Seminary of the Southwest Determination of Caffeine in Excedrin Lab Report

Determination of CaffeineAmount in Medicine by HPLC
Experiment 8 – CHEM 416
Caffeine Amount in Excedrin
Excedrin contains three active ingredients:
●
●
●
Aspirin
Acetaminophen
Caffeine
aspirin
acetaminophen
caffeine
You will measure the amount of caffeine in the drug sample by HPLC analysis (note:
all compounds are conjugated)
2
High Performance Liquid Chromatography (HPLC)
3
Basic HPLC Components
1. Solvents
2. Gradient valve
3. High pressure pump
4. Sample injector
5. Column
6. Detector
7. Display
Schematic of high pressure liquid chromatography system
4
Instrumentation
NEW!! Agilent 1260
Solvent tray
Column type: C-18 reverse-phase column
Degasser
(4.6 mm x 150 mm)
Sample tray and injector
Detector: UV detection (275 nm)
(diode array detector)
Solvent System: (experiment 8)
50% water w/ acetonitrile, 50% methanol
(isocratic)
Reverse phase column
Column
Detector
Pump
5
Columns
Normal-phase chromatography: packed with polar particles
● The LEAST polar solute elutes first
Reverse-phase chromatography: stationary phase is hydrophobic
● the MOST polar solute elutes first
Stationary Phase
Mobile Phase
Normal Phase
Polar
Nonpolar
Reverse Phase
Nonpolar
Polar
6
Amount of Caffeine in Excedrin
Procedure
7
Starting Materials
ALL solutions use the same diluent as
solvent: 50:50 mixture of H2O/acetonitrile
and methanol
Prepare three 10 mL standard solutions
containing 1 mg/mL:
1. Aspirin
2. Acetaminophen
3. Caffeine
8
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric
flask
10 mL
9
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric flask
Fill part way to dissolve solid with diluent, then fill
to the mark. Mix well.
10 mL
10 mL
10 mL
aspirin
(1 mg/mL)
acetaminophen
(1 mg/mL)
caffeine
(1 mg/mL)
10
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric flask
Fill part way to dissolve solid with diluent, then fill
to the mark. Mix well.
Transfer to screw cap vial for safe keeping.
10 mL
10 mL
10 mL
aspirin
(1 mg/mL)
acetaminophen
(1 mg/mL)
caffeine
(1 mg/mL)
Label ALL containers
11
Standard Solutions (0.1 mg/mL)
1 mL of 1 mg/mL stock solution diluted to 10 mL
2. 0.1 mg/mL standard solutions:
○
○
○
○
Transfer 1 mL of stock solution to 10 mL flask
Fill to mark with diluent, mix well
Transfer solution to sampling vial for HPLC analysis
Samples are analyzed to measure retention time
10 mL
10 mL
10 mL
aspirin
(0.1 mg/mL)
acetaminophen
(0.1 mg/mL)
caffeine
(0.1 mg/mL)
Aspirin
Acetaminophen
(0.1 mg/mL)
(0.1 mg/mL)
Caffeine
(0.1 mg/mL)
12
Calibration Curve Standards
4. Caffeine standards for calibration curve:
○
○
○
○
Caffeine stock solution
(1 mg/mL)
100 𝜇L
0.5 mL
0.7 mL
1 mL
0.01 mg/mL
0.05 mg/mL
0.07 mg/mL
0.1 mg/mL*
* already prepared caffeine standard
ALL standard volumes = 10 mL
10 mL
10 mL
10 mL
10 mL
caffeine
(0.01 mg/mL)
caffeine
(0.05 mg/mL)
caffeine
(0.07 mg/mL)
caffeine
(0.1 mg/mL)
Note: already prepared 13
Excedrin Sample
Already prepared Excedrin extract
(1 Excedrin Tablet + Diluent)
3. Excedrin dilution:
○
○
○
Excedrin tablet already dissolved in 10 mL of diluent
Transfer 100𝜇L of this solution to 10 mL volumetric
flask, fill to the line with diluent and mix well
Transfer diluted Excedrin solution to sampling vial for
analysis
10 mL
Dilute 100 𝜇L to 10 mL
Diluted Excedrin Extract
10 mL
Transfer to sample vial for
HPLC analysis
Diluted Excedrin Extract
