High Performance Liquid Chromatography (HPLC) Analysis of

Common Analgesic Compounds

Chemistry Outreach 2019

Introduction 

Liquid chromatography was first described in the early 1900s by a Russian botanist named Mikhail
Tsvet. He successfully illustrated the separation of plant pigments using a particle-packed column and
a solvent. He noticed that the different coloured compounds separated into bands along the column.
The pigments that interacted more with the particles in the column moved slower, whereas the
pigments more strongly attracted to the solvent moved quicker. He reported his findings and coined
the term “chromatography”, from the Greek words “chroma”, meaning colour, and “graph”, meaning
writing. 

Since Tsvet’s work, liquid chromatography has been expanded upon and is one of the most widely
used and powerful analytical techniques in chemistry. Pumps were added in 1970 to generate a high-
pressure flow of solvent through the column, leading to the development of high-pressure liquid
chromatography, or HPLC. Since then, improvements have been seen in the columns, detectors,
injectors, and pressure levels. This has led to the renaming of the HPLC acronym to high performance
liquid chromatography. In a typical HPLC set-up, the solvent, or mobile phase, is housed in a reservoir
and the flow rate is controlled by a high-pressure pump. The liquid sample is introduced at the injector.
The mobile phase moves past the injection site and carries the sample to the column. The column is
packed with material that will allow separation of the analytes. This chromatographic material in the
column is termed the stationary phase. The detector monitors the analytes as they are eluted from the
column and produces an electrical signal. A chromatogram is produced from data, showing relative
areas of signal intensity, and the retention time of the compound. The retention time is the amount of
time it takes for the compound of interest to travel from injection, through the column, to the detector. A
schematic of a HPLC system is shown below:

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 Figure 1 – Schematic of HPLC (Modified from http: www.waters.com/en_CA/Identifying-and-Quantitating-Compounds/nav.htm?
cid=1004906&localeen_CA)

Some common detectors that are used for HPLC include UV-VIS, mass spectrometry, conductivity,
and fluorescence. There are also a variety of columns that can be used. The two most popular
columns used for HPLC are normal phase and reverse phase.  

When performing an HPLC analysis, it is important to note that several factors can affect separation.
These include:

 Column selection: material, column size, particle size, particle shape, packing uniformity,
column efficiency
 Solvent selection: which solvent(s), concentrations
 Mode of separation: isocratic (mobile phase constant) or gradient (gradual change in mobile
phase composition)
 pH of mobile phase
 Flow rate
 Temperature
 Column load
 Sample volume

Test your understanding!

Discuss with your partner and pick any 3 of the factors mentioned above and explain how they would
affect separation
Factor How is Separation Affected?

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Scenario

You are working in a hospital lab. The hospital compounding pharmacy was tasked with preparing an
IV solution of acetaminophen for an ill child that was admitted. During their stay, the child developed
Reye’s Syndrome, in which the liver and brain were damaged. The doctors need to rule out whether
the IV prescription was accidentally made with acetylsalicylic acid (ASA), a substance shown to greatly
increase the risk of Reye’s Syndrome. You are given a sample of the prescribed medicine to analyze.
The hospital pharmacy admits that there were three white solids in use the day of manufacture: ASA
(active pharmaceutical ingredient (API) in aspirin), acetaminophen (API in Tylenol®), and caffeine (a
common additive in pain relievers). You need to determine what solution the pharmacist prepared, and
in what concentration. 

You decide to analyze the solution using your reverse phase HPLC, with isocratic separation and a
UV-VIS detector. You must first determine the ideal solvent composition for analysis, by varying the
amounts of water and methanol in the mobile phase. To effectively analyze the compounds of interest,
you must monitor the wavelength that gives the maximum absorption. For acetylsalicylic acid and
caffeine that is 275 nm, and for acetaminophen it is 250 nm. Once an ideal mobile phase is
determined, you will determine the retention times of each pure substance. You will then be able to run
a sample of the medication to determine which API it contains. A standard calibration curve will be
generated to determine the concentration (mg/mL) of the API in the prescription.  

