Advanced Chromatographic Techniques > Section 2 > Flashcards

Section 2 Flashcards

Fundamental Resolution Equation- Chromatographic Performance (69 cards)

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1
Q

Whats the Fundamental Resolution Equation?

What does resolution depend on?

What is this equation called?

A

Purnell equation :
To various degrees, resolution is dependent on
efficiency (N), retention factor (k) and
selectivity(α). (Note: k is the retention factor of the
second peak in the calculation and N is the average
of the two peaks).

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2
Q

What does N stand for in an equation?

A

Efficiency

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3
Q

What does k stand for in an equation?

A

Retention Factor

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4
Q

What does a stand for in an equation?

A

Selectivity

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5
Q

What is important to note about k and N?

A

k is the retention factor of the
second peak in the calculation and N is the average
of the two peaks

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6
Q

What was the retention factor previously called?

A

Capacity factor

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7
Q

What does k (retention factor) measure?

A

Measures the retention of an analyte on the chromatographic column

Ratio of time which an analytes spends in
the stationary phase relative to the mobile
phase

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8
Q

The higher the retention factor…

A

the longer the
analyte spends in the stationary phase

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9
Q

Is the retention factor independent?

A

Unlike retention time, the retention factor
is independent of small differences in flow
rate and column dimensions allowing
comparison between different HPLC
systems

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10
Q

What are 3 influences on retention factor?

A
  • Mobile phase strength
  • Mobile phase pH (for ionisable compounds only)
  • Column Temperature
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11
Q

What must be the same in regards retention factor (k)

A

The stationary phase and
mobile phase composition must be the
same

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12
Q

Whats the most important equation in chromatography?

A

Fundamental Resolution Equation

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13
Q

What is the cut off number for retention?

A

10

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14
Q

Whats the best one to look at for HPLC?

A

Selectivity- bank for book

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15
Q

More selectivity for HPLC=

A

Much bigger impact on resolution = better separation

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16
Q

What does water do to retention factor?

A

More water= higher retention factor

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17
Q

What does organic solvent do to the retention factor?

A

Lowers retention factor

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18
Q

What does the selectivity factor measure?

A

The selectivity (or separation) factor (α) is a
measure of the ability of the
chromatographic system to ‘chemically’
distinguish between sample components.

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19
Q

How is the selectivity factor calculated?

A

Calculated as the ratio of the retention
factors

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20
Q

What should the selectivity factor be to have a separation for
1- GC
2- HPLC

A

Selectivity must be greater than 1 to have
separation

In GC must be at least 1.01 -1.02

In HPLC must be at least 1.5 -1.9

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21
Q

What does a high selectivity factor (a) not directly indicate?

A

a high α value does not directly indicate a high resolution value. It does
not take into account peak shape

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22
Q

What are the 5 things that influence the Selectivity Factor?

A

All parameters (α is dependent on chemistry
of analyte, stationary phase and mobile
phase)

  • Stationary phase type
  • Mobile phase organic solvent
  • Mobile phase pH (ionisable compounds)
    7
  • Column temperature
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23
Q

What is GC driven by?

A

Affinity

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24
Q

What is HPLC driven by?

