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Preparation of calibration standards:
Stock solution (labelled STD 4) was prepared by weighing Paracetamol (0.2296g), Aspirin (0.3576g) and Caffeine (0.0504g) into separate weigh boats and transferring each to a 100ml volumetric flask - each boat wash washed with approx 10ml of methanol into the flask. Approx 50ml of methanol was added and the solution vigorously shaken to ensure that all 3 compounds were fully dissolved. Finally, the volumetric flask was made up to the mark with methanol and gently shaken and capped.
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The other standards were prepared by pipetting an aliquot of STD 4 into a 5ml volumetric flask and making to the mark with methanol.
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Standard Volume of STD 4 (ml)
​STD 1 3.50
STD 2 4.00
STD 3 4.50
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Calculate the concentration of each compound, mg/ml to 4 decimal places - input the values in the table below.
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Vial
ID
Paracetamol mg/ml
Aspirin mg/ml
Caffeine mg/ml
51
STD 1
52
STD 2
53
STD 3
54
STD 4
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Preparation of quality control standard QC 1:
Paracetamol (0.0992g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol.
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Calculated the concentration of QC 1, mg/ml to 4 decimal places - input the value in the table below.
Vial
ID
Paracetamol mg/ml
61
QC 1
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Preparation of quality control standard QC 2:
Aspirin (0.1486g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol.
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Calculated the concentration of QC 2, mg/ml to 4 decimal places - input the value in the table below.
Vial
ID
Aspirin mg/ml
62
QC 2
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Preparation of quality control standard QC 3:
Caffeine (0.0224g) was weighed directly into a 50ml volumetric flask and made to the mark with methanol.
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Calculated the concentration of QC 3, mg/ml to 4 decimal places - input the value in the table below.
Vial
ID
Caffeine mg/ml
63
QC 3
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Paper chromatography:
Select the correct inputs that highlight the practical differences between paper chromatography and HPLC.
Q. What does the abbreviation HPLC refer to?
HPLC:
(reverse phase)
Q. What is the difference between normal phase and reverse phase HPLC?
Using an C18 column as an example discuss differences in chemical structure, surface polarity of SP and the polarity of the MP's used. How many methylene (-CH2) groups and methyl (-CH3) groups are there in each C18 chain?
Q. Retention time (RT)
What is meant by the term 'retention time' ?
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Q. Retention time (RT)
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Explain why reproducible RT measurements are an important requirement for a successful, validated HPLC method.
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HPLC method development:
Product A, an energy drink currently on the market, undergoes HPLC quality control checks at the manufacturing plant using method LC-25A based on:
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Reverse phase column : C18
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Mobile phase: 60% A (water) and 40% B (methanol).
Using this method baseline separation is achieved for all 3 active ingredients, ensuring accurate quantitative QC analysis.
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The manufacturer is developing a new Product B which contains the same 3 active ingredients as A, plus a 4th new ingredient. Initial analysis of B using the existing HPLC method LC-25A show that the peak for the new ingredient is co-eluting with one of the other peaks.
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Q. Explain what is meant by the terms 'baseline' separation and 'co-eluting' peaks. What changes to the method would you investigate to solve the problem ? - explain your reasoning.
Select a module to build an HPLC system that requires the minimum length of tubing connecting each module.
Q. Why do we need a high pressure pump for HPLC?
Q. Why is the HPLC column housed inside an oven?
Q. Describe the function of the autosampler, including the steps performed to inject an aliquot of sample onto the column.
Q. Explain how the UV detector is used to detect compounds that elute from the HPLC column. Include a short discussion about the chemical structural features required to generate a UV signal.