14
HPLC Vial Prep
HPLC Autosampler Tray
Transfer each solution to clean sample vial
● Fresh pipette with each sample
● Fill at least ⅔ full
● Cap MUST have a rubber septum
● Label each vial – do NOT use marking tape
P1-A1 position
Place ALL sample vials in P1-A1 position, run HPLC trial for
● diluent blank first,
● 0.1 mg/mL standards (acetaminophen, aspirin & caffeine)
● Excedrin dilution
● Calibration curve standards
Sample vial
15
HPLC Samples
Vial
Contents
Concentration
1
Diluent
NA
2
Aspirin
0.1 mg/mL
3
Acetaminophen
0.1 mg/mL
4
Caffeine
0.1 mg/mL
5
Diluted Excedrin
unknown
6
Caffeine
0.07 mg/mL
7
Caffeine
0.05 mg/mL
8
Caffeine
0.01 mg/mL
16
HPLC Instrument Control Panel
Flow rate = 0.5 mL/min
Diode array detector
UV detector (275 nm)
Injection volume = 10 𝜇L
System pressure ≅ 240 bar
Mobile phase composition
50% water/acetonitrile & 50% MeOH
Column temperature ≅ 25°C
17
Sample Information
You will need to create a file for each run:
Signal 1: initials, run, sample, date
Sample name: sample (e.g. diluent)
Comments: more info
(e.g. 50:50 H2O/acetonitrile & MeOH)
Preset in advance for you:
Path: C:\Users\Public\ChemStation\1\Data\
Subdirectory: 416-F21-MONDAY or 416-F21-TUESDAY
Method: C:\Users\Public\Documents\ChemStation\1\Chem 416 Isocratic M
Sample Location: P1-A1 (do not change)
18
HPLC Chromatogram
0.05 mg/mL caffeine
(50:50 water & MeOH solvent)
caffeine
At constant pressure, flow rate and
temperature….
peak area is directly proportional to
solute concentration
plot peak area vs concentration to
construct a calibration curve
peak area
19
Calibration Curve
Use your calibration curve to determine the caffeine concentration in the diluted Excedrin sample
Use the calibration curve to determine the
caffeine concentration in the Excedrin
sample analyzed, then correct for dilutions
to determine the amount in the drug.
Figure 1: Peak area vs caffeine concentration calibration curve
20
Time Management
Arrive with a plan, follow these steps:
● Good record keeping will help avoid mistakes
● Label all containers
● Prepare the diluent for HPLC analysis first, then start to prepare your stock
solutions while running this sample
● Record the exact masses measured for each stock solution (all calculations are
based on these measured amounts)
● Prepare calibration curve standards while running 0.1 mg/mL aspirin,
acetaminophen and caffeine samples
● Plot your data for the calibration curve as you collect the points
● Double check that the unknown caffeine peak area falls within the range of the
21
calibration curve
Post Lab Question 5
Definitions:
1) Retention factor (k): used to compare migration rates of solutes in columns. Values less
than one mean the analyte elutes near the dead time, whereas values above 20,
retention times are too long
2) Selectivity factor (𝞪): describes the retention behavior of two solutes in a
chromatographic system. The higher the selectivity factor, the larger the difference in
retention times between the two peaks which implies better separation. (note: selectivity
is always equal to or greater than one)
3) Plate Count (N): The number of theoretical plates helps quantify the separation
efficiency. Maximizing the number of theoretical plates optimizes separation efficiency.
4) Length (L): Length of the column.
5) Retention time (tr ): The time required for the analyte to reach the detector after
injection.
