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PMY2106 Medicinal Substances > Chromatographic methods > Flashcards

Chromatographic methods Flashcards

1
Q

What is chromatography?

A

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other mobile phase moves in a definite direction.

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2
Q

What 3 things can be obtained in chromatography?

A

separation, identification, quantification

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3
Q

How does liquid-liquid chromatography work?

A

Mobile phase - liquid

Stationary phase - liquid coated/immobilized on a solid

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4
Q

How does gas-liquid chromatography work?

A

Mobile phase - inert gas

Stationary phase - high boiling liquid

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5
Q

How does gas-solid chromatography work?

A

Mobile phase - gas

Stationary phase - suitable adsorbent

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6
Q

How does solid-liquid chromatography work?

A

Mobile phase - liquid

Stationary phase - solid immobilised on a solid

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7
Q

How are the phases chosen?

A

By making sure components of the sample have differing solubilities in each phase.

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8
Q

If a component is soluble in the stationary phase but insoluble in the mobile phase, what will the speed be like?

A

Component more soluble in stationary phase means it will take longer to travel through the column.

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9
Q

If a component is insoluble in the stationary phase but soluble in the mobile phase, what will the speed be like?

A

Component more soluble in mobile phase means it will travel faster through the column.

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10
Q

With regards to the plate model theory, how is greater separation obtained?

A
  • greater number of theoretical plates (N)

- as plate height (H) becomes smaller

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11
Q

What is the equation relating L, N and H. Define these.

A 
L = NH
L = length of column
N = number of theoretical plates
H = height of plates (or HETP = height equivalent to a theoretical plate)
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12
Q

What does it mean when a column produces sharp peaks?

A

An efficient column will produce sharp peaks which means there is effective separation taking place and higher resolution.

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13
Q

What is the problem with plate model theory?

A

It neglects the concepts of solute diffusion and flow paths.

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14
Q

Describe and define the Van Deemter equation.

A 
H = A + B/u + Cu
H = height of plates
A = multi-path or eddy diffusion
B = molecular diffusion
C = resistance to mass transfer
u = average velocity of the mobile phase
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15
Q

Describe what the A term is.

A

A term is called the eddy diffusion or multi-path term.
The term accounts for the effects of packing size and geometry.
- solute molecules take different paths through the stationary phase and this will cause broadening of the solute band because different paths are different lengths.

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16
Q

What can be done to reduce the A term?

A

Once the column is packed nothing can be done but it can be reduced before packing:

  • effect reduced by using regular sized packing
  • small diameter packing
  • firmly packed material
  • no dead space in the column
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17
Q

Describe what the B term is.

A

B term is called the molecular diffusion term.
The effect of the B term is flow dependent - as the flow is increased, the time for diffusion is reduced.
- conc of the analyte is lower at the edges of the band than at the centre of the peak as the analyte diffuses out from the centre to edges - cause band broadening. If velocity of mp is high - less time spent in column - less diffusion.

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18
Q

Describe what the C term is.

A

C term is called the resistance to mass transfer.
Resistance occurs as analyte is being pushed through a tube packed with a solid stationary phase - mass pushes against the analyte = resistance.
Even if flow velocity of mp is high, if analyte has affinity for sp then analyte in mp will move ahead result in broadened band.

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19
Q

How can the effect of the C term be minimised?

A
  • use thin coatings of the stationary phase on a solid support
  • use less viscous phases
  • keep flow as low as possible
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20
Q

Explain the principle of HPLC.

A

……………………………………….. from slides

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21
Q

How are drugs separated?

A

………………book

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22
Q

What is normal phase chromatography?

A

……..

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23
Q

What is reversed phase chromatography?

A

,,,,,,,,,,,

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24
Q

What is adsorption chromatography?

A

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25
Q

What is ion chromatography?

A

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26
Q

What is size exclusion chromatography?

A

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27
Q

What is the major disadvantage of using NPC?

A

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28
Q

What are some applications for IEC?

A

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29
Q

HPLC instrumentation is made up of 8 components, list these.

A

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30
Q

What is the purpose of the pump/solvent delivery system?

A

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31
Q

What types of detectors can be used in hplc?

A

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32
Q

How does the sample affect HPLC results?

4 points

A

iiiiii

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33
Q

How does mobile phase affect HPLC results? (4 points)

A

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34
Q

How does the detector affect HPLC results? (2 points)

A

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35
Q

How does the column affect HPLC results? (4 points)

A

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36
Q

When preparing MP, it is important to have HPLC grade reagents and highly purified buffer salts. What is the effect of any present impurities?

A

iiiiiii

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37
Q

Why is it important to degas solvents before use in hplc?

A

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38
Q

What 4 factors can affect the performance of MP?

A

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39
Q

In what 3 ways can degassing be done?

A

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40
Q

What is the most economic way to remove any gas/air bubbles for degassing procedure? Describe how it works.

A

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41
Q

The mobile phase has a powerful influence on the separation and retention of the sample. This depends on the solvent strength. What does this mean?

A

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42
Q

What is the effect of increasing solvent strength?

A

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43
Q

What is the effect of using organic solvents compared to using water in RPC?

A

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44
Q

What is the effect of using organic solvents compared to using water in NPC?

A

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45
Q

pH of MP affects elution and retention of solute/drug.

A

Drug molecule can be ionised by making the pH of the MP higher. Ionisation of drug can lead to it being eluted faster as it cannot interact with the hydrophobic stationary phase and so lower retention. Whereas, if the pH of MP is lowered, the drug will be unionised hence interacts more with the stationary phase therefore higher retention and slower elution.

46
Q

Remember the percentage ionised equation:

A
47
Q

What is the typical flow rate?

A

0.5-2ml/min

48
Q

What is the effect of higher column temperatures?

