution in tube “4”
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Glucose
Standard”
4.00 mL
Dilute
HK Reagent
+
1.00 mL “4”
Test Tube “4 ”
Test Tube “5 “
4.00 mL
Dilute
HK Reagent
+
1.00 mL
“Patient
Sample XX”
Test Tube “XX”
DO THIS FOURTH
IMPORTANT!
Configure (set-up)
spectrometer to
read at 340 nm
before taking any
readings!
DO THIS FIFTH
Absorbance
readings
with Logger
Pro
Use as the
“blank” in
calibrating the
spectrometer
Read the
absorbance of
dilution in tube
“1”
Read the
absorbance of
dilution in tube
“2”
Read the
absorbance of
dilution in tube
“3”
Read the
absorbance of
dilution in tube
“4”
Read the
absorbance
of
“5”
Read the
absorbance of
“XX”
IMPORTANT! Patient Sample
“XX” must be read at the same
time as the calibration dilutions!
A vs. C calibration
plot with Logger Pro
DO THIS LAST
“0 ” to “5” readings are used to construct
the calibration plot (Absorbance vs.
Concentration)
Find
slope
(L)
Patient “XX”’s glucose conc. is
determined via Beer’s Law using the
slope of the A vs. C calibration plot
Patient “XX”’s
glucose
concentration
Let′s think!
GenChem
Creating a Calibration Plot
In your groups review your responses to the following
pre-lab question and come up with a consensus answer.
Write a detailed procedure for how you expect to go about preparing your
standard and sample solutions. This procedure must include answers to the
following questions
 how much of each solution should be made?
 what glassware should be used to measure the volumes?
GenChem
Divvying up tasks
Decide in your group who is
going to do what.
Performing the
reactions safely.
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You are required to wear
gloves when performing this
experiment.
GenChem
Let′s think!
Performing the Test Reaction
Reminders
1)
Syringes are provided for this (note that this is a change from the
guide).
2)
Use DI water
3)
DO NOT shake the test tubes (this could damage the enzymes in
the reagent). Leave the parafilm® in place to avoid evaporation.
4)
Once the reagents have been mixed, allow the reaction to proceed
undisturbed for 10 minutes at room temperature.
Yet one more
waste stream.
GenChem
Syringe needles are
sharps waste and
need to be disposed
of accordingly.
GenChem
Let′s explore!
Your Challenge
Prepare your standards
Prepare your samples
Perform the reactions
Measure their absorbances.
Available resources:
 Standard glucose solution.
 HK reagent.
Design and implement
an experimental
procedure!
 automatic pipet and various plastic
syringes.
 various glassware.
 Spectrometer.
You have
90 minutes
Let′s think!
Data Evaluation
GenChem
 Inspect your absorbance values. Are they suitable?
Should any be discarded?
 Plot your calibration data.
 Inspect your calibration plot. Is it suitable? Are more data
points required?
 Inspect your sample value. Is it within the range of your
standard values?
 Do you have the data you need or are additional
measurements required?
Make a list of additional
solutions to be prepared and
measurements to be made
GenChem
Let′s explore!
Followup Challenge
Prepare additional standards and samples
as required.
Measure their absorbance
Available resources:
 Standard glucose solution.
 HK reagent.
Design and implement
an experimental
procedure!
 automatic pipet and various plastic
syringes.
 various glassware.
 Spectrometer.
You have
30 minutes
GenChem
Wrap-up
Determine the [glucose] in your patient samples.
You will report your results in a progress report
constructed next week in class.
1.2
Absorbance
1
y = 0.0475x + 0.015
R2 = 0.9946
0.8
0.6
0.4
0.2
0
0
5
10
15
Concentration (ppm)
20
25