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EXPERIMENT 7
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC). THE
DETERMINATION OF CAFFEINE AND ASPIRIN IN A TABLET
Safety information
The chemicals listed below will be used in this experiment. The likely hazards associated with
each of the chemicals are noted and recommended procedures for handling are given. You must
read this page and the experimental description carefully before starting the experiment and
before coming into the laboratory. Note any potential hazards and adopt precautions as your safe
lab practice. When you are satisfied that you understand any possible difficulties that might arise
and the recommended procedures for dealing with them, sign the declaration and have it initialled
by a demonstrator. This must be done prior commencing lab work. At the beginning of the lab
session demonstrators will quiz you about the safety information and experimental procedure in
order to identify your ability to work safely and efficiently. If you fail to prove ability for safe
and efficient work you will not be allowed to start lab practical. Please note, that it is your own
responsibility to complete the lab practical during time that is allocated to you. Be sure to request
information or help if you are in doubt on any point.
| Chemical | Hazard | Precautions |
| Caffeine | Harmful, irritant | Do not ingest, avoid skin/eye contact, wear gloves |
| Aspirin | Harmful, irritant | Do not ingest, avoid skin/eye contact, wear gloves |
| Phenacetin | Harmful, irritant and possible carcinogen |
Do not ingest, avoid skin/eye contact, wear gloves |
| Methanol | Extremely flammable and very toxic. Toxic by inhalation, in contact with skin and if swallowed |
Do not breathe vapour, do not ingest, avoid skin contact, wear gloves. Keep away from source of ignition, keep container tightly closed |
| Acetic acid | Corrosive, to eyes, respiratory tract, skin, lungs and mucous membranes |
Do not breathe vapour, do not ingest, avoid skin contact, wear gloves. Keep away from source of ignition, keep container tightly closed |
AssignmentTutorOnline
Declaration – I have read and understood the contents of the safety information sheet and the
script for the experiment
Signed (student): ……………………………………………………..
Checked (demonstrator): ………………………………………….. Date: ………………………
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EXPERIMENT 7
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC). THE
DETERMINATION OF CAFFEINE AND ASPIRIN IN A TABLET
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LEARNING AIMS
To gain familiarity with the use of laboratory HPLC equipment
To gain understanding of the need for system suitability measurements in HPLC analysis
To extend data manipulation and presentational skills
To assess reproducibility of experimental results
To continue developing GLP skills
LEARNING OUTCOMES
To critically discuss the use of HPLC in Pharmaceutical analysis
To manipulate HPLC instrumentation
To calculate the amount of ingredients in raw materials and end products
To discuss quality of HPLC data obtained and quality of the product
DIRECTED READING
OMED 0104 – LECTURE NOTES
British Pharmacopoeia (BP 2007)
Pharmaceutical Analysis 2nd Ed; Watson (2005) Elsevier, ISBN: 0443074453; Chapter 12, pp:
267-315 (nice introduction to HPLC)
Quantitative Chemical Analysis 7th Ed; Harris (2007) W. H. Freeman, ISBN: 0716770415;
Chapter 5, pp: 78-96; Chapter 23, pp: 501-528; Chapter 25, pp: 566-588 (very good explanation)
Contemporary Instrumental Analysis, Rubinson and Rubinson (2000) Prentice-Hall, ISBN:
0137907265; Chapter 14, pp: 628-672 (advanced HPLC)
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INTRODUCTION
High Performance Liquid Chromatography is widely used as an analytical tool in pharmaceutical
sciences. The majority of assay, dissolution, related substances and degradation products
Pharmacopoeial methods are based on separation followed by the detection of compounds using
HPLC.
The goal of an HPLC analysis is to achieve the best possible separation in the shortest
experimental time. In order to achieve the goal a variety of approaches, experimental conditions
and equipment set-ups can be used.
A basic HPLC system consists of the mobile phase reservoir, pump, injector, column, and
detector. However, other parts such as a degasser, auto-sampler, column compartment (oven) and
fraction collector can be connected to the HPLC system.
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EXPERIMENTAL
Familiarise yourself with the operation of the system and if you have any questions ALWAYS
ask a demonstrator.
Check that the liquid chromatograph is set up correctly and make a note of the experimental
conditions.
Chromatographic conditions
| Detector | UV – 224 nm | |
| Column | C18 (LiChrospher RP-18, ODS-2), 5µ, 125 x 4.6 mm | |
| Flow Rate | 1.0 ml/min | |
| Sample loop | 20 µl | |
| Temperature of column | 25°C or room temperature | |
| Mobile Phase | Methanol | 49.5 % |
| Water Acetic Acid |
49.5 % 1 % |
Preparation of a mobile phase
Mix equal amounts of methanol (HPLC quality) and deionised water in a big clean vessel. Filter
the mobile phase through a fine membrane filter and transfer the filtrate into a clean and dry
mobile phase reservoir (bottle). The mobile phase should be degassed before use only if the
system is not equipped with a degassing unit.
Setting up the HPLC system
Find an appropriate column and attach it to the system. Bear in mind that part of the column to
which the pre-column is added should be connected to the pump using metal or PEEK tubing.
The other end of the column should be connected to the detector.
Once the system is completely connected and the experimental parameters are set up correctly the
mobile phase should run through the system to equilibrate the column. The flow rate of the
mobile phase should be increased gradually from low flow rates to the final flow rate envisaged.
