Chromatography Flashcards by Gleb Butyagin

What is the key fact about chromatography?

This is about spendint time in phases

Analytes spend the same amount of time in the mobile phase. Only the time spent in the stationary phase changes

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What are the 5 ways of separating analytes?

Adsorption
Partition
Ion-exchange
Size exclusion
Affinity

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Define dead time

Time for unretauned solute to go through the column

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Define adjusted retention time

Retention time - dead time

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Define retention factor

Adjusted retension time / dead time

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What is the column separation factor?

A fraction >= 1 showing how well the sample separate
Retention factor (A) / Retention factor (B)

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Define resolution

Ratio between two peak maxima and their average width at base

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Define partition coefficient

Equilibrium between mobile and stationary phases

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What does peak fronting signify?

Column overloading

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What does peak tailing signify?

Uneven stationary phase

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What causes peak tailing?

Some sites retaining the solute mole strongly than others

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Define Plate

Represents the theoretical distance required for one adsorption-desorption step of a solute between the stationary and mobile phases

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Define peak capacity

Maximum theoretical number of solutes that can be baseline resolved on a given column

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Purnell equation

Which three factors does the equation combine?

Efficiency
Retention factor
Selectivity

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Purnell equation

How can efficiency be increased?

2 ways

Increasing column length
Using more efficient column

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Purnell equation

Which factor affects the resolution most?

Selectivity

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What is the principle underlying optimising resolution by adjusting retention factor?

Make solute spend less time in the mobile phase and more in the stationary

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How to increase retention factor in GC?

Lower the temperature

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What is the general elution problem?

Adjusting the retention factor to improve the resolution between one pair of solutes increases the retention times of later solutes

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3 methods to improve resolution

Adjust retension factor (k’)
Adjust column selectivity (alpha)
Adjust column efficiency (N)

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How to adjust column selectivity (alpha) in GC?

Change stationary phase

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How to adjust column selectivity (alpha) in LC?

Change mobile phase

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3 factors affecting peak broadening

Multiple paths (eddie diffusion)
Longitudinal diffusion
Resistance to mass transfer

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What is a key contributor towards Eddie diffusion?

Particle size. For larger particles the solute needs to travel further to go around it

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How can logitudinal diffusion be reduced?

Make the solute go through the column faster

# GB How does gas mass affect the diffusivity

The lighter the gas the greater the diffusivity

# GC What are the requirements for mobile phase?

1. Chemically inert 2. Must not significantly influence analyte partition coefficient between phases

# GC Name the two types of columns

1. Packed 2. Capillary (tubular)

# GC Which column is longer?

Capillary

# GC Which column is broader

Packed

# GC Which column needs less material?

Capillary

# GC What is the disadvantage of needed less material?

Difficult to inject the same amount of sample reproducubly

# GC Which column has no Eddie diffusion?

Capillary

# GC Which column has taller plates?

Packed

# GC Which phase has a greater contol over partitioning?

Stationary

# GC What 2 factors does the elution order depend on?

1. Boiling point of the analyte (aka vapour pressure) 2. Interaction between the analyte and the stationary phase

# GC What are the requirements for the stationary phase? ## Footnote 5 points

- Partitions the analyte without reacting - Does not react to mobile phase - Thermally stable - Low vapour pressure - Polarity appropriate to sample’s components

# GC What happens to gas viscosity as temperature increases?

Increases

# GC What can happen to column as it is heated?

It starts to bleed

# GC How is bleeding observed on a spectrum?

Constantly raising base line

# GC Why does column bleeding happen?

The stationary phase degrades and elutes as the column is heated

# GC 2 ways to prevent column bleeding

- a **bonded stationary phase** that is chemically attached to the capillary’s silica surface - a **cross linked stationary phase** that chemically links together separate polymer chains

