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PHAR 20100 > Chromatography > Flashcards

Chromatography Flashcards

1
Q

What is chromatography used for in pharmaceutics

A

To analyse, identify and purify samples

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2
Q

Why is solid phase extraction SPE used for sample preparation?

A

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

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3
Q

Advantages of SPEnover LLE (liq-liq extraction)?

A 
Quick 
Less labour 
Less solvent 
More selective 
Easier to automate
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4
Q

How does SPE work

A

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

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5
Q

Different interactions with stationary phase

A
  • Non polar —> van der waals
  • Polar —> dipole dipole / h bond
  • Electrostatic —> ionic
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6
Q

Examples of non polar

A

Reverse phase silica

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7
Q

Examples of polar

A

Normal chromatography

Normal phase silica

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8
Q

Examples of electrostatic

A

Ion exchange chromatography

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9
Q

Types of absorbent used for reversed phase

A

C18

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10
Q

Different types of chromatography?

A 
Column chromatography 
Thin layer chromatography 
Gel filtration 
Ion exchange 
Hugh pressure liquid chromatography 
Gas chromatography
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11
Q

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

A

YES

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12
Q

what is a stationary phase?

A

Solid

Liquid supported on a solid

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13
Q

What is a mobile phase?

A

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

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14
Q

Do all components travel at same rates?

A

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

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15
Q

What happens in column chromatography?

A

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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16
Q

What is column chromatography used for?

A

Used to separate mixtures

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17
Q

What are the stationary phases used in CC?

A

Silica gel - most common

Alumina- less common

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18
Q

Do molecules travel at different rates?

A

YES- depending on their polarities

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19
Q

What travels up .?

A

The parts dissolved in the solvent

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20
Q

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

A

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

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21
Q

In TLC where are the polar and non polar molecules?

A

Most polar—> bottom

Non polar—> top

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22
Q

Where are polar and non polar molecules in CC ?

A

Polar—> top

Non polar—> bottom

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23
Q

Column chromatography process?

A

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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24
Q

Which molecules take longer to travel through?

A

Molecules with high affinity for stationary phase

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25
Q

What is the most polar solvent

A

Water

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26
Q

Why is the polar to least polar solvents list designed?

A

As different drugs have different polarities

To find rf value

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27
Q

TLC means?

A

Thin Layer Chromatography

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28
Q

What does column chromatography consist of?

A

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

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29
Q

What does stationary phase in CC do?

A

Adsorbs and separates the compound passing through it

30
Q

What is the eluant?

A

Mobile phase- liquid

31
Q

What is the eluate?

A

Solute- solid

32
Q

The most common stationary phase in CC?

A

Silica gel- high surface area

33
Q

Why does non polar come out first in CC?

A

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

34
Q

What is flash column chromatography used for?

A

To purify mixtures

35
Q

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

A

Pressure with a pump or N gas

36
Q

Is the particle size smaller in FCC?

A

YES

37
Q

In TCC as polarity increases, molecules move more…?

A

Slowly

38
Q

Can TCC pack a lot of silica?

A

YES

39
Q

In Thin Layer Chromatography, is silica in a column?

A

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

40
Q

Is TLC driven by gravity?

A

NO- capillary action takes the solvent up

41
Q

Which stationary phase is usually used in TLC?

A

Silica

42
Q

How does polarity affect TLC?

A

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

43
Q

Separation in TLC depends on?

A

polarity of solute and stationary phase

44
Q

Can silica fluoresce?

A

YES

45
Q

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

A

NO- different rates

46
Q

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

A

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

47
Q

What colour does the plate turn under UV light?

A

Green

48
Q

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

A

NO- sample turns dark

49
Q

Why does the sample in TLC not turn green?

A

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

50
Q

Different substances used to develop spots in TLC?

A
  • UV - dark spots
  • Iodine water- brown spots
  • potassium permanganate- detects sugars
  • ninhdydrin- purple/ print spots for amines
  • Alkaline tetrazolium- blue with corticosteroids
51
Q

How to calculate rf value?

A

Distance spot travels / distance solvent travels

use centre of the spot

52
Q

Uses of TLC?

A
  • quality control and purity evaluation
  • basic ID check
  • used on BP as qualitative ID test on pure substances
53
Q

What does gel filtration chromatography include?

A

Matrix with beads

Separates large molecules from small

54
Q

Can the beads have variable pore sizes in GFC?

A

YES

55
Q

Smaller beads come out before of after larger on GFC?

A

AFTER- trapped in beads

56
Q

Most common stationery phase in GFC?

A

Dextran and agarose

57
Q

Do the large molecules fit in the beads in GFC?

A

No- eluted in space between beads

58
Q

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

A

The size above which proteins will elute space between beads

59
Q

Vo (void volume) is?

A

Volume outside gel matrix

60
Q

Ve (elation volume) is?

A

Volume of buffer required to elute any given substance

61
Q

Use of GFC?

A

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

62
Q

What is Ion exchange chromatography?

A

Separates based on charge

The stationary phase (solid) binds to oppositely charged ions in sample

63
Q

Is IEC done in water?

A

YES

64
Q

What charge does stationary phase have in IEC?

A

+ve or -ve

65
Q

Anion exchange in IEC?

A

Negatively charged molecules attracted to positively charged solid

66
Q

Cation exchange in IEC?

A

Positively charged molecules attracted to negatively charged solid

67
Q

Which ions elute from the column first in IEC?

A

Molecules with the weakest ionic interactions

68
Q

Examples of anion exchangers and their strength?

A

Quaternary ammonium- strong
Tertiary ammonium- intermediate
Diethyaminoethyl- weak

69
Q

Cation exchangers and their strength?

A

Sulphonate- strong
Carboxylate- intermediate
Carboxymethyl- weak

70
Q

3 Factors affecting elution in IEC?

A

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

71
Q

Uses of IEC?

A
  • water purification

- isolation of metabolites from biological fluids

PHAR 20100 (14 decks)

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