A

Lowers the viscosity of the mobile phase and reduces retention time.

49
Q

What is the purpose of a pump or solvent delivery system?

A

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50
Q

What are the typical pressures of the pump?

A

6000-9000 psi

51
Q

What does isocratic and gradient mean in terms of the pump delivering the solvent?

A

Isocratic means that a constant fixed proportion of mobile phase is being pumped through whereby gradient means that the ratio of the mobile phase (strength of MP) inceases throughout analysis. Gradient is more useful when analysing a sample containing several molecules which have different solubilities.

52
Q

What are the applications of isocratic mobile phase solvent?

A

Isocratic mobile phase solvent composition remains constant with time.

  • used for simple separations
  • used in qc applications
53
Q

What are the applications of gradient mobile phase solvent?

A

Gradient mobile phase composition increases with time.

  • used for complex samples
  • used in method of development for unknown mixtures
  • linear gradients are typical (graph)
54
Q

Compare the typical peaks obtained for isocratic and gradient mobile phase compositions.

A
  • Run time for isocratic is longer than gradient.
  • Some peaks are not clearly separated so hard to distinguish - less resolution.
  • Clear separation can be obtained in gradient.
55
Q

What is the HPLC column like?

A

Stainless steel tube packed with small particles.

56
Q

What are the materials used inside the column?

A

Silica, polymer or hybrid.

57
Q

What is silica usually like and how can it be converted to hydrophobic?

A

Its surface is usually hydrophilic with OH groups on surface. But in RPC, since hydrophilic stationary phase is required, this can be done by end-capping of polar OH groups by silylation reactions.

58
Q

Why are reactions between drug and column weak?

A

To ensure drug gets eluted from the column.

59
Q

What interactions occur in RPLC?

A

Hydrophobic interactions
Polar interactions
Ion-exchange interactions

60
Q

What is a HPLC detector?

A

The detector is part of the chromatograph that can sense the presence and measure the amount of a sample component in the mobile phase.

61
Q

What are the characteristics of an ideal detector?

A 
High sensitivity
Negligible baseline noise
Large linear dynamic range
Stable over a longer period of time
Convenient and reliable to operate
Inexpensive to purchase and operate
Response should be independent of MP composition
62
Q

What is derivatisation in HPLC?

A

Derivatisation in HPLC is used to enhance detectability - used when a drug does not show a high enough intensity of peak.

63
Q

How is derivatisation done?

A

Pre-column derivatisation is done before separation and post-column derivatisation is done after separation.
The absorbance of the drug is increased by chemically reacting the drug with a compound with a higher degree of absorbance.

64
Q

What is retention time (tR)?

A

The time between sample injection and the peak maximum.

65
Q

What is the equation used to find the retention time?

A 
tR = tR' + tM
tM = void time
tR' = time between the first peak and highest peak
66
Q

What is retention volume (VR)?

A

VR is the volume of the MP needed to elute the analyte at a given flow rate (F)

67
Q

What is void volume (VM)?

A

VM is the total volume of the liquid MP contained within the column.

68
Q

What is peak volume?

A

Peak volume/bandwidth is the volume of the MP containing the eluted peak.

69
Q

What does tailing and fronting mean?

A

Tailing is when the second part of the peak is extended whereas fronting is when the first part of the peak is extended.

70
Q

What is the ideal tailing factor range?

A

0.5

71
Q

What is the ideal asymmetry factor range?

A

0.9

72
Q

What can cause peak tailing?

A

Caused by adsorption or when sample moves slowly taking longer to elute out of column.

73
Q

What causes peak fronting?

A

Caused by column overloading or chemical reaction of the analyte during chromatography.

74
Q

What is peak resolution (Rs)?

A

Measure of the degree of separation (required for quantitation) between two adjacent analytes.

75
Q

What is the minimum separation required for quantitation? Give the value of Rs.

A

Rs = 1.0

Ideal range = 1.5 - 2.0

76
Q

Know the equation for resolution

A
77
Q

What is N?

A

N is the plate number or number of theoretical plates and it is a measure of the efficiency of the column.

78
Q

In hplc what is the main factor controlling H?

A

particle diameter of the packing controls H/HETP.

79
Q

What value is H in a well packed column?

A

2.5

80
Q

What is capacity factor/retention factor/partition ratio (k)?

A

Measure of the degree of retention of the solute by the column.

81
Q

What do the values of k mean and what is the ideal value?

A

k = capacity factor
k = 0 = solute is unretained by stationary phase
k > 20 = component is highly retained
k = 1-20 = sufficient to interact with stationary phase

82
Q

How can quantitative analysis be done?

A

by determination of peak area and peak height.

83
Q

What is an internal standard (IS)?

A

An IS is an exogenous compound which is added to both calibrators and samples at the same concentration to obtain an accurate reading.

84
Q

What are the characteristics of an ideal IS?

A
  • never be found in sample
  • well-resolved
  • stable
  • available in pure form
  • structure similar to analyte
85
Q

Why is it important that the IS has a similar structure to the analyte?

A

So that elution happens at similar times and to prevent manipulation of the whole method.

86
Q

What is polarity?

A

Number and H-bonding strength of OH groups present in the molecule.

87
Q

What is the ideal pH for the mobile phase?

A

Between pH 2 to 8.5

88
Q

What are the two most important features of the MP?

A

pH of MP

ratio of organic solvent:water

89
Q

What are the advantages of HPLC?

A
  • rapid and precise quantitative results
  • automated operation
  • high-sensitivity detection so drugs can be detected at low levels
  • amenable to diverse samples
  • quantitative sample recovery
90
Q

What are the limitations of HPLC?

A
  • there is no universal detector
  • less separation efficiency than capillary GC
  • difficult for beginners

PMY2106 Medicinal Substances (9 decks)

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