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.

HPLC Instrumentation Overview

Principle Pattern An Example
Solvent Reservoirs Controller Solvent Cabinet Vacuum Degasser Binary Pump

Autosampler Thermostatted Column Compartment Detector

Solvent Filters

Guard column Injector Precolumn Filter

Solvent Inlet Filter

Analytical Column

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.
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Vacuum Degassing

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.

Multichannel Gradient Valve

Determines mobile phase composition. Largest solvent plug fills first. Agilent 1100 and 1200 quaternary pump.

Dual Piston Parallel Pump

Check Valves Pumphead Piston A B

Rotary Switching Valve

Single Combined Piston Delivery Delivery

Piston 'A' Advancing

Piston B Retracting

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.

Ballvalves for Reciprocating Piston Pumps

Gold Seal Sapphire Insert

Ruby Ball Spring Insert

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Pump Seals and Pistons

1 2 3 4 5

1. 2. 3. 4. 5.

Piston Support Rings Seal Keepers Seals Wear Retainers

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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.
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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.
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Gradient Formation

Low Pressure Gradient

High Pressure Gradient

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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

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Summary Video

Click picture to activate video.

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Sample Injectors

Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Two major designs: Automatic Injectors or Manual Injectors

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Manual Injectors
Sample Loop

Load - Inject

Front View

Rear View

Inject

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Manual Injectors
Sample Load
From Pump To column
Solvent in Solvent out Sample in

From Pump To column

Solvent in Solvent out

Sample in

Sample Inject
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Automatic Injectors

Step 1

Step 2

Step 3
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Rotor Seals

Rotor Seal found within valve

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Column Oven

Constant temperature for solvent and column is required to perform reproducible results.
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Common HPLC Detectors

UV-VIS Diode Array Multiple Wavelength Variable Wavelength Mass Spectrometers Refractive Index Fluorescence Light Scattering Electrochemical Radioactivity Conductivity

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WL 241/394
Py re ne C hr ys en e

Necessity for More Than One Detector Sensitivity

PAH's extracted from soil; Sup.LC-PAH 150x4.6mm; Solv.: H2O/CH3OH= 10:90

WL 270/388 WL 248/411
Be nz o( Pe e)p yr ry e le ne ne Be nz o( k) f lu or Be an nz th o( en a) e py re ne

WL 302/420

WL 247/504

Be nz o( gh i)p er In de no (

yle ne 12 3c

d) py re ne

Fluorescence

UV-signal

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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

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Necessity for More Than One Detector Qualitative Information

Qualitative Information Chlortoluron ? Atrazine ?

Take peak spectrum (UV)

Take peak spectrum (MS)

200 58 215 44 68 96 104
60 80 100 120

172 132 138158

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

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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

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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.
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UV-Vis Detectors
Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beers Law.
Detector Flow Cell

I0

c b

Log I0 = A = abc
I

Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).

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UV-Vis Detectors - Design Principles

UV Lamp

Cut-off filter Holmium oxide filter Slit

Variable Wavelength Detector

Sample diode

Mirror 1 Grating

Single wavelength detection of multi wavelength detection possible. Wavelength calibration is done automatically using a holmium filter.

Flow cell Mirror 2 Reference diode

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UV-Vis Detector with Spectral Capability

Vis Lamp Achromatic Lens UV Lamp Homium Filter Optical Slit Grating Diode Array

Detector Flow Cell

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.

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Online Spectra - UV-Vis Detector

Absorbance

Spectra

Wavelength

Time

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Fluorescence Detection
Trigger pack

Xenon flash Lamp, 15 W Exitation Monochromator, signal & spectra mode

Lens (condensor EX) Slit EM Slit EX

Emission Monochromator signal & spectra mode

Slit PMT

PMT detector Lens (condensor EM)

Mirror

Diffuser

Reference Diode 8 l Flow Cell, auto-recognition

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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.
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HPLC-MSD API- Electrospray

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Refractive Index Detector Design

The Refractive Index Detection is strongly influenced by:
Pressure changes Temperature changes Flow pulse

Gradient elution is not possible!

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Light Scattering Detector

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Conductivity Detectors

Schematics
F

Applications
water

cell C

fixed resistor Balance control Ions Acids in Bases Salts

soap products detergents

A ref.capacitor

soft drinks blood plating baths nuclear fuel reprocessing streams

variable resistances

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