22
Post Lab Question 5
Useful Equations:
a)
Retention factor (k):
b)
Selectivity factor (𝞪):
c)
Plate count (N):
Where:
K = distribution constant
Vs = volume of stationary phase
Vm = volume of mobile phase
R = resolution
H = plate height
d)
Length (L):
e)
Retention time (tr ):
𝜇 = linear velocity
tr = retention time
tm = dead time
23
Important Chromatographic Quantities
Principles of Instrumental Analysis by Holler, Skoog and Crouch
24
Important Chromatographic Relationships
Principles of Instrumental Analysis by Holler, Skoog and Crouch
25
Determination of Caffeine
Amount in Medicine by HPLC
Experiment 8 – CHEM 416
Caffeine Amount in Excedrin
Excedrin contains three active ingredients:
●
●
●
Aspirin
Acetaminophen
Caffeine
aspirin
acetaminophen
caffeine
You will measure the amount of caffeine in the drug sample by HPLC analysis (note:
all compounds are conjugated)
2
High Performance Liquid Chromatography (HPLC)
3
Basic HPLC Components
1. Solvents
2. Gradient valve
3. High pressure pump
4. Sample injector
5. Column
6. Detector
7. Display
Schematic of high pressure liquid chromatography system
4
Instrumentation
NEW!! Agilent 1260
Solvent tray
Column type: C-18 reverse-phase column
Degasser
(4.6 mm x 150 mm)
Sample tray and injector
Detector: UV detection (275 nm)
(diode array detector)
Solvent System: (experiment 8)
50% water w/ acetonitrile, 50% methanol
(isocratic)
Reverse phase column
Column
Detector
Pump
5
Columns
Normal-phase chromatography: packed with polar particles
● The LEAST polar solute elutes first
Reverse-phase chromatography: stationary phase is hydrophobic
● the MOST polar solute elutes first
Stationary Phase
Mobile Phase
Normal Phase
Polar
Nonpolar
Reverse Phase
Nonpolar
Polar
6
Amount of Caffeine in Excedrin
Procedure
7
Starting Materials
ALL solutions use the same diluent as
solvent: 50:50 mixture of H2O/acetonitrile
and methanol
Prepare three 10 mL standard solutions
containing 1 mg/mL:
1. Aspirin
2. Acetaminophen
3. Caffeine
8
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric
flask
10 mL
9
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric flask
Fill part way to dissolve solid with diluent, then fill
to the mark. Mix well.
10 mL
10 mL
10 mL
aspirin
(1 mg/mL)
acetaminophen
(1 mg/mL)
caffeine
(1 mg/mL)
10
Stock Solutions (1 mg/mL)
1. 1 mg/mL stock solutions:
○
○
○
○
Mass 0.01 g of solid
Transfer quantitatively to 10 mL volumetric flask
Fill part way to dissolve solid with diluent, then fill
to the mark. Mix well.
Transfer to screw cap vial for safe keeping.
10 mL
10 mL
10 mL
aspirin
(1 mg/mL)
acetaminophen
(1 mg/mL)
caffeine
(1 mg/mL)
Label ALL containers
11
Standard Solutions (0.1 mg/mL)
1 mL of 1 mg/mL stock solution diluted to 10 mL
2. 0.1 mg/mL standard solutions:
○
○
○
○
Transfer 1 mL of stock solution to 10 mL flask
Fill to mark with diluent, mix well
Transfer solution to sampling vial for HPLC analysis
Samples are analyzed to measure retention time
10 mL
10 mL
10 mL
aspirin
(0.1 mg/mL)
acetaminophen
(0.1 mg/mL)
caffeine
(0.1 mg/mL)
Aspirin
Acetaminophen
(0.1 mg/mL)
(0.1 mg/mL)
Caffeine
(0.1 mg/mL)
12
Calibration Curve Standards
4. Caffeine standards for calibration curve:
○
○
○
○
Caffeine stock solution
(1 mg/mL)
100 𝜇L
0.5 mL
0.7 mL
1 mL
0.01 mg/mL
0.05 mg/mL
0.07 mg/mL
0.1 mg/mL*
* already prepared caffeine standard
ALL standard volumes = 10 mL
10 mL
10 mL
10 mL
10 mL
caffeine
(0.01 mg/mL)
caffeine
(0.05 mg/mL)
caffeine
(0.07 mg/mL)
caffeine
(0.1 mg/mL)
Note: already prepared 13
Excedrin Sample
Already prepared Excedrin extract
(1 Excedrin Tablet + Diluent)
3. Excedrin dilution:
○
○
○
Excedrin tablet already dissolved in 10 mL of diluent
