Automated chromatography

Question 1

What does HPLC stand for?

Answer 1

High performance liquid chromatography

Question 2

What is the basic set up of HPLC?

Answer 2

One pump transports solvent mix

Manual injection of sample through injector port

Question 3

Types of HPLC?

Answer 3

analytical method

Semi-preparative - collect fractions

Question 4

What is sophisticated HPLC?

Answer 4

Automated and controlled by a PC and specialist software

Question 5

What does the PC control in sophisticated HPLC?

Answer 5

Gradient elution

Question 6

What are the HPLC columns like?

Answer 6

Stainless steel

The stationary phase (normal phase) is made of silica

Question 7

What are the differences between analytical columns and semi-preparative columns?

Answer 7

Semi preparative are larger, have a larger flow rate and have a larger sample size

Question 8

Can you reuse the HPLC columns?

Answer 8

Yes

They are washed after every time

Question 9

How does the particle size of SiO2 affect the reaction?

Answer 9

The smaller the particle, the greater the surface area
If they are smaller there are increased interactions between stationary phase and analyte
There is better separation of the components of the mixture

Question 10

in HPLC, is the eluent polar or non-polar?

Answer 10

Non-polar

Question 11

How does the price of eluents for HPLC compare to other chromatography methods?

Answer 11

They are more expensive

Question 12

Which type of compounds elute faster in HPLC?

Answer 12

Non-polar compounds

Question 13

What is different in reversed phase HPLC?

Answer 13

There is a different stationary phase

Question 14

What is the particle size like for SiO2 in HPLC?

Answer 14

2-10 μm

Question 15

What is the particle size like for the stationary phase in the reversed phase HPLC?

Answer 15

2-8 μm

Question 16

How is the stationary phase different in the reversed phase HPLC?

Answer 16

The silica is modified

It is given a non polar surface with hydrocarbon chains attached

Question 17

What is the mobile phase like in reversed phase HPLC?

Answer 17

Polar

Question 18

What are some examples of the polar mobile phases used?

Answer 18

Methanol

Acetonitrile

Question 19

What eluent is used in reversed phase HPLC?

Answer 19

Water

Question 20

What eluent is used for gradient elution in reversed phase HPLC?

Answer 20

Acetonitrile / water

Question 21

Which compounds elute first in reversed phase HPLC?

Answer 21

Polar

Question 22

Where is there attraction between in reversed phase HPLC?

Answer 22

Attraction between polar solvent and polar molecules and flow quickly through the column

Question 23

What do the non-polar compounds interact with?

Answer 23

Hydrocarbon groups and elute slowly through the column

Question 24

What is retention time?

Answer 24

Time taken for a compound to travel through the column to the detector

Question 25

Where is retention time measured from?

Answer 25

Injection to detection

Question 26

What do different compounds have?

Answer 26

Different retention times

Question 27

What does retention time depend on?

Answer 27

- Solvent flow rate - Stationary phase - composition of the solvent - Temperature of the column

Question 28

What do you do to a HPLC chromatogram?

Answer 28

Integrate to obtain concentrations of each component

Question 29

What is the detector usually in HPLC?

Answer 29

a UV spectrometer

Question 30

In detection, what does the absorption depend on?

Answer 30

The amount of compound detected

Question 31

Solvents also absorb light, what do you need to do to do this?

Answer 31

Select a suitable wavelength | Background noise set to zero

Question 32

What is the output from the detector in HPLC?

Answer 32

A series of peaks Each represents a compounds Use values to identify the components of the molecule

Question 33

When would you have to use a modified column instead of a normal HPLC column?

Answer 33

To separate mixtures of very polar compounds | To separate enantiomers

Question 34

In gas chromatography, what is the stationary phase?

Answer 34

The column

Question 35

In gas chromatography, what is the mobile phase?

Answer 35

Inert carrier gas

Question 36

What are the parts of gas chromatography?

Answer 36

Injection port Oven Detector Signal processing software

Question 37

Why do you need high temperatures in gas chromatography?

Answer 37

Makes sure there is a vapour of the molecule travelling through

Question 38

What is the process of gas chromatography?

Answer 38

The sample is introduced and vaporised Carrier gas transports vapours Column separates vapour mixture

Question 39

What should the solvent do in gas chromatography?

Answer 39

Completely dissolve the sample

Question 40

What should the sample not do in gas chromatography?

Answer 40

It shouldnt react with the sample or stationary phase it should not elute at the same time as the sample It should not be used in excess

Question 41

What happens when the constant temperature is too low in gas chromatography?

Answer 41

All components are separated | Peak diffusion with time

Question 42

What happens when the constant temperature is too high in gas chromatography?

Answer 42

Mixture of components co-elute

Question 43

What happens when there is a programmed temperature increase in gas chromatography?

Answer 43

Separated signals and they are all sharp and clear

Question 44

What is LC-MS?

Answer 44

Liquid chromatography is coupled to mass spectrometry

Question 45

What are the features of LC-MS?

Answer 45

Reverse phase HPLC separates components Eluent is vapourised using a high voltage Ions are detected for each fraction separately

Question 46

What is the output for LC-MS?

Answer 46

HPLC trace and mass spectra