Using a Standard Curve to Determine the Concentration of Blood Glucose in an Unknown Sample
Introduction:
Blood glucose levels are a predictor of the disease diabetes. Diabetes is when a person is unable to maintain
homeostasis of blood glucose due to a problem with the ability to produce or use insulin. There are 3 types of diabetes,
Type I which is an autoimmune disease, Type II which is a resistance to insulin caused by poor diet, and Type III
which occurs during pregnancy. Prolonged high blood sugar can lead to organ failure, so it is important to diagnose
and treat diabetes.
If diabetes is suspected, blood glucose testing is usually done after fasting for 8 hours. A fasting glucose test is one of
the first steps taken to begin to determine if a person has trouble maintaining blood glucose. This would be followed
up by a glucose tolerance test. In this test, the patient would ingest a given amount of glucose, and a series of
measurements would be taken over time to look at the body’s ability to stabilize blood glucose levels.
The purpose of this investigation is to determine the concentration of glucose in an unknown sample. This information
could be then used to inform a patient about their health and suggest whether further testing for diabetes might be
necessary. A standard curve using known glucose concentrations will be used to determine the concentration in an
unknown sample.
Hypothesis:
If an unknown sample of blood glucose is reacted with potassium permanganate, then its approximate concentration
can be determined by comparing to known concentrations using a standard glucose curve.
Methods:
Known concentrations of glucose were chosen to mimic the concentrations found in the blood. The concentrations
range from 0 mg/dl to 200 mg/dl in increments of 25 mg/dl. In order to assay for reactivity, the reducing properties of
glucose were used. Glucose was reacted with pink potassium permanganate. As the glucose donates electrons to
reduce the permanganate, the solution goes from pink to colorless. The time it takes for the solution to change is
directly proportional to the concentration of glucose.
In this lab the independent variable was the patient sample of glucose. The dependent variable was measured by
comparing it to a glucose standard curve to estimate concentration in mg/dL. The known concentrations of glucose
were measured and the unknown sample test were performed at room temperature. The following reaction was used:
MnO4- + 8H+ + 5e- --> Mn2+ + 4H2O
Purple pink in
Colorless in
solution
solution
For each reaction, sulfuric acid and potassium permanganate were taken from the same 25 ml stock solution. Ten ml
of each glucose solution (and the unknown) were put into a test tube using a pipetter. 5 ml of sulfuric acid was added
as a catalyst for the redox reaction. 2 ml of the potassium permanganate were added to the test tube and the time that it
took to turn colorless was recorded in seconds.
Data and Analysis:
0
Glucose
Concentration
(mg/dL)
5
Time (s)
25
50
75
100
125
150
175
200
12
19
26
30
34
42
46
51
This table shows the standard curve for glucose. The unknown glucose solution took 32 seconds to turn clear.
Glucose Concentration Standard Curve
60
Seconds to turn KMnO4 clear
y = 0.2267x + 6.7778
2
50
R = 0.993
40
30
20
10
0
0
50
100
150
200
250
mg/dL of Glucose
This graph shows the standard curve for glucose. The R2 value on the graph shows that the line of best fit is within the
acceptable range. The unknown glucose solution took 32 seconds to change. When this is put into the equation for the
line of best fit, the X value is 111.25 mg/dL. Therefore, the patient’s blood glucose is 111.25 mg/dL. This is within
the high range for fasting glucose.
Conclusions:
The standard curve for glucose allowed for the concentration of the glucose in the patient’s sample to be determined as
111.25 mg/dL. This was determined by comparing the time in seconds it took the unknown sample to clear to the
standard curve of 9 known glucose concentrations. The line of best fit has an r-squared value of 0.993 which suggests
that the standard curve is a reliable measure of glucose concentration. Based on these results, the patient should be
tested further for a better understanding of the reason for elevated blood glucose.
This result is within the high range for fasting glucose. The range for fasting blood glucose should be between 70 and
100 mg/dL. Blood glucose levels without fasting should be between 70 and 125 mg/dL. Further tests would need to
be given to this patient to determine the cause of increased blood glucose levels. A timed glucose tolerance test might
be ordered next. For this test, the patient would be given a concentrated glucose drink. Then, for several hours the
amount of blood glucose would be monitored in order to determine if the person has the ability to bring blood glucose
levels back to homeostatic levels (70-100 mg/dL). If the results of this second test showed high blood sugar, the
physician could diagnose a pre-diabetic condition. The patient could begin a diet that minimized high sugar content
foods. After changing dietary habits, the patient would then be retested. If they could still not manage blood glucose,
then the possibility of using insulin to regulate blood glucose could be discussed.
Sources of error in this investigation include the reliability of the standard curve. Based on the r-squared value being
.993 this data is reliable. R-squared values closer to one show the reliability of the data. Since .993 is very close to 1,
the data shows a reliable direct correlation between blood glucose concentration and time it takes to turn potassium
permanganate clear. Since this determination is being done by judging the color change, there is some room for
variation. A more reliable method would be to use a colorimeter to determine if the solution is indeed clear. When
performing the blood glucose test in the clinical setting, it is very important to know if the person fasted for 8 hours.
Consuming food could elevate the blood glucose, and if a person did not fast, 111 mg/dL would be an acceptable blood
glucose level. Therefore, it is always important to interview the patient before the test is completed.