Transfer 100𝜇L of this solution to 10 mL volumetric
flask, fill to the line with diluent and mix well
Transfer diluted Excedrin solution to sampling vial for
analysis
10 mL
Dilute 100 𝜇L to 10 mL
Diluted Excedrin Extract
10 mL
Transfer to sample vial for
HPLC analysis
Diluted Excedrin Extract
14
HPLC Vial Prep
HPLC Autosampler Tray
Transfer each solution to clean sample vial
● Fresh pipette with each sample
● Fill at least ⅔ full
● Cap MUST have a rubber septum
● Label each vial – do NOT use marking tape
P1-A1 position
Place ALL sample vials in P1-A1 position, run HPLC trial for
● diluent blank first,
● 0.1 mg/mL standards (acetaminophen, aspirin & caffeine)
● Excedrin dilution
● Calibration curve standards
Sample vial
15
HPLC Samples
Vial
Contents
Concentration
1
Diluent
NA
2
Aspirin
0.1 mg/mL
3
Acetaminophen
0.1 mg/mL
4
Caffeine
0.1 mg/mL
5
Diluted Excedrin
unknown
6
Caffeine
0.07 mg/mL
7
Caffeine
0.05 mg/mL
8
Caffeine
0.01 mg/mL
16
HPLC Instrument Control Panel
Flow rate = 0.5 mL/min
Diode array detector
UV detector (275 nm)
Injection volume = 10 𝜇L
System pressure ≅ 240 bar
Mobile phase composition
50% water/acetonitrile & 50% MeOH
Column temperature ≅ 25°C
17
Sample Information
You will need to create a file for each run:
Signal 1: initials, run, sample, date
Sample name: sample (e.g. diluent)
Comments: more info
(e.g. 50:50 H2O/acetonitrile & MeOH)
Preset in advance for you:
Path: C:\Users\Public\ChemStation\1\Data\
Subdirectory: 416-F21-MONDAY or 416-F21-TUESDAY
Method: C:\Users\Public\Documents\ChemStation\1\Chem 416 Isocratic M
Sample Location: P1-A1 (do not change)
18
HPLC Chromatogram
0.05 mg/mL caffeine
(50:50 water & MeOH solvent)
caffeine
At constant pressure, flow rate and
temperature….
peak area is directly proportional to
solute concentration
plot peak area vs concentration to
construct a calibration curve
peak area
19
Calibration Curve
Use your calibration curve to determine the caffeine concentration in the diluted Excedrin sample
Use the calibration curve to determine the
caffeine concentration in the Excedrin
sample analyzed, then correct for dilutions
to determine the amount in the drug.
Figure 1: Peak area vs caffeine concentration calibration curve
20
Time Management
Arrive with a plan, follow these steps:
● Good record keeping will help avoid mistakes
● Label all containers
● Prepare the diluent for HPLC analysis first, then start to prepare your stock
solutions while running this sample
● Record the exact masses measured for each stock solution (all calculations are
based on these measured amounts)
● Prepare calibration curve standards while running 0.1 mg/mL aspirin,
acetaminophen and caffeine samples
● Plot your data for the calibration curve as you collect the points
● Double check that the unknown caffeine peak area falls within the range of the
21
calibration curve
Post Lab Question 5
Definitions:
1) Retention factor (k): used to compare migration rates of solutes in columns. Values less
than one mean the analyte elutes near the dead time, whereas values above 20,
retention times are too long
2) Selectivity factor (𝞪): describes the retention behavior of two solutes in a
chromatographic system. The higher the selectivity factor, the larger the difference in
retention times between the two peaks which implies better separation. (note: selectivity
is always equal to or greater than one)
3) Plate Count (N): The number of theoretical plates helps quantify the separation
efficiency. Maximizing the number of theoretical plates optimizes separation efficiency.
4) Length (L): Length of the column.
5) Retention time (tr ): The time required for the analyte to reach the detector after
injection.
