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 Analytical
Filter Column
Solvent Inlet Filter

Solvent Inlet Filer Precolumn Filter

 Stainless Steel or  Used between the injector and
glass with 10 micron guard column.
porosity.  2 to 0.5 micron
 Removes particulates  Removes particulates from sample
from solvent. 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
Rotary
Switching
Valve
Pumphead

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. Piston
1 2. Support Rings
3. Seal Keepers
4. Seals
2 5. Wear
3 Retainers
4
5

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
Solvent in
Solvent out Sample in
To column

From Pump
Solvent in
Solvent out Sample in
To column

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
Necessity for More Than One Detector -
Sensitivity

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UV-signal

248/411
270/388
241/394

247/504
302/420
WL
WL

WL

Fluorescence

WL
WL
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 Atrazine
? ?

Take peak spectrum Take peak spectrum

(UV) (MS)

200

58

215
44
68 172

96 104 132 138158

60 80 100 120 140 160 180 200 220

Wavelength (nm) 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 e.g.,RSD<10%, S/N > 20

MDL e.g., S/N > 3

Intercept

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

I0 c I

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 Holmium oxide

filter
Detector Slit
Sample
diode Mirror 1

 Single wavelength detection of Grating

multi wavelength detection
Flow cell
possible.
Mirror 2
 Wavelength calibration is done
automatically using a holmium Reference diode
filter.

31
UV-Vis Detector with Spectral Capability

Vis
Lamp
Achromatic
Lens
Detector Diode Array
Flow Cell
UV
Lamp

Homium
Filter
Grating
Optical
Slit

 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

Absorbance
Spectra

Wavelength

Time

33
Fluorescence Detection

Trigger pack Emission

Monochromator
signal &
Lens spectra mode
(condensor EX)
Xenon Slit EM Slit PMT
flash Lamp, Slit EX
PMT detector
15 W

Lens (condensor EM)

Exitation
Mirror Diffuser
Monochromator,
signal & Reference Diode
spectra mode

8 µl Flow Cell, auto-recognition

34
Electrochemical Detectors

Thin-layer design Wall-jet design Porous flow-

through 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
r fixed soap products
cell
resistor
detergents
C Ions

A ref.capacitor D
Balance
control E
Acids in
Bases
Salts
} soft drinks
blood
plating baths
nuclear fuel reprocessing
streams
B

~
variable
resistances

39