A

Selectivity

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25
What is efficiency (N) a measure of?
A measure of the dispersion (broadening) of the analyte band as it moves through the chromatographic system
26
If you get a thinner peak what does that translate to?
Higher efficiency
27
If your peak is fronting or tailing what is the issue there?
Its harder to get proper Wb (With of peak at base)
28
What drives efficiency?
Theoretical plates , each TP describes interactions between s phase and m phase
29
What effects Efficiency (N)? (4)
Column length Particle Size Flow Rate Temperature/Viscosity
30
What is Resolution a measure of? (Rs)
A measure of the ability of a column to separate two analytes
31
If the peaks are Gaussian (normally distributed), a resolution of ?? should give baseline resolution (but not if there is tailing)
1.5
32
What's another way of saying peak shape?
“Peak asymmetry” or “Peak Tailing factor”
33
What is 3 causes of peak tailing?
* Instrument dead volume * Secondary adsorptive effects with the column (e.g.) residual silanols for basic compounds) * Quality of column packing
34
What are two causes of fronting?
* Sample concentration too high * Channelling in column
35
What is more common?? Peak tailing or fronting?
Peak tailing is more common than peak fronting so more likely B>A
36
What's an acceptable peak shape?
As= 1.2
37
Whats an excellent peak shape?
As= 1.0 - 1.05
38
Whats an unacceptable peak shape?
As= 2
39
Whats a very poor peak shape?
As= 4
40
What causes instrument dead volume?
Plumbing between injector and column
41
What causes quality of coulmn packing?
If column is not packed well vs peak packing
42
What is packing columns?
silica slurry poured into columns and packed under high pressure into column with no gaps = well produced column
43
What has a massive impact on how well we can pack a column?
Particle size
44
What are causes of peak distortion or tailing? (8)
* “Bad” column (plugged frit or void) * Contaminated column (build-up of strongly retained sample contaminants) * Column overload (too large a sample) * Sample solvent that is too strong * Extra-column peak broadening * Silanol interactions (including contamination of the column packing by metals) * Inadequate or inappropriate buffering * Use of a higher column temperature with cold (inadequately thermostatted) mobile phase
45
How does “Bad” column (plugged frit or void) occur?
If something gets stuck on the front of the column it gives us a disturbance in flow.
46
How does a Contaminated column (build-up of strongly retained sample contaminants) occur?
It stays in column and causes issues overtime.
47
How does the use of a higher column temperature with cold (inadequately thermostatted) mobile phase have issues arising?
When pumping at increased flow rates, the temperature can change in the column and can cause issues with peak shape
48
When increasing K what is the largest gain?
The largest gain in resolution is achieved when the k value is between 1 and 5. (But lectures are happy with 10-15 too. Once we get past 10/15, there is no point in increasing the retention factor further. Increasing retention further broadens peaks and decreases efficiency.
49
In RP chromatography a increase in k is achieved by :
reducing the concentration of strong solvent in the mobile phase. (for HPLC)
50
Increasing a : What have influences on increasing a? (5)
* All parameters (α is dependent on chemistry of analyte, stationary phase and mobile phase) * Stationary phase type * Mobile phase organic solvent * Mobile phase pH (ionisable compounds) * Column temperature
51
What is the easiest one to increase and get good?
a - selectivity factor. Easy one to get good selectivity for is mobile phase
52
Is increasing selectivity a expensive?
Relatively cheap
53
The biggest change in α can be brought about by changes in the type of
stationary phase
54
What all play a critical role in the physicochemical interaction with the analyte ?
using orthogonal column stationary phase hydrophobicity, polarity, and nature of the base silica all play a critical role in the physicochemical interaction with the analyte.
55
If we reduce particle size, we...
Increase efficiency
56
If we reduce particle size we increase efficiency but what could become an issue?
could be an issue in change in back pressure
57
What increases efficiency?
Ultra high-pressure chromatography increases pressure.
58
What is increasing N related to?
Increasing column efficiency through column length or reduction in particle size.
59
doubling the number of theoretical plates increases what?
As the resolution is proportional to the square root of the number of theoretical plates in equation, doubling the number of theoretical plates increases the resolution by a factor of √2 = 1.41
60
Example of doubling plates:
For example if we use efficiency to go from a resolution of 0.8 at 10,000 plates to a resolution of 1.5, we need 36 000 plates. This would require a column 3.6 times as long and increase the run time by X 3.6 also. * A better way to increase efficiency is to reduce particle size but this increases back pressure
61
What is efficiency (N) based on?
Width of peak
62
What controls the number of plates?
Length of column
63
Doubling of plated only increases solution by what?
1.41
64
Similar particle size does what?
Increases efficiency
65
To have a good peak shape you need to be working with between ??? numbers of plates
10-35,000
66
Separation power principles:
Selectivity -Chemical separation power, created by the physicochemical competition for compounds between the packing material and the mobile phase Selectivity is a measure of chemical separation power
67
What is selectivity a measure of?
Chemical separation power
68
Selectivity has a stronger impact on...
resolution than column efficiency
69
What is the primary cause of peak tailing ?
Its the occurrence of more than one mechanism of analyte retention. In reversed-phase separations, analyte retention is usually achieved through nonspecific hydrophobic interactions with the stationary phase. However, polar interactions with any ionised residual silanol groups residing on the silica support surface are also common. Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups, producing tailing peaks. This is illustrated in the figure shown below at a mobile phase pH >3.0.
Advanced Chromatographic Techniques
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