22
Post Lab Question 5
Useful Equations:
a)
Retention factor (k):
b)
Selectivity factor (𝞪):
c)
Plate count (N):
Where:
K = distribution constant
Vs = volume of stationary phase
Vm = volume of mobile phase
R = resolution
H = plate height
d)
Length (L):
e)
Retention time (tr ):
𝜇 = linear velocity
tr = retention time
tm = dead time
23
Important Chromatographic Quantities
Principles of Instrumental Analysis by Holler, Skoog and Crouch
24
Important Chromatographic Relationships
Principles of Instrumental Analysis by Holler, Skoog and Crouch
25
Name: _________________________________
Score: ________
CHEM 416 Exp 8 HPLC (25 points)
❏ PreLab questions/protocols (3 pts)
Experimental Section (4.5 pts)
❏ Brief intro/purpose statement
❏ Reagent table
❏ Instrument info (model, column, mobile phase and detector and parameters,
e.g. pressure, flow rate, injection volume, column temperature)
❏ Protocols for prep and spectral analysis
❏ Diluent
❏ Caffeine stock solution
❏ Acetaminophen stock solution
❏ Drug “extracted sample”
❏ Caffeine standards
Data and Results (10 pts)
❏ Standard stock solutions w/ chromatograms (Figures 1 – 4)
❏ Acetaminophen stock, dilution prep and retention time
❏ Aspirin stock, dilution prep and retention time
❏ Caffeine stock prep (w/ caffeine mass), dilution prep and retention time
❏ ES drug dilution & DF
❏ Table 1 w/ concentration, peak area, tR and λmax
❏ Table 2 w/ calibration curve solution concentration & dilutions
❏ Table 3 w/ calibration curve concentration, peak area and tR
w/ example chromatogram for each standard (Figures 5 – 7 labeled)
❏ Calibration curve, line equation and R2 w/ correct range (Figure 8)
❏ Concentration of diluted “extracted sample”
❏ Concentration of “extracted sample”
❏ Mass of caffeine in Excedrin tablet
Discussion/Conclusion (2 pts)
❏ Summary statement
❏ Unknown sample tested and target compound
❏ Analysis by HPLC w/ column type, solvent system & UV wavelength
❏ Relative retention times of acetaminophen, aspirin and caffeine
❏ Caffeine amount in Excedrin with % error
Questions (5.5 pts)
❏ Q1: Why reverse phase column?
❏ Q2: Peak height vs peak area
❏ Q3: Elution order
❏ Q4: Retention factor (k)
❏ Q5: Retention factor, selectivity factor, theoretical plates,
column length and retention times
½ pt
½ pt
1 pt
½ pt
½ pt
½ pt
½ pt
½ pt
½ pt
½ pt
½ pt
1 pt
1 pt
1 pt
1.5 pt
1.5 pt
1 pt
½ pt
1 pt
2 pt
1 pt
1 pt
½ pt
½ pt
½ pt/each
Name: _________________________________
Score: ________
Deductions:
a) 0.5 pt for poor organization
b) 0.5 pt for not following report template
c) 0.5 pt for not labeling figures and tables
d) 0.5 pt for failing to use results when responding to post lab questions as appropriate
e) 0.5 pt for using incomplete sentences and poor grammar/spelling.
f) 0.5 pt for missing units
g) > 10 pts for plagiarism
Name:
Experiment 8 – Determination of Caffeine in Excedrin by HPLC
I. Introduction/purpose
II. Experimental
A. Reagent Table
B. Instrument and conditions
C. Sample Prep Protocols
■
Diluent (blank)
■
Acetaminophen sample
■
Aspirin sample
■
Caffeine samples
■
Drug sample
III. Data and Results
■ Acetaminophen standard
Concentration of stock acetaminophen solution =
______ mL of the stock solution is diluted to 10 mL.
Concentration of diluted acetaminophen stock solution =
Retention time of acetaminophen =
Figure 1. Acetaminophen chromatogram (0.1 mg/mL).
■
Aspirin standard
Concentration of stock aspirin solution =
______ mL of the stock solution is diluted to 10 mL.
Concentration of diluted aspirin stock solution =
Retention time of aspirin =_
Figure 2. Aspirin chromatogram (0.1 mg/mL)
■
Caffeine standard
__________ of caffeine is weighted out and put in a _______ mL volumetric flask, then filled to
the mark with diluent.
Concentration of stock caffeine solution =
______ mL of the stock solution is diluted to 10 mL
Concentration of diluted caffeine stock solution =
Retention time of caffeine =
Figure 3. Caffeine chromatogram (0.1 mg/mL)
1
Experiment 8
Name:
■
Drug sample
One Excedrin tablet is dissolved in enough diluent to make a 10 mL extract solution. ________
mL of the original extract solution is added to the diluent to make a ________ mL solution.
