CHEM 6041 – Manual 1
EXPERIMENT MANUAL HPLC-LCMS
CHEM6041 Week 1 labs
Student learning outcomes:
At the end of the Week 1 experiments you will have an understanding of
repeatability and reproducibility for preparing solutions, different ways to
extract compounds from solid samples, and an understanding of what data is
available from SEM.
Aims:
Overall, the goals of these experiments are to:
1. Learn what information can be obtained via SEM, and what samples
are required
2. Provide experience preparing standard solutions
3. Understand repeatability and reproducibility, and the different types of
each.
4. Understand different extraction techniques and how they can provide
different information
Expectations:
We expect you to:
1. Follow safety precautions
2. Be prepared for your labs
3. Carry out all three labs
Note: This lab has 3 parts. The sample preparation for part A must be
complete in the first lab so they can run overnight on the autosampler. You
should also aim to have the data collected for part B. Part C and the data
analysis for part A and part B will be done in the second lab of the week.
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EXPERIMENT MANUAL HPLC-LCMS
Week 1: Part A – Extracting Caffeine in Tea
Specific Aims of this Particular Lab:
1. To learn to extract an analyte from a solid sample for the
purpose of quantifying an unknown in a relatively complex
mixture.
2. To understand the purpose of standards.
Specific Expectations of this Particular Lab:
1. Be able to extract and quantify an analyte in solid chemical
mixture.
2. Identify the most appropriate sample technique to determine the
amount of caffeine in tea:
a. As drunk by a person from a “standard cup of tea” (ISO
3103)
b. For use as a natural source of caffeine for the production of
caffeine pills.
Solutions prepared by technician in advance of lab:
1. Mobile phase (0.1% formic acid in 40% acetonitrile, 60% water)
2. Stock solution of caffeine in methanol 500 mg/L
3. Unknown tea samples (green tea, black tea, decaffeinated black
tea)
Reagents:
1. Redistilled methanol
2. Water
Equipment needed for sample preparation:
1. 10 ml Volumetric flasks,
2. 100-1000 µl and 500-5000 μl autopipettes,
3. Autopipette tips,
4. syringes
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EXPERIMENT MANUAL HPLC-LCMS
5. syringe filters
6. sample vials
7. conical flasks
8. Hotplate
Methods:
1. Prepare a series of standard calibration solutions of caffeine from
about 1 mg/L to 50 mg/L (e.g. 1 mg/L, 2 mg/L, 5 mg/L, 10 mg/L,
20 mg/L, 50 mg/L) by diluting the provided stock solution. You
should need no more than 10 mL of the provided caffeine stock
solution, so please transfer 10 mL of the stock solution into a
sample tube for your use to avoid contamination. Each standard
solution should be made up with mobile phase.
2. Choose a type of tea, ensuring your group covers all three
provided types. For each type, accurately weigh and record ca.
0.5 g of tea (three times per student – once for methanol
extraction, once for cold water extraction and once for hot water
extraction), and work quickly to avoid the adsorption of water
from the air.
3. Prepare the solutions as required for each extraction technique
a. For each of the methanol and water extractions, transfer
the tea to a large sample vial and add 15 ml of solvent.
Shake well and sonicate for 5 minutes.
b. For the hot water extraction, place some water in a conical
flask, cover with a watch glass and use a hotplate to boil
the solution. Add 15 ml of hot water to the tea sample in a
large sample vial and allow to sit for 5 minutes before
chilling using an ice bath.
4. Filter a portion of each solution into separate small sample vials,
using a syringe and syringe filter. To do this, draw the sample into
the syringe, attach a syringe filter to the syringe, and then expel
the fluid from the syringe, ensuring the filter is held firmly in
place.
5. For each sample, pipette 1 mL of filtrate into a 10 mL volumetric
flask and make to volume with mobile phase.
6. Transfer to a HPLC sample vial and add to the batch queue.
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EXPERIMENT MANUAL HPLC-LCMS
Data analysis:
1. Follow software guide to print out the chromatograms with tables
of retention times and peak area.
2. Calculate the concentration of caffeine in each tea sample and
with each analysis method using Brynn Hibbert’s excel sheet (posted
online to the CHEM6041 Moodle page)
Student Reflections:
Are the results reproducible? How much does the determined value of
caffeine vary between groups?