Prior to any analysis, the chromatographic system should be equilibrated by passing mobile phase
through the column. To achieve equilibrium of the system the amount of mobile phase that passes
through the system should equal 30 column volumes.
For example, if the volume of a column is 1 ml then 30 ml of mobile phase are required to
establish equilibrium between the mobile and stationary phases in a column. Therefore at a flow
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rate of 1 ml/min, the mobile phase should run through the system for about 30 minutes before any
experimental work is performed.
Preparation of a Phenacetin internal standard solution
Weigh accurately about 25 mg of phenacetin in to a 25.00 ml volumetric flask, add 15.0 ml of
methanol and stir until completely dissolved (use an ultrasonic bath if necessary). Make up to the
mark with distilled water.
Note: -Use of an ultrasonic bath to aid dissolution does increase the temperature of a solution
hence any solution should be cooled down to room temperature before filling up to the mark of a
volumetric flask.
Acetylsalicylic acid standard solution
Weigh accurately about 25 mg of acetylsalicylic acid in to a 25.00 ml volumetric flask. Add 30.0
ml of methanol and stir until completely dissolved (use ultrasonic bath if necessary). Make up to
the mark with distilled water.
Caffeine standard solution
Weigh accurately about 40 mg of caffeine in to a 25.00 ml volumetric flask. Add 15.0 ml of
methanol and stir until completely dissolved (use ultrasonic bath if necessary). Make up to the
mark with distilled water.
Standard solution
Transfer 5.00 ml of the phenacetin internal standard solution, 5.00 ml of the acetylsalicylic acid
standard solution and 5.00 ml of caffeine standard solution in to 50.00 ml volumetric flask and
make up to the mark using mobile phase (methanol/water – 50:50).
Sample stock solution
Find out the average weight of tablets by weighing accurately 10 “Phensic” tablets. Powder 5
tablets using a pestle and mortar. Make sure that obtained powder is homogeneous without large
unbroken particles. Measure accurately a quantity of tablet powder equivalent to ONE “Phensic”
tablet and transfer in to a 50.00 ml volumetric flask. Add 30.0 ml of methanol and try to dissolve
the powder (use ultrasonic bath if necessary). Some amount of powder may not be dissolved due
to the presence of insoluble binders. Do not worry about this, just let the powder settle at the
bottom of the volumetric flask. Make up to the mark with distilled water. Filter the solution
before using it further if necessary.
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Sample solution
Transfer 5.00 ml of the phenacetin internal standard solution and 5.00 ml of sample stock
solution in to a 50.00 ml volumetric flask and make up to the mark using the mobile phase
(methanol/water – 50:50).
Phenacetin
Transfer 1.00 ml of phenacetin solution in to 10.00 ml volumetric flask and make up to mark
with mobile phase (methanol/water – 50:50).
Acetylsalicylic acid
Transfer 1.00 ml of acetylsalicylic acid solution in to 10.00 ml volumetric flask and make up to
mark with mobile phase (methanol/water – 50:50).
Caffeine
Transfer 1.00 ml of caffeine solution in to 10.00 ml volumetric flask and make up to mark with
mobile phase (methanol/water – 50:50).
Inject solutions and record chromatograms in the following order:
1. Phenacetin
2. Acetylsalicylic acid
3. Caffeine
4. Standard solution – 3 times
5. Sample solution – 3 times
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EVALUATION OF RESULTS
Find out the retention times for all three compounds of the standard solution from the first three
HPLC runs.
Calculate the concentration of acetylsalicylic acid and caffeine in the standard solutions (express
it as mg/ml). Use average values from three replicates. How close are those values?
Using the equation below, calculate the concentrations of both compounds in the sample solution.
Again for the area of a compound in the sample solution use the average of three replicates.
A(ist1) is area of internal standard in standard solution
A(ist2) is area of internal standard in sample solution
.( )
( )
( )
( 2)
( 1)
.( ) Conc known
Area known
Area unknown
A istd
A istd
Conc unknown = × ×
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Area(known) is area of compound in a standard solution
Area(unknown) is area of compound in a sample solution
Conc.(known) is concentration (mg/ml) of compound in standard solution
Conc.(unknown) is concentration (mg/ml) of compound in sample solution.
Work out the amount of each of the compounds in the stock solution and express the final result
as mgs of compounds per one tablet. Compare your results with the stated amounts of active
compounds in the “Phensic” tablets. The manufacturing tolerances for these tablets are:
acetylsalicylic acid (95 – 105 %) and caffeine (90 – 110 %) of the stated amount. In addition,
compare your results to the values obtained by inserting the relevant numbers in the following
equation:
W(compound) is mass of compound (i.e. caffeine) used for the preparation of standard solution
W(powder) is mass of powder used for the preparation of sample solution
W(tablet) is average mass of “Phensic” tablet
Would you be able to use a step by step approach to develop above equation?
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QUESTIONS
1. What are the differences between HPLC grade and ordinary lab solvents?
2. Why is it beneficial to filter and degas a mobile phase?
3. What would happen if you changed the mobile phase to 50% methanol and 50% water?
4. What would happen if you increased the mobile phase flow rate?
| ( ) W tablet |
( ) Area known |
( 2) A istd |
= | × | × × |
( ) ( )
2
( )
( 1)
( )
W compound W powder
Area unknown
A istd
Compound mg per tablet ×