High Performance Liquid Chromatography (HPLC)

Question 1

What is the application of HPLC

Answer 1

Analysis of mixtures of non-volatile or thermally unstable compounds that cannot be analysed by GC

Question 2

Why do we require clean and pure HPLC grade solvents?

Answer 2

this is to prevent column degradation with impurities and to minimize detector background signals from contaminants

Question 3

What are the degassing methods?

Answer 3

Vaccum devices, ultra sonication (high frequency vibration drives gasses out of solvent), heating (decreases solubility of gases) He sparging (dissolved gases are swept out of a solvent)

Question 4

Why is high pressure needed in HPLC?

Answer 4

to improve separation power and create a desired solvent flow

Question 5

What is isocratic elution?

Answer 5

The composition of the mobile phase remains constant during the separation

Question 6

What is gradient elution?

Answer 6

Solvent composition of the mobile phase is changed with time during the separation

Question 7

What is the example of a gradient?

Answer 7

Starting at 10% methanol and ends at 90% methanol after 20 minutes

Question 8

What are the components of the mobile phase?

Answer 8

in mobile phase there are two solvents which is solvent A, the weak solvent that allows solute to elute only slowly and solvent B, the strong solvent that rapidly elites the solutes from the column

Question 9

What are the examples of Solvent A and Solvent B?

Answer 9

Solvent A is usually water meanwhile Solvent B is usually solvent that is immiscible with water such as acetonitrile, methanol, THF and isopropanol

Question 10

What are the functions of the organic solvent in mobile phase?

Answer 10

To reduce the retarding strength of the aqueous component

Question 11

What are the advantages of the gradient elution?

Answer 11

Decreases the retention of the later eluting component so they elute faster, giving narrower and taller peaks for most components. Next, it improves the peak shape for tailed peaks.

Question 12

What are the requirements for HPLC pumping system?

Answer 12

Pressures up to 6000 psi, pulse free, prevents remixing of solutes, control flow rate from 0.1 to 10mL/min

Question 13

What are the advantages of the reciprocating pump?

Answer 13

it provide constant flow rates and independent of solvent viscosity or column backpressure

Question 14

What is are the functions of guard column?

Answer 14

protect and increase the lifetime of the analytical column by retaining debris, particulate matter and contaminants that would clog the changing column efficiency

Question 15

Discuss the effect of the particle size on the separation performance in packed column liquid chromatography.

Answer 15

as the size decreases, the A term is smaller because with small particles the differences in path lengths is less prominent.

the C term is smaller because the equilibrium between phases is more faster/efficient such as the large surface area

these result in the entire curve being shifted downward

Question 16

Why do we use silica for stationery phase?

Answer 16

Silica is inert to most compounds, has a high surface activity which can be modified easily with water and other agents, and can be used to separate a wide variety of chemical compounds.

Question 17

What are the limitation of silica stationary phase?

Answer 17

it only operates on pH range of 2-8, it dissolves above pH 8 and the siloxane bond to the stationary phase hydrolyses below pH 2

Question 18

What are the types of detector?

Answer 18

UV absorbance, fluorescence, electrochemical, refractive index, and mass spectrometric

Question 19

Since UV or visible detector are most widely used, what is the UV region and Vis region?

Answer 19

UV region (100-400 nm) and Vis region (400-780 nm)

Question 20

Why is the UV region usually set at 254 nm?

Answer 20

This will detect any pi to pi* transitions so it would detect anything with the benzene ring.

Question 21

What is the function of absorbance detector DIODE ARRAY (DAD)

Answer 21

To measure absorbance across a broad spectrum of wavelengths simultaneously and allows for the recording of the entire spectrum of each solute as it passed through the diode array detector

Question 22

What is mass spectrometry (MS)

Answer 22

to examine a compound that will provide the structural information of each compounds match from the library of mass spectra of known compounds stored on the computer and thus identify the compound

Question 23

Why is the connection of MS to LC complicated?

Answer 23

LC operates in the liquid phase whereas MS is a gas phase method and analytes are non volatile and may be thermally labile but must be presented in gaseous form.

Question 24

What is the difference between MS and UV detector?

Answer 24

MS can identify components based on structure using the information from computer meanwhile UV detector can detect components based on their absorbance.

Question 25

What are the advantages of the MS detector?

Answer 25

it provides the mass spectra of the compound, from the mass spectra we can obtain the structural information of the compound which can be used to identify the compound

Question 26

What are the types of liquid chromatography?

Answer 26

Partition chromatography (most common), adsorption chromatography, size exclusion chromatography and ion exchange chromatography

Question 27

Definition of partition chromatography.

Answer 27

chromatographic technique in which solutes are separated based on their partition between a liquid mobile phase and liquid stationary phase coated on a solid support.

Question 28

What are the criteria to perform partition chromatography or liquid-liquid chromatography?

Answer 28

when we’re separating highly polar materials, the stationary phase is a second liquid that is immiscible with the liquid mobile phase.

Question 29

About liquid-liquid chromatography

Answer 29

SP is a solvent held in place by adsorption on the surface of the packing particles. The liquid coat might itself be soluble in the MP which will cause the column to bleed.

Question 30

How do we prevent the loss of SP in liquid-liquid chromatography?

Answer 30

use liquid-bonded-phase partition chromatography

Question 31

What is the characteristic of SP in liquid-bonded chromatography?

Answer 31

SP is an organic species that is chemically attached to the surface of the packing particles by chemical bonds, more sturdy and increases the lifetime of the column