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PHAR 20100 > HPLC > Flashcards

HPLC Flashcards

1
Q

HPLC stands for?

A

High Performance Liquid Chromatography

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2
Q

Composition of HPLC?

A
  • mobile phase flowing over a stationary phase packed in a column
    solvents–> pump–> injection valve–> column–> detector
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3
Q

What is the stationary phase in HPLC?

A

fine silica particles

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4
Q

What is the column made of in HPLC?

A

stainless steel

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5
Q

Can air bubbles be present? Why?

A

NO- will interfere with analysis and detection- huge peaks detected

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6
Q

How to get rid of bubbles?

A

Degas

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7
Q

method to set up the HPLC?

A
  1. solvent prep- filtered via 0.45microm pore & degassed
  2. mixing valve
    3- pump control- deliver mobile phase
    Solvents—> pump—> injection valve—> column—> detector—> out
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8
Q

How to perform separation in HLPC?

A
  • Equilibrate column with starting solvent
  • inject analyte sample using injection valve
  • analytes separated be interaction with stationary phase inside column
  • detection E.g . UV achieved
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9
Q

Two ways to achieve separation?

A

Normal phase

Reverse phase

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10
Q

What is normal phase HPLC?

A

the mobile phase is non polar- organic
Stationary phase- polar
Solute is retained by interaction of its polar func groups with polar groups on surface on stationary phase

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11
Q

What is reverse phase HPLC?

A

Mobile phase is water- polar

Stationary phase Is hydrophobic- non polar

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12
Q

What elutes first in reverse phase?

A

Most polar analyte

Others follow in order of decreasing polarity

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13
Q

What’s commonly used as stationary phase in NP?

A

Microporus silica

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14
Q

What’s commonly used as mobile phase in NP?

A

Non polar solvent E.g. hexane, chloroform

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15
Q

What elutes first in NP HPLC?

A

Least polar comes first- doesn’t stick

Followed by others in order of inc polarity

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16
Q

Is NP good for water soluble analytes?

A

NO- retained too strongly

17
Q

Uses of NPC?

A

Separation of analytes with low polarity and high solubility in low polarity solvents such as hexane

18
Q

Analyte examples for NPC?

A

Lipids
Oils
Phospholipids
Prostaglandins

19
Q

Most common method of separation and analysis in pharm?

A

Reverse phase HPLC

20
Q

How can the stationary phase be made less polar

A

Adding more alkyl groups

21
Q

How to Analyse drugs using RPC?

A

Partitions lipophilic portion of drug molecule to bonded stationary phase
Forms van der waals
When conc of organic modifier = high enough to break interactions—> partitions into mobile - elutes

22
Q

How can resolution/ performance/ peaks be improved?

A

Longer column length
Slower flow rate
Smaller stationary phase particles- higher SA
less steeper gradient

23
Q

Can the peak be assymmetrical?

A

NO- affects calculation when area under curve is measures

24
Q

How to perform quantitative analysis of HPLC?

A

Measuring peak height

Measure in peak areas

PHAR 20100 (14 decks)

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