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Analytical methods > HPLC > Flashcards

HPLC Flashcards

1
Q

Describe the HPLC system

A

Consist of a solvent connected to a intricite system of tubes.
- System includes (in order) a pump, Injector (introduces sample), HPLC coloumn, detector

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2
Q

Purpose of the solvent?

A

Used as a mobile phase

  • Often have one aqueous and one organic
  • Can have a single mixed solvent
  • Can use isocratic or gradient elution
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3
Q

What are some general addatives to solvent in HPLC?

A

Addatives:

  • Buffers for pH control
  • Salts
  • Ion-pair reagents e.g. ion pair reagent allow hydrophobic stationary phase to bind onto ionic compounds (hydrophilic)

Form MS:

  • Need something to promote adducts
  • Acids because MS require analyte ions to be able to detect
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4
Q

How many pumps are used in an ioscratic gradient?

A

Only one

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5
Q

What is a typical HPLC coloum?

A

Stainless steel that can withstand high pressures, with packing material inside. Packing material is held in place by a frit

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6
Q

What is a guard coloum?

A

Protects the more expensive analytical coloum from particulates. May contain packing material or just a filter

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7
Q

HPLC particles are usually…..

A. 80-150um
B. 45-80nm
C. 2.5-15um
D. 300-400um

A

C

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8
Q

What are some potential functional groups added to the particles?

A

C8, C18, C4, phenyl, amide, CN, PFP, HILIC

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9
Q

With examples to the phases how can resolution improved?

A

Via the mobile phase:

  • Composition
  • Flow rate
  • Gradient/isocratic
  • pH
  • Temp

Column

  • Phase
  • Length - theoretical plates
  • Particles and pore size - affect eddy diffusion
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10
Q

How can the mobile phase be manipulated to increase to optimisation of chromatography?

A

Isocratic - constant composition

Gradient - composition changes throughout the run

Gradient mobile phase is more attractive in when there are multiple analytes

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11
Q

What are some variables to the gradient?

A

Range- % organic at start and end

Time - shallow or steep gradient

Always important to ensure to reequilibriate the column before next sample

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12
Q

Non-polar compounds need what % of organic solvent to elute?

A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%

A

D

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13
Q

Low/medium polarity compounds need what % of organic solvent to elute?

A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%

A

B

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14
Q

Polar compounds need what % of organic solvent to elute?

A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%

A

C and may require specialised chromatography - HILIC

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15
Q

What is the eluotropic series?

A

Not all solvents are equal and can be classified according to polarity

More non-polar compounds will require a more non-polar sovlent for elution from the stationary phase.

A good starting point is a medium polarity and go up or down the series as required

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16
Q

Why does pH need to be optimised?

A

pH affects ionisable analytes as two different ions will exist in the same sample depending where the pH is relative to pKa of the analyte.

17
Q

How does temperature affect the column?

A

AS temp increase the efficiency of the column as the resistance to mass transfer (C) decrease

So therefore more mass transfer will occur in the column

Note. Mass transfer is the time it takes to equilibriate between the phases by the analyte

18
Q

What are some polar groups?

A

Hydroxyl, carboxyl, amino, phosphate, carbonyl, sulphydryl (R-CH2-SH)

19
Q

Examples of polar analytes

A

Metanephrine, normetanephrine, tobramycine

20
Q

Name some non-polar groups?

A

Methyl

21
Q

Example of non-polar analyte

A

25-OH-VitD

Some parts are polar but majority is non-polar

22
Q

With example why is it important to regard the analytes size?

A

For chromatography using pores.
- 99% of chromatographic surface is inside the pores

Mobile phase must be allowed into the pore in order for chromatographic retention of the analyte to take place

23
Q

How is pore size adjusted to enhance chromatographic results?

A

Especially important for analysing larger molecules, e.g. peptides

If the analyte is too large then chromatography will simply not occur and analyte will elute.

24
Q

How is column length adjusted to enhance chromatographic results?

A

Remember the more theoretical plates the better resolution.

  • BUT increased column length results in higher back pressure and longer run times
  • The run time can be decreased by increasing flow rate, but causes an increase to back pressure

The increased back pressure can severily impact the lifetime of the column

25
Q

How can the the column efficiency be adjusted?

A

By decreasing the particle size the number of particle plates increase.
- Thus, increasing column efficiency

Smaller more regular particles decrease eddy diffusion- good to decrease peak broadening

26
Q

What characteristics of the analytes are useful in choosing column type?

A 
Ionic groups  
Polar groups
Hydrophobicity
Double bonds 
Size 
Benzene rings
27
Q

What is the most common column type in clinical setting?

A

C18 column (very common)

  • Hydrophobic phase good for retention of non-polar analytes, e.g. testosterone
  • C18 has many variations with end-capping and other side chains which increase pH stability or analyte retention. E.g. Butyl side chains improve the peak shape
28
Q

When is a C8 column more favourbale than C18?

A
  • Good for analytes that are retained for too long on C18

e. g. 25-OH Vit D

29
Q

When is a phenyl column applicable?

A
  • Used for the seperation of aromatic compounds
  • Seperate based on hydrophobic and pi-pi interactions
    e. g. retinol or vit E
30
Q

Describe one modification of the phenyl column.

A

Penta-fluoro phenyl column

Four different retention mechanism:

  • Polar interactions
  • Dipole-dipole
  • pi-pi
  • Hydrophobic

Beware - acetonitrile suppresses pi-pi interactions so avoid using with PFP column

31
Q

How does a cyano column achieve seperation?

A

By hydrophobic interactions (propyl group), dipole interactions with CN group

  • Can be used for normal or RP chromatography
  • In past, this phase was not very robust
32
Q

What is a hydrophobic interaction liquid chromatography (HILIC)?

A

Used for retention of very polar compounds that are not retained well in reverse phase chromatogrpaphy

  • An acetonitrile-rich mobile phase is used in the inital condition; analytes are eluted using aqueous mobile phases
  • Elution pattern is similar to normal phase chromatography but the organic/aqueous mobile phase combination used is similar to reverse phase
33
Q

Why might amide columns be more appropriate than HILIC?

A

Provide wider pH and temp stability and suitbale for use with 100% aqueous mobile phases

Analytical methods (15 decks)

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