What was the effect of the method of analyte extraction (methanol vs
cold water vs hot water)?
Which extraction method is more indicative of the amount of caffeine
present in a standard cup of tea for human consumption?
Discuss the amount of caffeine in team samples relative to the amount
indicated in the samples based on literature articles and any literature
from the manufacturer(s).
What can be done to ensure your analysis produces a reliable result?
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EXPERIMENT MANUAL HPLC-LCMS
Week 1: Part B – Preparing calibration solutions
Specific Aims of this Particular Lab:
1. To gain further experience in making standard solutions.
2. To examine the repeatability and reproducibility of preparing
standards.
Specific Expectations of this Particular Lab:
1. Be able to independently make a calibration solution directly
from a stock solution and by using serial dilution
2. Be able to calculate the standard deviation for multiple
calibration solutions
Solutions prepared by technician in advance of lab:
1. Stock solution of caffeine in methanol 500 mg/L
Reagents:
1. Redistilled methanol
2. Water
Equipment needed for sample preparation:
1. 10 ml Volumetric flasks,
2. 100-1000 µl and 500-5000 μl autopipettes,
3. Autopipette tips
4. Cuvettes
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Methods:
Note: All spectra should be from 200 to 350 nm. You will need to
record the absorbance of the peak at ~275 nm.
1. Prepare a standard calibration solution of caffeine at 20 mg/L.
2. Prepare a second calibration solution of caffeine at 2 mg/L. Half of
your group will do this by directly using the 500 mg/L stock
solution, the other half will use the prepared 20 mg/L solution.
3. Using the UV-Vis instruments obtain at least 2 absorbance spectra
for the 20 mg/L solutions. Use the following variations (divided
among your group, each person does 1):
a. Obtain the 2 spectra without removing the cuvette from
the instrument.
b. Obtain 1 spectrum, then rinse the cuvette with water and
prepare another solution in the same cuvette, using the
same instrument.
c. Obtain 1 spectrum on one instrument, then using a
different cuvette and instrument and obtain another
spectrum.
4. Using the same instrument and cuvette as your first spectrum
above, obtain a single spectrum of your 2 mg/L solution.
Data analysis:
1. Tabulate the absorbance of each measurement, and add this to
the class document. Each person should have a row listing the 2
separate absorbance measurements for their 20 mg/L solution, how
they obtained their 2 spectra, the absorbance for their 2 mg/L solution,
and how they prepared the 2 mg/L solution.
2. Using the data for the entire class calculate the standard
deviation for:
a) The 20 mg/L data
b) The entire 2 mg/L data
c) The 2 mg/L data, where the solution was prepared by
serial dilution
d) The 2 mg/L data, where the solution was prepared directly
from the 500 mg/L standard.
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3. For each of the 20 mg/L pair of measurements, calculate the
absolute value of the difference in measurements, and then for each of
the 3 possible measurement options, calculate the average and
standard deviation of these differences.
3. For each set of measurement, calculate the ratio of the
absorbance of the 2 mg/L solution to the 20 mg/L solution (first
measurement) and then for each of the 2 preparation methods,
calculate the average and standard deviation of these ratios.
Student Reflection: Discuss the reproducibility of the measurements.
What method provides the greatest reproducibility? Discuss the
accuracy of the 2 mg/L solutions. Which preparation method is the
most precise? Which preparation method is the most precise in terms
of the ratio of the measurements?
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EXPERIMENT MANUAL HPLC-LCMS
Week 1: Part C – SEM
Note: This lab will include a significant amount of assistance from a
demonstrator to guide you through the use of the SEM.
Specific Aims of this Particular Lab:
1. To gain a basic understanding of the information you can obtain
from an SEM
Specific Expectations of this Particular Lab:
1. Be able to identify when an SEM can provide useful information
Samples prepared by technician in advance of lab:
1. A stub with a piece of metal foam attached to the surface.
Methods:
1. Obtain images of the metal foam using all three modes (SE, BSE
and MIX).
2. Use the SEM software to obtain measurements of the length,
width and angle of something in your image.
3. Perform an EDS analysis, of 2 points in your sample, and obtain
a 2D map of your sample.
Student Reflection: What information do the different modes provide?
What information can be obtained from an SEM with EDS?