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MEDL-210 MB > Module 12: Chromatography > Flashcards

Module 12: Chromatography Flashcards

1
Q

what is chromatography used for

A

separation, detection and quantitation of a complete mixture of analytes in a sample

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2
Q

What is separation based on

A

different physical interactions between individual components and the stationary phase of the system

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3
Q

2 phases

A

mobile and stationary

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4
Q

Mobile phase

A

carries the sample through the stationary phase

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5
Q

sample compounds that have a greater affinity for the mobile phase will prefer to

A

stay in it

causing them to travel faster than those samples that have a greater affinity to remain in the stationary phase

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6
Q

Ion exchange

A

solute mixtures are separated by the charge of ionic species

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7
Q

Ion exchanger

A

stationary phase

porour solid whose surface is covered with ionic sites (pos or neg)

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8
Q

2 types of Ion exchange

A

cation: stationary phase carries neg charge - selects cations from sample
anion: stationary phase carries pos charge - selects anions from sample

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9
Q

Steric exclusion

A

gel filtration/gel permeable
solute mixtures separated based on size and shape
stationary phase is porous and traps molecules inside pores

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10
Q

Adsorption

A

based on competition between the components in the sample and the mobile phase for adsoptive sites on the solid stationary phase
components stick to sites on surface of stationary phase “velcro”

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11
Q

Partition

A

separation is based on polarity
Normal or reversed
As sample moves through column with mobile phase, separation is obtained as the polar or non polar compounds interact with he two phases

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12
Q

normal partition

A

mobile phase is NONPOLAR

Stationary phase is POLAR

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13
Q

Reverse partition (more common)

A

mobile phase is POLAR

Stationary phase is NONPOLAR

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14
Q

Affinity

A

separation based on specific interaction between one kind of solute molecule and a second molecules that is immobilized on a stationary phase
Immobilizing molecule called a ligand

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15
Q

ligand

A

immobilizing molecule in affinity chromatography
may be an antibody, protein or specific resin group intended to bind a specific analyte
most selective type of chromatography used
Can be used to separate glycosylated hemoglobin (hba1c) from other hemoglobins
binds to boronate group of stationary phase

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16
Q

2 large groups of chromatography

A

plane and column

17
Q

plane chromatography

A

paper

thin layer chromatography

18
Q

paper chromatography

A

sheet of paper (filter paper) is used as the stationary phase
aliquot of sample near bottom of paper, placed in jar with small amount of liquid in the bottom.
Liquid mobile phase will migrate up paper by capillary action and pick up components of the sample that are more soluble in the mobile phase
Paper is visualized suing stains, iodine vapour or UV light

19
Q

Rf

A

racing factor
distance a component migrated, compared with the distance the solvent front moves

rf = Distance leading edge of component moves / total distance solvent front moves
Each sample component Rf is compared with he Rf of standards to identify the component

20
Q

Thin layer chromatography (TLC)

A

similar to paper chromatography
thin layer of sorbent is coated onto a glass or plastic plane (acts as stationary phase)
separated based on preferential solubility

21
Q

Column chromatography

A

stationary phase is packed into a tube called a column
mobile phase carries sample through column
1) mobile phase reservoir (additional pump required for liquid system)
2) injector port with septum
3) column
4)detector
5)display output

High pressure liquid chromatography
Gas chromatography

22
Q

High pressure liquid chromatography

A

liquid mobile phase is driven through the column using the pressure generated by a pump
As liquid passes through column, the ELUATE is collected at the end and passed through a detector (UV spec)
Used for glycosylated hemoglobins

23
Q

Gas chromatography

A 
Mobile phase is an inert gas
Sample used must be volatile 
sample is injected, temp of infection port must be above the boiling point of the components
Often used for alcohol analysis
Often coupled with a mass spec
24
Q

Types of detectors for Hight pressure liquid and gas chromatography

A

Flame Ionization Detector (FID) Most common
Thermal Conductivity Detectors
Electron Capture detectors (most sensitiv)
UV spec
Linear Diode Arrays- consist of a line of detection diodes arranged in a line. Each diode is dedicated to a few wavelengths. Advantage is that multiple different compounds can be detected at once

25
Q

automated chromatography process

A

liquid sample injected through septum into the column using a needle and glass syringe
Each component elutes from the column at a different time and is recorded as an individual peak by detector

26
Q

chromatogram

A

record of all separate components that have been detected

Each component is recognized by separate

27
Q

retention time

A

the time it takes for each component peak to reach the detector after sample injection
Is used for the ID of each specific component
Area under peak is used to determine quantity of the individual components in a sample

28
Q

Resolution

A

relative separation of 2 separate chromatographic peaks

29
Q

Internal standard

A

structurally similar reference compound that is added in constant concentration to help account for system variations such as low them or flow rate
n-propanol used for gas chromatography

30
Q

Errors: no peaks

A

leaky septum, detector not responding/not lit

31
Q

Errors: ghost peaks

A

contaminated syringe
Not waiting for previously injected sample to elute
Poor injection technique

32
Q

Errors: irregular shaped peaks

A

poor injection, overloading the column, poor column prep

33
Q

Peaks with long tails

A

water contamination of column

MEDL-210 MB (13 decks)

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