Chromatography 2 - HPLC

Question 1

What is the correct order of components in HPLC?

Answer 1

pump
injector
column
detector

Question 2

What are the properties of the solvent reservoir?

Answer 2

is made up of glass bottles with a filtered inlet
solvents are degassed prior to use

filtered inlet - stops the pump from drawing up large particles or debris that could damage the system

Question 3

What are examples of solvents that can be used in HPLC?

Answer 3

methanol
water
acetonitrile

Question 4

What are the properties of the HPLC pump?

Answer 4

high pressure pumps are required to force the solvent through the packed stationary phase

as the plunger moves out it sucks in solvent from the solvent reservoir
as the plunger moves in it pushes solvent in to the column as the doors/valves open

Question 5

What are the advantages of smaller particles being used in HPLC?

Answer 5

higher resolution
faster analysis
increased sample load capacity

Question 6

What is the difference between injection into gas chromatography and high performance liquid chromatography?

Answer 6

GC
- injection is into a gas flow with low pressure
= only requires a rubber septum

HPLC
- injection is into high pressure
= requires a rheodyne valve

Question 7

What is a rheodyne valve?

Answer 7

a special type of valve that allows the sample to be introduced into the high pressure flow
- are able to withstand/sustain pressures unto 10,000 psi

injection volume can be changed by changing the valve position

Question 8

What are the properties of the HPLC column?

Answer 8

is the region where separation takes place

- can be normal or reverse phase

Question 9

What are the different methods of separation within the HPLC column?

Answer 9

normal phase

• stationary phase = polar - e.g. silica
• mobile phase = non-polar - e.g. hexane, ethylacetate

reverse phase

• stationary phase = non-polar - e.g. C18 modified silica
• mobile phase = polar - e.g. methanol, acetonitrile

reverse phase is more useful for pharmaceutical compounds

Question 10

How can a chromatogram be made?

Answer 10

records signal vs time

signal is produced by the absorption of UV by compounds which have eluted

Question 11

What is retention time?

Answer 11

time elapsed from introduction of the sample to time it leaves the chromatogram

usually taken from the apex of the peak in a Gaussian profile

Question 12

What causes poor peaks to form in a Gaussian profile?

Answer 12

poor chromatography
poor column choice
poor solvent
overloading - solvent is too concentrated

Question 13

What is a diode array?

Answer 13

method which produces a 3D plot of a chromatogram

as the eluent comes out, all lights of different wavelengths are shone through
light is separated by a dispersion device
mini detector measure absorbance at a specific wavelength

dispersion device = prism, diffraction grating

Question 14

What is the difference between a diode array and a UV detector?

Answer 14

diode array

• can detect multiple wavelengths at once
• produces a chromatogram with 3D plots

UV

• uses a singe detector that only detects at a specific wavelength
• produces a chromatogram with single peaks

Question 15

What are the advantages of using a diode array?

Answer 15

produces UV profiles for each sample/compound = allows the difference between them to be seen

shows the wavelengths at which unknown compounds absorb at

Question 16

What are the types of analysis? What is the difference between them?

Answer 16

qualitative = what is in there
- runs known standards and compares retention times with those of the unknown

quantitative = how much is there
- signal from the detector and the size of the peak are directly proportional to the amount of substance eluting

Question 17

How can quantitative analysis be done?

Answer 17

run a series of standard solutions of analytes with a known concentration and produce a calibration curve of peak area vs concentration

is used to predict unknown concentration of analytes
- must use a series of standards that are estimated to be similar to that of the unknown