Dilution factor =
Figure 4. Diluted Excedrin extract chromatogram
Table 1: Data Summary Table
Concentration
(mg/mL)
Peak Area
Retention time
(min)
𝛌max (nm)
Caffeine standard solution
Acetaminophen standard
solution
Aspirin standard solution
Caffeine in diluted extract
sample
■
NA
NA
Caffeine calibration curve standards
Concentration of the caffeine stock solution from part II =
Table 2: Preparation of Caffeine Standard Solutions
________ mg/mL caffeine stock solution
Caffeine standard concentration (mg/mL)
Volume of stock solution diluted to 10 mL
1
2
3
4
Table 3: Calibration Curve Data
Standard
Conc. of caffeine standard
(mg/mL)
Retention time
(min)
Peak Area
1
2
3
4
ES-dilution
▪
unknown
Calibration curve chromatograms
Figure 5. Caffeine chromatogram (0.01 mg/mL).
2
Experiment 8
Name:
Figure 6. Caffeine chromatogram (0.05 mg/mL).
Figure 7. Caffeine chromatogram (0.07 mg/mL).
▪
Calibration graph
Figure 8. Peak area versus caffeine concentration.
▪
Line equation for the calibration curve: y =
■
and R2 =
Amount of Caffeine in Drug Sample
○
Diluted extract sample concentration
Peak area =
Concentration of caffeine in the diluted extract sample =
Calculation:
●
Original extract sample concentration
Concentration of caffeine in the original extract sample =
Calculation:
●
One Excedrin tablet
Amount of caffeine in one Excedrin tablet =
Calculation
IV. Discussion/Conclusion
Remove these instructions from your report:
Write a brief conclusion (100 – 200 words, i.e. less than one page) containing a concise statement
summarizing the question answered and analysis method employed by this experiment to do so. Provide a
summary of collected data (with units and correct significant figures) such as relevant concentrations,
chromatographic data (retention times), observations etc…. with experiment results and comparison to
expected value included. You must include the following items:
1. The compound of interest
2. Brief summary of instrumentation, analysis conditions and property measured
3. Retention times of caffeine, acetaminophen and aspirin
4. Amount of caffeine in one Excedrin tablet
5. Percent error (you will need to look up the amount of caffeine in an Excedrin tablet)
V. Answers to Post-lab Questions 1 – 5
1. Explain the rationale for using a reverse phase C18 column for the determination of caffeine.
2. Would the construction of a calibration curve based on peak height (rather than area) give
accurate results for the determination of caffeine in this experiment? Explain.
3. For a reversed-phase separation, predict the order of elution for: n-hexane, n-hexanol, benzene.
Justify your answer.
3
Experiment 8
Name:
4. Given that the dead time (tM) for this experiment is 1.1 min, find the retention factor (k) for
caffeine in this experiment. (Note: retention factor can also be called the capacity factor (k′))
5. From distribution studies, species M and N are known to have distribution constants between
water and hexane of KM = 5.01 and KN = 5.31 (where KM = [M]H2O/ [M]hex and sometimes referred
to as the partition coefficient). The two species are to be separated by elution with hexane in a
column packed with silica gel containing adsorbed water. The ratio V s/VM (where Vs is the
volume of the stationary phase and VM is the volume of the mobile phase) for the packing is
known to be 0.422. (Show ALL equations and calculations for credit)
(a) Calculate the retention factor (k) for each of the solutes.
(b) Calculate the selectivity factor (α).
(c) How many plates (N) will be needed to provide a resolution (R) of 1.5?
(d) How long a column (L) is needed if the plate height (H) of the packing is 2.2 ⋅ 10-3 cm?
(e) If a linear velocity (μ) of 20.5 cm/min is employed, what time will be required to elute
(i.e. the retention time) the two species.
4
Experiment 8
Name:
Experiment 8 – Determination of Caffeine in Excedrin by HPLC
I. Introduction/purpose
II. Experimental
A. Reagent Table
B. Instrument and conditions
C. Sample Prep Protocols
■
Diluent (blank)
■
Acetaminophen sample
■
Aspirin sample
■
Caffeine samples
■
Drug sample
III. Data and Results
■ Acetaminophen standard
Concentration of stock acetaminophen solution =
______ mL of the stock solution is diluted to 10 mL.
Concentration of diluted acetaminophen stock solution =
Retention time of acetaminophen =
Figure 1. Acetaminophen chromatogram (0.1 mg/mL).
■
Aspirin standard
Concentration of stock aspirin solution =
______ mL of the stock solution is diluted to 10 mL.
Concentration of diluted aspirin stock solution =
Retention time of aspirin =_
Figure 2. Aspirin chromatogram (0.1 mg/mL)
■
Caffeine standard
__________ of caffeine is weighted out and put in a _______ mL volumetric flask, then filled to
the mark with diluent.