Note: Before starting the experiment, make sure to familiarise yourself with the health and
safety information provided in the Appendix.

Glassware/Equipment Required

 1 x 50 mL beaker
 20 mL scintillation vials
 100-1000 µL Eppendorf pipet and tips
 Reagents provided:  

 40% methanol/4% acetic acid solvent                                        

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  ASA, acetaminophen, and caffeine standards

Procedure
A. Preparation of Solutions 

1. A 40% methanol/ 4% acetic acid solvent was pre-prepared by combining 200 mL of ultra-pure
water, 200 mL of HPLC grade methanol, and 20 mL of glacial acetic acid. It was then topped up to 500
mL with ultra-pure water.

2. Three stock solutions (ASA, Acetaminophen, and Caffeine) have been pre-prepared and filtered.

3. Take 4 clean, dry 20 mL vials and label them: Mix #1, Mix #2, Mix #3 and Mix #4.

4. For Mix #1, combine:

 0.5 mL of Filtered ASA Stock Solution

 0.25 mL of Filtered Acetaminophen Stock Solution
 0.25 mL of Filtered Caffeine Stock Solution
 4.0 mL of solvent

5. For Mix #2, combine:

 1.0 mL of Filtered ASA Stock Solution

 0.5 mL of Filtered Acetaminophen Stock Solution
 0.5 mL of Filtered Caffeine Stock Solution
 3.0 mL of solvent.

6. For Mix #3, combine:

 1.5 mL of Filtered ASA Stock Solution

 0.75 mL of Filtered Acetaminophen Stock Solution
 0.75 mL of Filtered Caffeine Stock Solution
 2.0 mL of solvent.

7. For Mix #4, combine:

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  2 mL of Filtered ASA Stock Solution
 1.0 mL of Filtered Acetaminophen Stock Solution
 1.0 mL of Filtered Caffeine Stock Solution
 1.0 mL of solvent.

8. The samples are now ready to be analyzed on the HPLC. There will be a total of 12 injections:

 3 injections of Mix #4, to observe the effect of different solvent compositions, to optimize
separation
 3 injections, one of each individual component, to determine the component retention times
(using optimal solvent mix from this point on)
 4 injections, one of each mixture, to create a calibration curve
 2 injections of an unknown

Solvent Composition Comparison for Optimizing Separation 

1. Three different solvent mixtures have been prepared:

 Pump A = 40 % Methanol, 4 % Acetic Acid, in water

 Pump B = 20 % Methanol, 4 % Acetic Acid, in water
 Pump D = 55 % Methanol, 4 % Acetic Acid, in water
 Flow rate is 1.5 mL/min

2. The Lab Assistant has already set up the Chromeleon 7 software connected to the HPLC
equipment

3. Using the Wizard, create a sequence. For the injection name use the following format:
Date_Name_Sample_Solvent (e.g. May16_IronMan_Mix4_20MeOH)

4. Select the appropriate method: Chrom//HPLC-UV/Chromeleon Local/Inst

Data/Ultimate/3000/IntroHPLC/40MeOH (Note: for the other solvent compositions, use “55MeOH” and
“40MeOH”)

5. Processing method is: Chrom//HPLC-UV/Chromeleon Local/Inst Data/Ultimate/3000/IntroHPLC/

Basic 2
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6. For <Report Template>, enter the following: Chrom//HPLC-UV/Chromeleon Local/InstData/
Ultimate/3000/IntroHPLC/220L 

7. For the <Object Name>, enter Mix #4.

8. Click on <Add to Queue>, then click <Start>…the bar will go green.

9. Inject at least 20 µL of the Mix #4 sample into the 20 µL loop in the LOAD position three times.
(LOAD position = handle should be up). Do not inject any air bubbles, and do not remove the syringe
after your third 10 µL injection – it will stay in the injection port until step 12.

 NOTE: you are overloading the loop with sample so when you inject, your volume is EXACTLY
20 µL. Any excess sample will be carried to waste.

