Transcript HPLC 4

HPLC Instrumentation
In This Section, We Will Discuss:
 General components of a high performance liquid chromatograph.
 HPLC solvent delivery systems.
 How automatic injectors work.
 Common HPLC detectors.
2
HPLC Instrumentation Overview
Principle Pattern
An Example
Solvent Reservoirs
Controller
Solvent Cabinet
Vacuum Degasser
Binary Pump
Autosampler
Thermostatted
Column Compartment
Detector
3
Solvent Filters
Guard
column
Injector
Precolumn
Filter
Analytical
Column
Solvent Inlet Filter
Solvent Inlet Filer
 Stainless Steel or
glass with 10 micron
porosity.
 Removes particulates
from solvent.
Precolumn Filter
 Used between the injector and
guard column.
 2 to 0.5 micron
 Removes particulates from sample
and autosampler wear debris.
 Must be well designed to prevent
dispersion.
4
Vacuum Degassing
5
Functions of the Solvent Delivery System
The solvent delivery system has three basic functions:
1. Provide accurate and constant flow.
2. Provide accurate mobile phase compositions.
3. Provide the force necessary to push the mobile phase through
the tightly packed column.
6
Multichannel Gradient Valve

Determines mobile phase composition.

Largest solvent plug fills first.

Agilent 1100 and 1200 quaternary pump.
7
Dual Piston Parallel Pump
Check
Valves
Pumphead
Rotary
Switching
Valve
Piston
A
B
Single Combined
Piston Delivery
Delivery
Piston 'A' Advancing
Piston B Retracting
8
Dual Piston in Series Pump
 First piston displaces
solvent at twice the
speed and stroke volume
of the second piston.
 Provides constant flow and the
pressure necessary to get
through column.
9
Ballvalves for Reciprocating Piston Pumps
Gold Seal
Sapphire
Insert
Ruby Ball
Spring
Insert
10
Pump Seals and Pistons
1
2
3
4
5
1.
2.
3.
4.
5.
Piston
Support Rings
Seal Keepers
Seals
Wear
Retainers
11
Frits and Filters
Purge valve
PTFE Frit
Frits, Filters, and Sieves are used to protect
other parts of the LC from pump and seal material.
12
Damping Units
Damping
Unit
Pump
Ripple
2%
P/P
Pressure
 Filled with compressible liquid separated
from the mobile phase by a membrane.
 Pressure ripples reduced to < 2% original
value.
13
Gradient Formation
Low Pressure Gradient
High Pressure Gradient
14
Summary
The pump is the most critical piece of equipment for a
successfully operating HPLC.
Performance parameters for HPLC pumps:





