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Lecture 6

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Lecture 6

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Selectivity factor () and column resolution (Rs)

 The ability of a column to resolve two solutes is related to


the relative magnitude of the partition coefficients for the
two solutes. K = CS/CM
 The selectivity factor () for two solutes is defined as
K2
 =  is always greater than unity
K1
 Solute 2 is more strongly retained (larger retention time)
and solute 1 is less strongly retained (smaller retention
time).
 Since K2 = k2’ VM / VS and K1 = k1’ VM / VS , then
k2’ Where k2’ and k1’ are the capacity factors
 =
k1’ for solute 2 and solute 1 respectively.
Selectivity factor ()

 The selectivity factor () is equal to 1 when both the


solutes elute with identical retention time. k2’
 =
  is greater than 1 when tR2 is greater than tR1. k1’

k2’ = (tR2 - tM) / tM k1’ = (tR1 - tM) / tM

 Then, the selectivity factor () will be


tR2 - tM
 =
tR1 - tM

 The  value to be calculated from the chromatogram


experimentally.
Selectivity factor () and column resolution (Rs)

 The resolution, Rs, provides a quantitative measure of its


ability to separate two analytes.
 The resolution of column for two components is defined as
the ratio of the peak separation to the mean peak width.

2 (tR2 - tR1)
Rs =
W2 + W 1
Resolution
Poor resolution peaks
not well resolved, no
baseline to baseline
resolution

A resolution of 1.5
gives an essentially
complete separation of
A and B

Good resolution peaks


well resolved, baseline
to baseline resolution

4
Resolution
 The resolution for a given stationary phase can be
improved by lengthening the column.
2 (tR2 - tR)
Rs =
W2 + W

 The number of plates (N) increases.


 The time required for the separation increases.
Resolution
 the column resolution can be related to the number of
theoretical plates (N), the selectivity factor (), and the
retention factors of both species kA and kB

N    1  kB  N  kB  kA 
RS     RS   
4    1  kB  4  1  kB 

where kB = retention factor of the slower moving species


 = selectivity factor of a pair of solutes on the
column
 this formula is used to optimise the column performance

6
Resolution

N    1  kB 
RS    
4    1  kB 

k 2’ tR2 - tM
= k2’ =
k 1’ tM

Each factor can be calculated directly from the


recorded chromatogram and can be adjusted more or
less independently.
Resolution
N    1  kB 
RS    
4    1  kB 
 to obtain high resolution, the 3 terms must be maximised
• an increase in N, by lengthening the column  increase in
retention time & increase band broadening
• more desirable to reduce plate height H by reducing the
particle size of the stationary phase
(1) lowering the flow rate of the mobile phase,
(2) decreasing the particle size of the column packing,
(3) reducing the thickness of the film of liquid stationary phase and
(4) working at high temperatures for decreased viscosity of the
mobile phase.
8
Resolution N    1  kB 
RS    
4    1  kB 

 separations can be improved by controlling the retention


factor, k. This can be achieved by changing the temperature
(in GC) or the composition of the mobile phase (in LC)
 Increase in the value of capacity factor, k2’, enhances
resolution (better separation). However, large capacity
factors mean increased elution time.
 The capacity factor , k2’, can be increased by increasing the
stationary phase volume.
 A change in the capacity factor is an indication of
degradation of the stationary phase.

9
Resolution

 k2’ > 10 are to be avoid


because little increase in
resolution and markedly
increase in time for
separations.
Minimum elution time  Good resolution with
k’ = about 2 minimum elution time, lies
in the range of k’ = 1 to 5.
k’ = (tR - tM) / tM
Resolution
N    1  kB 
RS    
4    1  kB  Longer tR

 when selectivity factor,  close to unity, optimising k and


increasing N is not sufficient to give separation in
reasonable time
 we must first try to increase this selectivity factor  but
without changing the retention factor k too much
 Optimising column performance needs a lot of work!
 Most of the time, it’s just about experience, imagination,
trial & error

11
Narrow peak

Well separated

Selectivity factor = (tR2 – tM) / (tR1 – tM) = k2 / k1 = K2 / K1


Efficiency N = 16(tR/W)2
Retention factor k = (tR - tM)/tM
k’ = (tR - tM)/tM

Fig.26-13
Elution time for complete separation
 Obviously, what is desired in chromatography is the highest
possible resolution (Rs) in the shortest possible elapsed time (tR).
 The time for completion of a separation is determined by the
velocity (v) of the slower-moving solute 2 according to

v = L / tR2 Rearrangement of Rs:


2 2
Velocity of solute 2  1 + k2’
N = 16 Rs2
From, - 1 k2’
1
V = u x and L = N H Substituting into (tR2) gives:
1 + k2’
2 3
16 Rs H2 1 + k2’

N H (1 + k2’) tR2 =
tR2 = u  - 1 2
k2’
u

Which gives the time required for


the complete analysis
EXAMPLE:

Applications - Qualitative

tR=1.0 min tR=2.1 min


standard standard
Y Y

tR=2.1 min
tR=1.0 min
sample sample
containing containing
Y Y

on column A on column B 15
EXAMPLE:

Applications - Qualitative
Qualitative analysis
Soft Drink Components
Column: GENESIS FM10964E (C8EC, 2
4 m, 4.6 mm i.d. x 100 mm) Standard solution
1
Flow: 1.8 mL/min
Eluent: Isocratic, 25% MeOH in 40 mM 34
6
5 8
phosphate + 10 mM tetra-n-butyl- 7
ammonium hydrogen sulphate,
pH 3.1 2
Detection: Absorbance, 220 nm Cola Soft Drink
Peaks: 1. Uracil, 2. Caffeine, 3. Impurity, 4
8
4. Aspartame, 5. Acesulfame-K,
1 5
6. Sodium saccharin, 7. Potassium 3
sorbate, 8. Sodium benzoate
Chromatograms in association with C.
Walker, Brilvic Soft Drinks Ltd, UK.
16
EXAMPLE:

Applications - Quantitative
Quantitative analysis
For quantitative analysis, peak area is
usually used as a quantitative measure of
the amount of solute eluted
 operating conditions must be
standardised
 detector response for each sample
component must be determined
 the area of a chromatographic peak
must be directly proportional to the
amount of the solute eluted
17
• Describe different type of mobile and
stationary phases used in HPLC analysis

• Describe Normal and reverse phase HPLC??


Give appropriate examples in each phases??
• Exercise

1. Draw a schematic of an HPLC instrument.


Elaborate the functions of the instruments?

2. Explain how particle size in the packing


material of HPLC column effects the
efficiency and resolution of the
chromatographic peaks?

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