1 introduction hplc process lecture 1 yuri kazakevich seton hall university
Post on 21-Dec-2015
225 Views
Preview:
TRANSCRIPT
4
HPLC Retention
Major parameters, • VR is retention volume, depends on the column type,
size, and the instrument parameters
• Vo is dead volume, volume of the liquid phase inside the column
• k’ is retention factor (capacity factor),
independent of the column size and instrument setup
kV V
VR o
o'
RR tFV
5
Retention Characteristics
• Retention factor, k’• Selectivity, a • Efficiency, N
General recommendations:• Optimize retention factor between 1 – 10• HPLC selectivity should be > 1.2• Maximize efficiency
2
'1
'2
0
0 16;;'
b
RR
w
VN
k
k
V
VVk
RR tFV
6
Dead Volume
Vo, Dead volume is the volume of the liquid phase in the column
Simple rule: Column dead volume = 65% of the volume of empty column 4
65.02
0
LDV
e
10
Efficiency
2
16
b
R
w
VN
N
LHETP
Reduced HETP is a measure of how well the column is packed.
pd
HETPh
11
Column Efficiency
• Column length is a compromise between the efficiency and backpressure
• Column efficiency is proportional to the column length• Specific efficiency (# of particles per one plate) decreases
with an increase of column length
Length [cm]
Particle Dia. [um]
Efficiency, N
Specific Efficiency, h
10 3 11111 3 10 5 10526 1.9 15 5 13636 2.2 25 5 15625 3.2 25 10 10000 2.5
14
Parameters affecting efficiency:• Flow rate• Column length• Particle diameter• Particle size distribution
Parameters affecting retention factor:• Eluent type• Eluent composition• Stationary phase type• Analyte nature
Parameters affecting selectivity:• Stationary phase type• Analyte nature• Eluent additives• Temperature• Eluent composition (ionizable analytes)
Factors Influencing HPLC Separation
15
Reversed Phase Separation Principle
• Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)
• Difference in analyte sorption affinities results in their separation
• More polar analytes retained less
• Analytes with larger hydrophobic part are retained longer
• Almost no separation of structural isomers
1211109876543210Retention Time (min)
50
100
150
200
250
300
350
400
Respo
nse
1.6
8
2.2
3
5.9
4
9.0
5
9.8
4
16
Retention Process(Surface Equilibria)
SKVVR 0
KV
S
V
VVk R
0
0
0'
RT
GG
RT
GK eluentanalyteexpexp
0
ln)'ln(V
S
RT
GGk eluentanalyte
17
Retention Factor, k’
Retention factor is a measure of the analyte competitive interactions with the stationary phase
RT
G
RT
G
RT
Gk eluentanalyteexpexp'
0
5
10
15
20
25
30
0 2 4 6
alkylbenzenes, nc
70
80
90
100
-2
-1
0
1
2
3
4
0 1 2 3 4 5 6
nc
70%
80%
90%
100%
%MeCN
18
Reversed-Phase HPLC Retention: Neutral Analytes
Dependencies of retention of alkylbenzenes, alkylphenones, and alkylparabenes plotted against the number of carbon atoms in alkyl chain.
ln(k’)= m (#carbon atoms in alkyl chain) + b
19
Selectivity
– Eluent composition• Ideally does not have any effect on the selectivity
for neutral compounds
k
kk k
G
RT
G
RT
G
RT
G
RT
G
RT
G
RT
eluent eluent
'
'exp ln( ' ) ln( ' )
exp
exp
1
21 2
1 2
1 2
20
Eluent Composition Effect on Selectivity
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7
0
5
10
15
20
25
0 1 2 3 4 5 6 7
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6 7 8 9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
90% MeCN
80% MeCN
70% MeCN
60% MeCN
21
Eluent Composition Effect on Selectivity
c%
Peak numbers
2,1 3,2 4,3
70 1.75 1.6 1.45
80 1.72 1.58 1.4
90 1.7 1.65 1.43
100 1.71 1.62 1.42
02468
101214161820
70 80 90 100
Eluent composition
1
2
345
22
-4
-3
-2
-1
0
1
2
3
4
0 20 40 60 80 100
MeOH [v/v%]
Alkylpyridines vs. eluent composition on Luna-C18
Eluent Composition EffectMeOH/Water
top related