Chromatography and Mass Spectrometry

Question 0

What are the four types of separations?

Answer 0

Ion exchange:separation based on exchange of ions between surface and eluents.

Partition: separation based on solute partitioning between two liquid phases.

Adsorption: separation based on solute adsorption/desorption steps.

Size exclusion: separation based on molecular size.

Question 1

What is chromatography?

Answer 1

Physical process that separates compounds in a mixture (solute) as a result of their different distribution between the stationary and mobile phase.

Question 2

How does HPLC work?

Answer 2

The use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

Question 3

What sample type should be used for HPLC?

Answer 3

Liquid ones.

Question 4

What are the limitations of HPLC?

Answer 4

Solubility in the mobile phase, no thermal restrictions.

Question 5

What is normal phase chromatography?

Answer 5

Uses polar stationary phase
Less polar mobile phase
Uses silica (supporting medium) which is low cost and very well characterised.

Question 6

What are the problems with normal phase chromatography?

Answer 6

Local of sensitivity: compounds always eluted in the same order.
Water adsorbed into the strongest sites.

Question 7

What are the two mobile phases?

Answer 7

Isocratic elution: the use of a single solvent (with equal pressure power)
Gradient elution: altering the composition of the solvent mixture.

Question 8

What is reverse phase chromatography?

Answer 8

The most widely used mode of HPLC
Mainly for the separation of non-ionic substances because ionic, and hence strongly polar, compounds show very little affinity for the non-polar stationary phase.

Question 9

Name six detectors and their function.

Answer 9

Variable wavelength detector: based upon UV-visible light spectrophotometry. Capable of measuring absorbances down to 190nm.

Scanning wavelength detectors: have the facility to record the complete absorption spectrum of each analyte, thus aiding in identification.

Fluorescent detectors: great light sensitivity but limited since few analyses fluoresce.

Mass spectrometer detector: this enables the analyte to be detected and it’s structure determined simultaneously.

NMR detector: this gives structural information about the analyte that is complementary to that obtained via HPLC-MS.

Refractive index detector: this relies on a change of the refractive index of the eluate as analytes emerge from the column.

Question 10

What are automatic fraction collectors?

Answer 10

Designed either to collect a selected volume of eluate or to collect the eluate for a predetermined period of time before a new collection tube is placed in position automatically.

Question 11

What is detected in HPLC for diagnosis?

Answer 11

Vitamins

Determination of serotonin release from platelets in the diagnosis of heparin-induced thrombocytosis.

Question 12

What is mass spectrometry?

Answer 12

An extremely valuable analytical technique in which the molecules in a test sample are converted to gaseous ions that are subsequently separated according to their mass-to-charge ratio and detected. In the mass spectrum that is produced the relative amounts of the ions is displayed as their relative abundance on the y-axis and their m/z values on the x-axis.

Question 13

What are the essential features of MS?

Answer 13

Production of ions in the gas phase.
Acceleration of the ions to a specific velocity in an electric field.
Separation of the ions in a mass analyser
Detection of each species of a particular mass to charge ratio.

Question 14

What does MALDI-TOF mass spectrometry stand for?

Answer 14

Matrix assisted laser desorption ionisation - time of flight.

Question 15

How are proteins identified using MALDI?

Answer 15

After MS analysis, a peptide mass fingerprint is produced which is compared with peptide masses obtained from theoretical digestion in protein sequence databases (Swiss-Prot).

Question 16

MALDI is used as a diagnostic tool for what?

Answer 16

Tumour marker.

Metabolomics.

Question 17

There are a wide range of stationary phases for HPLC but what are they required to do?

Answer 17

Enable partition, adsorption, gel permeation, affinity and ion-exchange chromatography to be performed.

Question 18

The choice of mobile phases for HPLC depends on what?

Answer 18

Compatibility with the stationary phase: the mobile phase must not react chemically with the stationary phase or break the bond linking it to the supporting material.

Compatibility with the detection system: in any chromatographic analysis, the method of detection is determined by the nature of the analyte and the mobile phase used must not interfere with this system.

Polarity: the polarity of the mobile phase should be such that there is an effective partition of the analyte between the two phases, I.e the stationary and mobile phase, and not a complete affinity for only one.

Question 19

Why is polarity in HPLC important?

Answer 19

The polarity of a solvent is expressed as solvent strength, which is a measure of the ability of the solvent to break adsorptive bonds and elute a solute from an absorbent. High values indicate polarity. The major factor in selecting a mobile phase for separations based on partition or adsorption is the polarity of the analyte.

Question 20

What features are needed for the mobile phase for HPLC?

Answer 20

Must be available in a pure form and usually requires degassing before use.

Question 21

Why is HPLC versatile?

Answer 21

Lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography.

Question 22

How is ionisation achieved in MS?

Answer 22

To get positively charged species is the removal of an electron or the addition of one or more protons to give either molecular ions (M+) or protonated molecular species (M+nH)n+.

Question 23

What is time of flight?

Answer 23

The mass is related to the time taken to reach the detector.