Chromatography 2

Question 1

Chromatograms

Answer 1

These are graphs which show when different components reach the detector.

Ideally each component should be seen as a separate peak.

Question 2

Peak Area

Answer 2

The size of the peak produced for each component relates to the amount present in the original sample.

Computers attached to chromatographic equipment use the peak area to indicate the amount present.

The peak is seen where the response of the detector increases from the baseline, which represents the zero response of the instrument when no component is being detected.

Question 3

Co-elution

Answer 3

This is where two components have not been completely separated and some of each component reached the detector at the same time.

It is desirable to have at least some baseline between the peaks.

Question 4

Selectivity Factor and Resolution

Answer 4

Selectivity factor and resolution are two ways of expressing how well two components of a mixture are separated.

For qualitative analysis the components should be separated so that it is easy to see the different components and make identification using retention times.

For quantitative analysis, the components should be separated as the size of each peak related to how much was present in the sample analysed.

Question 5

Selectivity Factor

Answer 5

The separation is expressed using only the retention times of each component.

Problems occur as some components will produce sharp peaks and other broader peaks.

Question 6

Resolution

Answer 6

This utilises not only the retention time but also the point where each peak starts and ends.

This accounts for the difference in peak width.

Question 7

Chromatography Detectors

Answer 7

Two different types of analysis can be conducted with chromatography, firstly the identity of components can be established based on the retention time or retardation factor.

The quantity of an identified component in a mixture can be established, by comparison to standards of differing concentration or by standard addition.

Question 8

Different Detectors

Answer 8

Flame ionisation detector
Thermal conductivity detector
Nitrogen-phosphorous detector
Electron-capture detector
Atomic emission detector
Fourier-Transform Infra-Red detector
Mass spectrometer
UV-Vis detectors
Photodiode array dectors
Refractive index detector
Fluorescence detector
Electrochemical detectors

Question 9

Flame Ionisation Detector (FID)

Answer 9

Responds to all organic compounds, anything that combusts.

With this the components leaving the column are burnt in a small flame fed by a mixture of hydrogen and air.

The temperature of the flame is such that the combustion products are immediately broken into ions. This generates more ions in the flame.

Ions are measured by conductance between two electrodes.

Question 10

Thermal Conductivity (TCD)

Answer 10

This detector uses a heated filament immersed in the components leaving the column. Only the mobile phase is present, so the heat from the filament is constant.

When other components are present, the filament heats up. As the temperature changes, the electrical resistance does too.

Question 11

Nitrogen-Phosphorous Detector (NPD)

Answer 11

An alkali bead is heated in the flame of an FID, which emits electrons which are collected at an electrode above the bead.

When compounds containing nitrogen or phosphorous are present, these coat the surface of the bead and less electrons reach the electrode.

Question 12

Electron-Capture Detector

Answer 12

The detector uses a heated radioactive bead, which emits electrons and ionizes some of the carrier gas as it travels past, which produces a current between the two electrodes.

When compounds containing electronegative elements pass the bead, they absorb electrons and reduce the current between the electrodes.

Question 13

Atomic Emission Detector

Answer 13

Compounds enter a heated chamber containing a microwave element. The microwave destroys the compounds and the atoms are excited by the heat of the chamber.

Light emitted by the excited atoms is separated into individual lines, similar to a prism with normal white light.

A detector then records each wavelength simultaneously. The chromatogram produced shows the elemental composition simultaneously, each line representing a chemical element.

Question 14

Qualitative Use

Answer 14

Using detectors, retention time and retardation factor can be used to qualitatively identify components within a mixture.

Question 15

Quantitative Use

Answer 15

The linear ranges for different detectors relative to how they respond to different concentrations of compounds or elements.

The larger the linear range, the less sample preparation required.

The sensitivity and limit of detection indicate how low a concentration the detector will actually respond to.

Question 16

Qualitative Analysis

Answer 16

The identity of components can be established based on retention time/retardation factor.

Same analysis conditions means direct comparison of chromatograms is possible.

Question 17

Using Retention Times

Answer 17

Identification can often be made using retention times and analysis of reference standards of the suspected drug.

Once retention times in a sample have been found, they can be compared to reference standards.

Question 18

Quantitative Analysis (1)

Answer 18

The concentration of a component of interest can be established based on the response from an appropriate detector and comparison to standards of known concentration.

Each compound responds differently at the detector, so standards must contain different concentrations of the compound being analysed.

Question 19

Quantitative Analysis (2)

Answer 19

This relies on the response of detectors to be closely related to the amount of sample it’s detecting.

Question 20

Accounting for Sample Prep

Answer 20

Finding the original concentration requires knowledge of preparation taken.

Concentration may need converting if in mg/L, where other volumes are in ml.