Concentration of stock caffeine solution =
______ mL of the stock solution is diluted to 10 mL
Concentration of diluted caffeine stock solution =
Retention time of caffeine =
Figure 3. Caffeine chromatogram (0.1 mg/mL)
1
Experiment 8
Name:
■
Drug sample
One Excedrin tablet is dissolved in enough diluent to make a 10 mL extract solution. ________
mL of the original extract solution is added to the diluent to make a ________ mL solution.
Dilution factor =
Figure 4. Diluted Excedrin extract chromatogram
Table 1: Data Summary Table
Concentration
(mg/mL)
Peak Area
Retention time
(min)
𝛌max (nm)
Caffeine standard solution
Acetaminophen standard
solution
Aspirin standard solution
Caffeine in diluted extract
sample
■
NA
NA
Caffeine calibration curve standards
Concentration of the caffeine stock solution from part II =
Table 2: Preparation of Caffeine Standard Solutions
________ mg/mL caffeine stock solution
Caffeine standard concentration (mg/mL)
Volume of stock solution diluted to 10 mL
1
2
3
4
Table 3: Calibration Curve Data
Standard
Conc. of caffeine standard
(mg/mL)
Retention time
(min)
Peak Area
1
2
3
4
ES-dilution
▪
unknown
Calibration curve chromatograms
Figure 5. Caffeine chromatogram (0.01 mg/mL).
2
Experiment 8
Name:
Figure 6. Caffeine chromatogram (0.05 mg/mL).
Figure 7. Caffeine chromatogram (0.07 mg/mL).
▪
Calibration graph
Figure 8. Peak area versus caffeine concentration.
▪
Line equation for the calibration curve: y =
■
and R2 =
Amount of Caffeine in Drug Sample
○
Diluted extract sample concentration
Peak area =
Concentration of caffeine in the diluted extract sample =
Calculation:
●
Original extract sample concentration
Concentration of caffeine in the original extract sample =
Calculation:
●
One Excedrin tablet
Amount of caffeine in one Excedrin tablet =
Calculation
IV. Discussion/Conclusion
Remove these instructions from your report:
Write a brief conclusion (100 – 200 words, i.e. less than one page) containing a concise statement
summarizing the question answered and analysis method employed by this experiment to do so. Provide a
summary of collected data (with units and correct significant figures) such as relevant concentrations,
chromatographic data (retention times), observations etc…. with experiment results and comparison to
expected value included. You must include the following items:
1. The compound of interest
2. Brief summary of instrumentation, analysis conditions and property measured
3. Retention times of caffeine, acetaminophen and aspirin
4. Amount of caffeine in one Excedrin tablet
5. Percent error (you will need to look up the amount of caffeine in an Excedrin tablet)
V. Answers to Post-lab Questions 1 – 5
1. Explain the rationale for using a reverse phase C18 column for the determination of caffeine.
2. Would the construction of a calibration curve based on peak height (rather than area) give
accurate results for the determination of caffeine in this experiment? Explain.
3. For a reversed-phase separation, predict the order of elution for: n-hexane, n-hexanol, benzene.
Justify your answer.
3
Experiment 8
Name:
4. Given that the dead time (tM) for this experiment is 1.1 min, find the retention factor (k) for
caffeine in this experiment. (Note: retention factor can also be called the capacity factor (k′))
5. From distribution studies, species M and N are known to have distribution constants between
water and hexane of KM = 5.01 and KN = 5.31 (where KM = [M]H2O/ [M]hex and sometimes referred
to as the partition coefficient). The two species are to be separated by elution with hexane in a
column packed with silica gel containing adsorbed water. The ratio V s/VM (where Vs is the
volume of the stationary phase and VM is the volume of the mobile phase) for the packing is
known to be 0.422. (Show ALL equations and calculations for credit)
(a) Calculate the retention factor (k) for each of the solutes.
(b) Calculate the selectivity factor (α).
(c) How many plates (N) will be needed to provide a resolution (R) of 1.5?
(d) How long a column (L) is needed if the plate height (H) of the packing is 2.2 ⋅ 10-3 cm?
(e) If a linear velocity (μ) of 20.5 cm/min is employed, what time will be required to elute
(i.e. the retention time) the two species.
4
Experiment 8