# GC 3 ways of sample injection

* Direct vaporisation * Split/splitless injection * Cold on column injection

# GC Describe the principles of **direct vaporisation** | Used only for direct vaporisation

* Inject through a septum into a heated chamber * Rapid vaporisation to minimuse width of initial sample band

Describe the principles of **split/splitless injection**

* Similar to direct vaporisation, but has 2 outlets to avoid overloading * Amount of sample injected can be monitored

Describe the principles of **cold on-column injection**

* Inject cold onto column before volatilising the sample at a low T * Inject into uncoated silica to allow different droplets to collect together

# GC List some of the desired detector qualities ## Footnote 7 total, not all neeeded

- Large linear range - Universal, sensitive to all analytes or to specific class of analytes - Insensitive to flow rate or temperature change - Fast response - Non destructive of the analyte - Able to provide some information on the identity of an analyte

# GC Principle of work of **Thermal conductivity detector (TCD)**

- Mobile phase passes over Re-W filament - Its electrical resistance depends on T which depends on thermal conductivity of gas - When analyte elutes the thermal conductivity of the gas decreases causing the T of the filament and its resistance to increase

# GC Advantages of **Thermal conductivity detector (TCD)** ## Footnote 2 points

* Universal * Non destructive

# GC Disadvantages of **Thermal conductivity detector (TCD)** ## Footnote 1 point

Poor detection sensitivity

# GC Advantages of **Flame ionisation detector (FID)** ## Footnote 2 points

* Almost universal for organic compounds * Most inorganic and many gases are not detected, perfect for atmospheric and environmental samples

# GC Disadvantages of **Flame ionisation detector (FID)** ## Footnote 1 point

Destructive

# HPLC What does smaller packing material require?

The use of higher pressure to push the analyte through

Which phase is more common for HPLC? | normal/reverse

Reverse

What is the polarity of the **stationary** phase of **normal phase** chromatography?

More polar

What is the polarity of the **stationary** phase of **reverse phase** chromatography?

Less polar

# HPLC What is normal phase commonly used for?

Separation of compounds that engage in polar interactions

# HPLC What is the point of the secondary equilibrium

Introduce additional partitioning equilibria with the stationary phase and impart extra selectivity to the separation

# HPLC What is the solvent triangle used for?

Find the best separation for binary mixtures with three different mobile phases

# HPLC Why is the loop injector used instead of direct injection?

Due to high pressure

# HPLC Name 3 type of detectors

- Spectroscopic - Electrochemical - Mass spectrometer

# SFC Supercritical's fluid density is closer to liquids or gas?

Liquid

# SFC What has a greater diffusion? | Superctitical liquid/liquid

Supercritical fluid

# SFC What has a greater viscosity? | Superctitical liquid/liquid

Liquid

# Ion exchange What is the purpose of ion-suppressor

To get rid of solvent ions as both analyte and solvent are charged

# Size exclusion What material to be used for stationary phase?

Porous | eg: porous silica 5-400 nm

# Size exclusion Particles of what size elute first?

Larger

# Electrophoresis What separates the analytes

How they move through a conductive medium

# Electrophoresis What is used as a conductive medium?

An aqueous buffer in an applied electric field

# Electrophoresis Electrophoretic mobility

Movement of analyte in direct response to electric field; cations towards cathode (with E), anions towards anode (against E)

# Electrophoresis Electroosmotic flow

Movement of analyte because of buffer’s movement due to electric field, usually towards cathode (same for all analytes)

# Electrophoresis Why does buffer viscosity change?

As the current is applied the viscousity is increased

Advantages of Electrophoresis ## Footnote 4 points

- Greater resolution and peak capacity - Needs only small sample volumes - Fast - Needs only few micro litres of buffer

Disadvantages of electrophoresis

Detection limits 100-1000x poorer than GC and HPLC

Chromogentic agent

Analytes with weak or no absorbance in UV-Vis can be reacted with other specices to produce a stong absorption

Chromatography Flashcards

(75 cards)