Lecture 6
Lecture 6
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
4
Resolution
The resolution for a given stationary phase can be
improved by lengthening the column.
2 (tR2 - tR)
Rs =
W2 + W
N 1 kB N kB kA
RS RS
4 1 kB 4 1 kB
6
Resolution
N 1 kB
RS
4 1 kB
k 2’ tR2 - tM
= k2’ =
k 1’ tM
9
Resolution
11
Narrow peak
Well separated
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
Applications - Qualitative
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