HPLC Flashcards

1
Q

What is the mobile phase concentration like during isocratic elution?

A

The composition of the mobile phase remains the same for the entire time.

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2
Q

What is the mobile phase concentration like during gradient elution?

A

The composition of the mobile phase is adjusted according to a pre set schedule

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3
Q

What is the advantage of gradient elution over isocratic elution?

A

Gradient elution can reduce assay time and achieve better separation and peak shapes

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4
Q

What are the 2 most common detectors used for HPLC?

A

UV vis and Mass spectometry

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5
Q

What is the equation to measure absorbance from transmittance?

A

A = lg (1/T)

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6
Q

What is the equation to measure absorbance from light intensity?

A

A = lg (Io/I)

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7
Q

What are the two equations of beer lamberts law?

A

A = εbc
A = A1% bc

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8
Q

For the equation A = εbc, what are the units of each of the variables?

A

b: cm
c: mol/l

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9
Q

For the equation A = A1% bc, what are the units of each of the variables?

A

b: cm
c: g/100ml

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10
Q

What is the difference between normal phase and reverse phase HPLC?

A

Normal phase: the stationary phase is polar and the mobile phase is non polar
Reverse phase: the stationary phase is non- polar and the mobile phase is polar

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11
Q

What is usually used for the stationary phase for normal-phase HPLC?

A
  1. silica
  2. silica with polar organic moieties
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12
Q

What is usually used for the stationary phase for reverse-phase HPLC?

A
  1. silica with non-polar organic moieties
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13
Q

In reverse phase HPLC, what are the non-polar chains attached to the silica?

A

C8, C12, C18

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14
Q

What are the common solvents used in normal phase HPLC?

A

Hexane
Dichloromethane
Isopropanol
Methanol

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15
Q

What are the common solvents used in reverse phase HPLC?

A

Water (buffer)
Methanol
Acetonitrile
Tetrahydrofuran

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16
Q

How do intermolecular interactions affect retention time?

A

If the interactions between the molecule and the stationary phase of the column are strong, then the retention time is greater.

17
Q

What are common factors that determine elution strength?

A
  1. Mobile phase concentration
  2. Temperature
  3. (For reversed phase) pH and ionic strength
18
Q
A