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MEDL 210 > Module 12 > Flashcards

Module 12 Flashcards

1
Q

What are the different phases of chromatography?

A

Mobile- carries sample

Stationary

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2
Q

How are components separated in ion exchange?

A

Separated by magnitude and charge of ionic species.

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3
Q

What is the stationary phase in ion exchange?

A

Ion exchanger, porous solid with surface covered in ionic sites.

Cation exchange- carries -, selects for anions

Anion exchange- carriers +, selects for cations

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4
Q

What is ion exchange used for?

A

Purifying biological materials

Removing interfering substances from a solution

Separating mixes of charged molecules

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5
Q

What is steric exclusion and how does it work?

A

Separation based in size and shape.

Stationary phase is porous and traps molecules, larger ones are excluded, move more quickly.

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6
Q

What is steric exclusion also known as?

A

Gel filtration or permeation

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7
Q

What are the uses of steric exclusion?

A

Separation of antibodies, enzymes, proteins, triglycerides.

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8
Q

What is adsorption and how does it work?

A

Separation based on competition between sample components and the mobile phase for adsorptive sites on the stationary phase.

More soluble components remain in the mobile phase and move faster.

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9
Q

What is adsorption used for?

A

Analysis of drugs, vitamins, fats, pesticides.

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10
Q

What is partition and how does it work?

A

Separation based in polarity.

Normal- mobile phase is nonpolar, polar substances bind the stationary phase and nonpolar travel faster.

Reverse- mobile phase is polar, nonpolar substances bind the stationary phase and polar travel faster.

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11
Q

Which type of partition is more common and why?

A

Reverse- safer and the sample tends to be polar based

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12
Q

What is partition used for?

A

Porphobilinogen screen

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13
Q

What is affinity and how does it work?

A

Separation based on specific interaction between one kind of solute molecule and a secondary molecule immobilized on the stationary phase (ligand).

Analyte of interest binds with the ligand, very specific.

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14
Q

What can a ligand be?

A

Antibody, protein, specific resin group.

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15
Q

What is affinity separation used for?

A

Separates glycosylated Hb from other Hbs.

Binds to boronate group of the stationary phase.

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16
Q

What is plane chromatography?

A

Separation occurs on a flat plane.

Simple screening methods.

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17
Q

What are the types of plane chromatography?

A

Paper

Thin layer chromatography (TLC)

18
Q

How does paper chromatography work?

A

Sheet of paper = stationary phase

Small volume of liquid = mobile phase

Mobile phase migrates up by capillary action, picks up the more soluble components.

Separation is visualized.

19
Q

How can separation be visualized?

A

Stains, iodine vapour or UV light.

20
Q

What is Rf?

A

Distance a component migrates compared with the distance the solvent front moves.

Component/solvent front

Compared with Rf of standards

21
Q

How does TLC work?

A

Thin layer of sorbent is coated on a glass or plastic plate (stationary phase).

Placed in jar with liquid mobile phase.

Separation based on preferential solubility.

22
Q

What is column chromatography?

A

Stationary phase is packed in a tube.

Mobile phase is passed through the tube.

23
Q

What can packing material be for column chromatography?

A

Aluminum silica gel or resin beads.

24
Q

What are the components of column chromatography?

A

Mobile phase reservoir

Injector port with septum

Column

Detector

Display output- chromatogram

25
Q

What extra component do liquid column chromatography systems require?

A

Pump in the reservoir

26
Q

How does high pressure liquid chromatography (HPLC) work?

A

Automated

Mobile phase (liquid) is driven through the column.

Eluate is collected and passes through a detector.

27
Q

What is HPLC used for?

A

Heat labile components or nonvolatile substances.

Glycosylated Hb and some drugs.

28
Q

What is gas chromatography and how does it work?

A

Mobile phase is an inert gas.

Stationary phase is a liquid distributed over the surface of a porous inert solid.

Sample must be volatile.

Temperature must be higher than the component BP to vapourize them.

Analytes exit column based on different affinities.

Can be coupled with a mass spectrometer for a more definitive ID.

29
Q

What is gas chromatography used for?

A

Alcohol analysis

30
Q

What types of detectors are there for chromatography?

A

Flame ionization detector (FID)

Thermal conductivity detectors (TCD)

e- capture detectors (ECD)

UV spectrophotometers

Linear diode arrays

31
Q

How do FIDs work?

A

Carrier has enters and forms a combustible mix which is ignited.

Compounds enter and burn incompletely.

Ions are collected.

Current flow is proportional to the amount of component burned.

32
Q

How do TCDs work?

A

Carrier has flows over the electrically heated filament, dissipates heat.

When sample mixes the amount of heat changes causing a change in conductivity proportional to component concentration.

33
Q

What compounds does ECD work for?

A

Halogenated compounds

34
Q

What is a chromatogram?

A

Record of all separate components detected, recognized by separate peaks.

Area under peak determine quantity of components.

35
Q

What is retention time?

A

Time it takes for each peak to reach the detector, used to ID components by comparing to standards.

36
Q

What is resolution?

A

Relative separation of two separate peaks.

Higher- better ID and quantification

Affected by temp and column characteristics.

37
Q

What is an internal standard and why is it used?

A

A structurally similar reference compound added in constant concentration to all samples.

Account for system variations.

38
Q

What internal standard is often used in gas chromatography?

A

N-propanol

39
Q

What issues could be encountered with chromatography?

A

Drift/baseline wander

Noisy baseline

No peaks

Short retention time

Ghost peaks

Irregular peaks

Unsymmetrical peaks

Long tails

40
Q

What is chromatography used for?

A

Separation, detection and quantitation of a mix of analytes in a sample.

Based on different physical interactions between the analytes and stationary phase.

MEDL 210 (12 decks)

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