10. Pull the handle down to the INJECT on the column. Handle movement must be quick! (Please see
the diagram of the injection handle)

11. When the run is complete, the green bar will change to white.

12. Return the handle to the LOAD position. Again, handle movement must be quick. Remove the
needle from the injection port.

13. To see the results, double click on the Chem 220L Report Template.

14. Observe that 2 wavelengths are monitored simultaneously: 250 nm and 275 nm. Different
compounds will have different absorption maxima and it is important to choose the correct wavelength
for each compound to achieve maximum sensitivity. In your analysis, do not average the results seen
at both wavelengths – only use the data from the wavelength that shows maximum absorption.

15. Repeat the above steps for the other solvent compositions (55% and 40% MeOH). 

 NOTE: When changing solvents, you must allow the column to equilibrate for ~5 minutes.

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Determining Retention Times of Compounds

1. From the previous part of the experiment, determine the optimal mobile phase.
2. Using the correct method name and set up, inject each of the three standard solutions.
3. Make sure to use appropriate file names (e.g. May16_IronMan_ASA Standard_??MeOH).
4. Determine what the retention time is for each compound.

Generating Calibration Curve and Determining Unknown

1. Using the correct method and set up, inject each of the three mixture solutions, and the unknown.
Your unknown should be read twice.

2. Make sure to use appropriate file names (e.g. May16_IronMan_Mix1_55MeOH).

3. Before cleaning the station, ensure that you have all 12 files that you need:

 Mix #4 run with 20% MeOH, 55% MeOH, and 40% MeOH 
 ASA Standard
 Acetaminophen Standard
 Caffeine Standard
 Mixes #1-4 run with chosen solvent
 Unknown, two trials

Waste Disposal and Clean Up

 All waste should be disposed of in the labelled waste container in the fume hood.
 Benches/work areas need to be wiped down.
 Get a TA to check your lab book and equipment drawer before leaving. 

Calculations and write up

 In the Lab Notebook provided, include the chromatograms and subsequent calibration plots
you have generated using your data. Show the equations of the lines, the R 2 values, and how

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 you used the equation of the line to determine the concentration (in mg/mL) of your unknown.
Report the name and average concentration of your unknown.

 Get your calculated concentration checked by the assistant to see if it is within range.

Appendix

Material Safety Data Sheets

Acetic acid, glacial, concentrated (17.4 M) 

CH3COOH

MW = 60.05 g/mol

Physical Properties: clear, colourless liquid, with pungent odour (vinegar)

Health Concerns: Extremely hazardous in case of inhalation, is corrosive in nature and can cause
respiratory damage. Hazardous in contact with skin and eyes; can cause irritations and burns. 

LD50 (oral) = 3 310 mg/kg

LD50 (inhalation) = 5 620 ppm (1 hour)

Acetaminophen
C8H9NO2

MW = 151.17 g/mol

Physical Properties: white solid

Health Concerns: May cause irritation; avoid breathing dust and contact to skin and eyes.

LD50 (oral) = 2 400 mg/kg

Acetylsalicylic acid 

C9H8O4

MW = 180.16 g/mol

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Physical Properties: white, crystalline solid

Health Concerns: Skin and eye irritant; avoid breathing dust. In concentrated amounts this substance
is toxic to blood, lungs, and mucous membranes.

LD50 (oral) = 200 mg/kg

Caffeine

C8H10N4O2

MW = 194.19 g/mol

Physical Properties: bitter, white, crystalline solid

Health Concerns: Skin and eye irritant; avoid breathing dust or ingesting. In concentrated amounts this
substance is toxic to the heart, gastrointestinal tract and central nervous system.

LD50 (oral) = 150 mg/kg

Methanol
CH3OH

MW = 32.04 g/mol

Physical Properties: clear, colourless liquid with alcohol-like smell

Health Concerns: Skin and eye irritant; toxic to eyes; may be toxic to blood, kidneys, liver, brain, upper
respiratory tract, peripheral and central nervous systems

LD50 (oral) = 5 628 mg/kg. LD50 (inhalation) = 64 000 ppm (4 hours)