4103FSBMOL - Practical 4 - HPLC and ATR-FTIR. Flashcards
High Performance Liquid Chromatography (HPLC) and Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATR-FTIR) - Analysis of an unknown powder.
What does HPLC stand for?
High Performance Liquid Chromatography.
What does ATR-FTIR stand for?
Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy.
How does HPLC work?
It separates components of a mixture by the time taken for components to pass through the column containing a solid microparticulate stationary phase. It is based off the components’ affinities for the stationary and mobile phase.
What are the mixture of components transported through the column by?
A pressurised flow of liquid mobile phase.
What temperature does HPLC have to be carried out by?
Room Temperature - no need to volatise samples.
What are samples injected into the HPLC column injected via?
A valve and sample loop.
What does a higher affinity for the stationary phase, than the mobile phase mean?
Component is retained longer on the staionary phase and so will have a longer retention time.
What does a higher affinity for the mobile phase, than the stationary phase mean?
Component is retained shorter on the staionary phase and so will have a shorter retention time.
What happens when the sample elutes from the column?
What different types are there?
They get detected by a detector.
Mass Spectrometer (MS), UV, Refractive Index (RI), Fluorescence.
What do you measure to compare how long a component stays in the column in HPLC?
Retention Time.
How does IR help detect a component?
The absorption of IR radiation causes bonds to bend and vibrate which causes peaks on an IR Spectrum.
Is IR Spectroscopy destructive or non-destructive?
Non-destructive.
Is ATR-FTIR destructive or non-destructive?
Non-destructive.
What type of samples can ATR-FTIR machines analyse?
Solid and Liquid samples.
What is an ATR-FTIR machine made up of?
A high refractive index ATR Crystal (such as a Diamond).
What depth do the internal reflections penetrate into the sample?
0.5 to 5 microns.
What do you have to check before you weigh out the 10mg of unknown powder into a weighing boat?
That the analytical balance has been cleaned and tared with the weighing boat on first.
What do you have to check after you weigh out the 10mg of unknown powder into a weighing boat?
That all the doors on the analytical balance are closed to ensure the weight is correct.
What do you have to rinse the weighing boat into the 10ml volumetric flask using and then make it up using?
Methanol.
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What is the Brand of the ATR-FTIR Instrument?
Agilent Technologies.
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What is the Brand of the HPLC Instrument?
Agilent Technologies.
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What is the Separation Type of the HPLC instrument?
Reverse Phase.
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What is the Temperature of the HPLC instrument we used?
25°C.
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What is the Run Time of the HPLC instrument we used?
2.5 minutes.
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What is the Type of Column of the HPLC instrument we used?
Agilent C18.
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What is the Mobile Phase of the HPLC instrument we used?
Methanol/ Acetic Acid/ H2O.
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What is the Flow Rate of the HPLC instrument we used?
1ml/ minute.
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What is the Injection Volume of the HPLC instrument we used?
10µl.
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What is the Detector of the HPLC instrument we used?
UV at 254nm.
What do you have to clean the crystal with for ATR-FTIR?
Solvent and a Lens Cleaning Tissue which then is allowed to evapourate.
This is to ensure there is no contamination.
