4103FSBMOL - Practical 4 - HPLC and ATR-FTIR.

Question 1

What does HPLC stand for?

Answer 1

High Performance Liquid Chromatography.

Question 2

What does ATR-FTIR stand for?

Answer 2

Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy.

Question 3

How does HPLC work?

Answer 3

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.

Question 4

What are the mixture of components transported through the column by?

Answer 4

A pressurised flow of liquid mobile phase.

Question 5

What temperature does HPLC have to be carried out by?

Answer 5

Room Temperature - no need to volatise samples.

Question 6

What are samples injected into the HPLC column injected via?

Answer 6

A valve and sample loop.

Question 7

What does a higher affinity for the stationary phase, than the mobile phase mean?

Answer 7

Component is retained longer on the staionary phase and so will have a longer retention time.

Question 8

What does a higher affinity for the mobile phase, than the stationary phase mean?

Answer 8

Component is retained shorter on the staionary phase and so will have a shorter retention time.

Question 9

What happens when the sample elutes from the column?

What different types are there?

Answer 9

They get detected by a detector.

Mass Spectrometer (MS), UV, Refractive Index (RI), Fluorescence.

Question 10

What do you measure to compare how long a component stays in the column in HPLC?

Answer 10

Retention Time.

Question 11

How does IR help detect a component?

Answer 11

The absorption of IR radiation causes bonds to bend and vibrate which causes peaks on an IR Spectrum.

Question 12

Is IR Spectroscopy destructive or non-destructive?

Answer 12

Non-destructive.

Question 13

Is ATR-FTIR destructive or non-destructive?

Answer 13

Non-destructive.

Question 14

What type of samples can ATR-FTIR machines analyse?

Answer 14

Solid and Liquid samples.

Question 15

What is an ATR-FTIR machine made up of?

Answer 15

A high refractive index ATR Crystal (such as a Diamond).

Question 16

What depth do the internal reflections penetrate into the sample?

Answer 16

0.5 to 5 microns.

Question 17

What do you have to check before you weigh out the 10mg of unknown powder into a weighing boat?

Answer 17

That the analytical balance has been cleaned and tared with the weighing boat on first.

Question 18

What do you have to check after you weigh out the 10mg of unknown powder into a weighing boat?

Answer 18

That all the doors on the analytical balance are closed to ensure the weight is correct.

Question 19

What do you have to rinse the weighing boat into the 10ml volumetric flask using and then make it up using?

Answer 19

Methanol.

Question 20

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What is the Brand of the ATR-FTIR Instrument?

Answer 20

Agilent Technologies.

Question 21

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What is the Brand of the HPLC Instrument?

Answer 21

Agilent Technologies.

Question 22

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What is the Separation Type of the HPLC instrument?

Answer 22

Reverse Phase.

Question 23

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What is the Temperature of the HPLC instrument we used?

Answer 23

25°C.

Question 24

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What is the Run Time of the HPLC instrument we used?

Answer 24

2.5 minutes.

Question 25

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What is the Type of Column of the HPLC instrument we used?

Answer 25

Agilent C18.

Question 26

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What is the Mobile Phase of the HPLC instrument we used?

Answer 26

Methanol/ Acetic Acid/ H₂O.

Question 27

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What is the Flow Rate of the HPLC instrument we used?

Answer 27

1ml/ minute.

Question 28

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What is the Injection Volume of the HPLC instrument we used?

Answer 28

10µl.

Question 29

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What is the Detector of the HPLC instrument we used?

Answer 29

UV at 254nm.

Question 30

What do you have to clean the crystal with for ATR-FTIR?

Answer 30

Solvent and a Lens Cleaning Tissue which then is allowed to evapourate.

This is to ensure there is no contamination.