Flow Precision
Flow Range
Delay Volume
Pressure Pulse
Composition Precision
15
Summary Video
Click picture to activate video.
16
Sample Injectors
Requirements:
Reproducible introduction of the sample volume into the mobile
phase flow.
Two major designs:
Automatic Injectors or Manual Injectors
17
Manual Injectors
Sample Loop
Load - Inject
Front View
Rear View
Inject
18
Manual Injectors
Sample Load
From Pump
To column
From Pump
To column
Solvent in
Solvent out
Sample in
Solvent in
Solvent out
Sample in
Sample Inject
19
Automatic Injectors
Step 1
Step 2
Step 3
20
Rotor Seals
Rotor Seal
found within
valve
21
Column Oven
Constant temperature for solvent and column is required to perform
reproducible results.
22
Common HPLC Detectors
•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Light Scattering
•Electrochemical
•Radioactivity
•Conductivity
23
WL
247/504
WL
302/420
WL
248/411
WL
270/388
WL
241/394
In
de
no
(1
23
-c
d)
py
re
ne
Be
nz
o(
gh
i)p
er
yle
ne
Be
nz
o(
Pe e)p
yr
ry
e
le
ne ne
Be
nz
o(
k)
flu
or
Be
an
nz
th
o(
en
a)
e
py
re
ne
C
hr
ys
en
e
Py
re
ne
Necessity for More Than One Detector Sensitivity
UV-signal
Fluorescence
PAH's extracted from soil;
Sup.LC-PAH 150x4.6mm;
Solv.: H2O/CH3OH= 10:90
24
Necessity for More Than One Detector Selectivity
Flecainide in
Serum
UV signal
FL signal
Therapeutic concentration: 1.8mg/l, 20ul injected
UV and fluorescence signal
25
Necessity for More Than One Detector Qualitative Information
Qualitative Information
Chlortoluron
?
Take peak spectrum
(UV)
Atrazine
?
Take peak spectrum
(MS)
200
58
215
44
172
68
96 104
60
Wavelength (nm)
80
100
120
132 138158
140
160
180
200
220
Mass/Charge
26
HPLC Detector Characteristics
Detector performance characteristics:
 Sensitivity (LoD, LoQ)
 Selectivity
 Linearity
 Qualitative information
 Reliability
 Ease of use
 Universality
27
LOD
The limit of detection for a detector can be characterized by its
signal to noise ratio (S/N) for an analyte under a given set of
conditions.
Peak
Noise
28
Limit of Detection - Limit of Quantitation
Response
Linear range
Slope = sensitivity
MQL
MDL
Intercept
e.g.,RSD<10%, S/N > 20
e.g., S/N > 3
Amount
 Limit of detection (LOD) is a result of the whole chromatography system,
not only the detector performance
 Limit of quantification (LOQ) is a defined limit for a method used for a
specific purpose.
29
UV-Vis Detectors
Principles: The fraction of light transmitted through the detector cell is
related to the solute concentration according to Beer’s Law.
Detector Flow Cell
c
I0
I
b
Log I0 = A = abc
I
Characteristics: Specific, Concentration Sensitive, good stability,
gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).
30
UV-Vis Detectors - Design Principles
UV Lamp
Cut-off filter
Variable Wavelength
Detector
Holmium oxide
filter
Slit
Sample
diode
 Single wavelength detection of
multi wavelength detection
possible.
 Wavelength calibration is done
automatically using a holmium
filter.
Mirror 1
Grating
Flow cell
Mirror 2
Reference diode
31
UV-Vis Detector with Spectral Capability
Vis
Lamp
Achromatic
Lens
Detector
Flow Cell
UV
Lamp
Diode Array
Homium
Filter
Optical
Slit




Grating
Diode Array UV-Vis Detector allows online measurement of spectra.
Wavelength range 190 - 950 nm.
Wavelength Resolution: Up to 1 nm.
Wavelength calibration with Holmium oxide filter.
32
Online Spectra - UV-Vis Detector
Spectra
Absorbance
Wavelength
Time
33
Fluorescence Detection
T
r
i
g
g
e
r
p
a
c
k
E
m
i
s
s
i
o
n
M
o
n
o
c
h
r
o
m
a
t
o
r
s
i
g
n
a
l
&
s
p
e
c
t
r
a
m
o
d
e
L
e
n
s
(
c
o
n
d
e
n
s
o
r
E
X
)
X
e
n
o
n
l
i
t
P
M
T
S
l
i
t
E
M S
S
l
i
t
E
X
f
l
a
s
h
L
a
m
p
,
P
M
T
d
e
t
e
c
t
o
r
1
5
W
L
e
n
s
(
c
o
n
d
e
n
s
o
r
E
M
)
E
x
i
t
a
t
i
o
n
M
o
n
o
c
h
r
o
m
a
t
o
r
,
s
i
g
n
a
l
&
s
p
e
c
t
r
a
m
o
d
e
M
i
r
r
o
r D
i
f
f
u
s
e
r
R
e
f
e
r
e
n
c
e
D
i
o
d
e
a
u
t
o
r
e
c
o
g
n
i
t
i
o
n
8
µ
l
F
l
o
w
C
e
l
l
,
34
Electrochemical Detectors
Thin-layer design




Wall-jet design
Porous flowthrough design
Gold for carbohydrates.
Platinum for chlorite, sulfate, hydrazine, etc.
Carbon for phenols, amines.
Silver for chloride, bromide, cyanide.
35
HPLC-MSD API- Electrospray
36
Refractive Index Detector Design
The Refractive Index Detection is
strongly influenced by:
 Pressure changes
 Temperature changes
 Flow pulse
Gradient elution is not possible!
37
Light Scattering Detector
38
Conductivity Detectors
Schematics
Applications
F
water
fixed
resistor
r
cell
C
A ref.capacitor
D
Balance
control
E
soap products
Ions
Acids in
Bases
Salts
detergents
}
soft drinks
blood
plating baths
nuclear fuel reprocessing
streams
B
~
variable
resistances
39