HPLC Instrumentation by toVV2OW

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									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
                                   WL
                                                                                         Sensitivity


                                   241/394


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                                                                   o(




     PAH's extracted from soil;
     Sup.LC-PAH 150x4.6mm;
                                                              Pe e)p




     Solv.: H2O/CH3OH= 10:90
                                                                ry       yr
                                                                  le       e
                                                                    ne ne
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                                  302/420


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                                                                                         Necessity for More Than One Detector -




                                   Fluorescence
                                                  UV-signal




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

                                        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                                             Holmium oxide
                                                                  filter
  Detector                                                                Slit
                                   Sample
                                   diode                                               Mirror 1

 Single wavelength detection of                                      Grating
  multi wavelength detection
                                    Flow cell
  possible.

 Wavelength calibration is done                                                   Mirror 2

  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


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

                    ~
           variable
           resistances




                                                                                   39

								
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