Chromatography Flashcards by Hira Hammad

What is chromatography used for in pharmaceutics

To analyse, identify and purify samples

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Why is solid phase extraction SPE used for sample preparation?

To remove interferences from sample
More reliable results
Concentrating analyses to improve sensitivity

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Advantages of SPEnover LLE (liq-liq extraction)?

Quick 
Less labour 
Less solvent 
More selective 
Easier to automate

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How does SPE work

Sample filtered through absorbent particles
Analytes captured from liq matrix
Concentrated analytes eluted with solvent
Eluted sample collected

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Different interactions with stationary phase

Non polar —> van der waals
Polar —> dipole dipole / h bond
Electrostatic —> ionic

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Examples of non polar

Reverse phase silica

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Examples of polar

Normal chromatography

Normal phase silica

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Examples of electrostatic

Ion exchange chromatography

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Types of absorbent used for reversed phase

C18

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Different types of chromatography?

Column chromatography 
Thin layer chromatography 
Gel filtration 
Ion exchange 
Hugh pressure liquid chromatography 
Gas chromatography

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Do all chromatography techniques have a stationary phase and a mobile phase?

YES

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what is a stationary phase?

Solid

Liquid supported on a solid

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What is a mobile phase?

Liquid
Gas
Flows through the stationary phase and carries components with it

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Do all components travel at same rates?

NO- different rates dependent on their attraction to the mobile phase and the stationary phase

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What happens in column chromatography?

Column loaded dry and filled with mobile phase- which is then flushed through the column OR column loaded with a slurry of stationary and mobile phase together (avoid bubbles)

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What is column chromatography used for?

Used to separate mixtures

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What are the stationary phases used in CC?

Silica gel - most common

Alumina- less common

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Do molecules travel at different rates?

YES- depending on their polarities

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What travels up .?

The parts dissolved in the solvent

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What decides whether a molecule remains absorbed in solid or travels with solvent

Whether the molecule prefers to be with solid phase or partition into liquid

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In TLC where are the polar and non polar molecules?

Most polar—> bottom

Non polar—> top

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Where are polar and non polar molecules in CC ?

Polar—> top

Non polar—> bottom

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Column chromatography process?

1- load stationary phase material to column
2- equilibrate column stationary phase with mobile phase
3- load sample in as small sample as possible
4- add more mobile phase to column
5- collect sample fractions from column

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Which molecules take longer to travel through?

Molecules with high affinity for stationary phase

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What is the most polar solvent

Water

Why is the polar to least polar solvents list designed?

As different drugs have different polarities | To find rf value

TLC means?

Thin Layer Chromatography

What does column chromatography consist of?

Stationary phase- solid E.g. silica Mobile phase- liquid Compounds

What does stationary phase in CC do?

Adsorbs and separates the compound passing through it

What is the eluant?

Mobile phase- liquid

What is the eluate?

Solute- solid

The most common stationary phase in CC?

Silica gel- high surface area

Why does non polar come out first in CC?

Doesn’t have much interaction with the matrix- has more interaction with solvent

What is flash column chromatography used for?

To purify mixtures

What’s is used to speed up the FCC process?

Pressure with a pump or N gas

Is the particle size smaller in FCC?

YES

In TCC as polarity increases, molecules move more…?

Slowly

Can TCC pack a lot of silica?

YES

In Thin Layer Chromatography, is silica in a column?

NO- use TLC plate- plastic/glass container with silica TLC sheet in it

Is TLC driven by gravity?

NO- capillary action takes the solvent up

Which stationary phase is usually used in TLC?

Silica

How does polarity affect TLC?

The more polar the compound- the smaller distance it will travel ( adheres to adsorbent) - lower rf value

Separation in TLC depends on?

polarity of solute and stationary phase

Can silica fluoresce?

YES

Is every component carried at the same rate with the mobile phase in TLC?

NO- different rates

Once solvent reaches the top of the plate in TLC, what is done?

Solvent removed from beaker and solvent on plate is allowed to evaporate

What colour does the plate turn under UV light?

Green

Does the sample turn green as well as the plate in TLC under a UV?

NO- sample turns dark

Why does the sample in TLC not turn green?

The double bonds/ aromatics in sample take up the UV- don’t let it fluoresce

Different substances used to develop spots in TLC?

- UV - dark spots - Iodine water- brown spots - potassium permanganate- detects sugars - ninhdydrin- purple/ print spots for amines - Alkaline tetrazolium- blue with corticosteroids

How to calculate rf value?

Distance spot travels / distance solvent travels | use centre of the spot

Uses of TLC?

- quality control and purity evaluation - basic ID check - used on BP as qualitative ID test on pure substances

What does gel filtration chromatography include?

Matrix with beads | Separates large molecules from small

Can the beads have variable pore sizes in GFC?

YES

Smaller beads come out before of after larger on GFC?

AFTER- trapped in beads

Most common stationery phase in GFC?

Dextran and agarose

Do the large molecules fit in the beads in GFC?

No- eluted in space between beads

What is the upper limit (exclusion limit) of gel in GFC??

The size above which proteins will elute space between beads

Vo (void volume) is?

Volume outside gel matrix

Ve (elation volume) is?

Volume of buffer required to elute any given substance

Use of GFC?

Desalt proteins- proteins larger than salt- salt retained in beads

What is Ion exchange chromatography?

Separates based on charge | The stationary phase (solid) binds to oppositely charged ions in sample

Is IEC done in water?

YES

What charge does stationary phase have in IEC?

+ve or -ve

Anion exchange in IEC?

Negatively charged molecules attracted to positively charged solid

Cation exchange in IEC?

Positively charged molecules attracted to negatively charged solid

Which ions elute from the column first in IEC?

Molecules with the weakest ionic interactions

Examples of anion exchangers and their strength?

Quaternary ammonium- strong Tertiary ammonium- intermediate Diethyaminoethyl- weak

Cation exchangers and their strength?

Sulphonate- strong Carboxylate- intermediate Carboxymethyl- weak

3 Factors affecting elution in IEC?

1- size of charge - 2+ show more affinity than 1+ 2- intensity of the charge- small 1+ (H+) greater affinity than larger 1+ (K+) 3- conc of ions- high conc of low affinity ions can displace low conc of high affinity

Uses of IEC?

- water purification | - isolation of metabolites from biological fluids